sirolimus and Colorectal-Neoplasms

The mechanistic target of rapamycin (mTOR) integrates growth factor signals with cellular nutrient and energy levels and coordinates cell growth, proliferation and survival. A regulatory network with multiple feedback loops has evolved to ensure the exquisite regulation of cell growth and division. Colorectal cancer is the most intensively studied cancer because of its high incidence and mortality rate. Multiple genetic alterations are involved in colorectal carcinogenesis, including oncogenic Ras activation, phosphatidylinositol 3-kinase pathway hyperactivation, p53 mutation, and dysregulation of wnt pathway. Many oncogenic pathways activate the mTOR pathway. mTOR has emerged as an effective target for colorectal cancer therapy. In vitro and preclinical studies targeting the mTOR pathway for colorectal cancer chemotherapy have provided promising perspectives. However, the overall objective response rates in major solid tumors achieved with single-agent rapalog therapy have been modest, especially in advanced metastatic colorectal cancer. Combination regimens of mTOR inhibitor with agents such as cytotoxic chemotherapy, inhibitors of vascular endothelial growth factor, epidermal growth factor receptor and Mitogen-activated protein kinase kinase (MEK) inhibitors are being intensively studied and appear to be promising. Further understanding of the molecular mechanism in mTOR signaling network is needed to develop optimized therapeutic regimens. In this paper, oncogenic gene alterations in colorectal cancer, as well as their interaction with the mTOR pathway, are systematically summarized. The most recent preclinical and clinical anticancer therapeutic endeavors are reviewed. New players in mTOR signaling pathway, such as non-steroidal anti-inflammatory drug and metformin with therapeutic potentials are also discussed here.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Autophagy; Colorectal Neoplasms; Drug Resistance, Neoplasm; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; MAP Kinase Signaling System; Metformin; Models, Biological; Prognosis; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53; Wnt Proteins

There has been a strong preclinical rationale for studying mammalian target of rapamycin (mTOR) inhibitors as single agents or in combination, in multiple malignancies and colorectal cancer in particular.. The authors summarize the complete clinical experience to date of all trials, both published and in abstract form, of everolimus in colorectal cancer. While initial Phase I trials showed promise, further studies have confirmed that single agent everolimus is not active in advanced metastatic colorectal carcinoma with trials showing single agent tolerability, but without significant hints of efficacy in terms of either objective tumor responses or prolonged stable disease. Combination regimens, including combinations with cytotoxic chemotherapy, and inhibitors of VEGF, EGFR and HDAC have been tested specifically in the colorectal setting in Phase I and Phase II clinical trials. The authors discuss the potential reasons for mixed results and suggest future directions for the development of everolimus in colorectal malignancies.. Studies demonstrate limited clinical activity of everolimus for the treatment of advanced colorectal cancer and have been complicated by increases in toxicity. However, the central role of the PI3K/mTOR pathway in cancer biology suggests that other drug combinations with mTOR inhibition may still merit evaluation.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Colorectal Neoplasms; Disease-Free Survival; Drug Screening Assays, Antitumor; Everolimus; Humans; Molecular Structure; Sirolimus; TOR Serine-Threonine Kinases

Colorectal cancer is the third leading cause of cancer death in Japan and the United States and is strongly associated with obesity, especially visceral obesity. Several metabolic mediators, such as adiponectin, have been suspected to play a role in obesity-related carcinogenesis. In a previous human study, the existence of a significant correlation between the number of human dysplastic aberrant crypt foci (ACF) and the visceral fat area was demonstrated, and also that of a significant inverse correlation between the number of dysplastic ACF and the plasma adiponectin level. Other studies have investigated the effect of adiponectin under the normal and high-fat diet conditions in a mouse model of azoxymethane-induced colon cancer. Enhanced formation of both ACF and tumors was observed in the adiponectin-deficient mice, as compared with that in the wild-type, under the high-fat diet condition but not under the normal diet condition. Furthermore, that the 5'-AMP-activated kinase/mammalian target of rapamycin pathway is involved in the promotion of colorectal carcinogenesis in adiponectin-deficient mice under the high-fat diet condition was shown. Therefore, that the 5'-AMP-activated kinase/mammalian target of rapamycin signaling pathway may play an important role in colorectal carcinogenesis was speculated. Metformin, a biguanide derivative widely used in the treatment of diabetes mellitus, has been shown to exert a suppressive effect on ACF formation in both mouse models and humans. Therefore, metformin might be a promising candidate as a safe drug for chemoprevention of colorectal carcinogenesis. Further studies with high evidence levels, such as randomized, controlled studies, are needed to clarify these relationships.

Topics: Aberrant Crypt Foci; Adiponectin; AMP-Activated Protein Kinases; Animals; Cell Proliferation; Cell Transformation, Neoplastic; Colon; Colorectal Neoplasms; Diet, High-Fat; Disease Models, Animal; Epithelial Cells; Humans; Hypoglycemic Agents; Metformin; Mice; Obesity, Abdominal; Risk Factors; Signal Transduction; Sirolimus

To determine the maximum tolerated dose (MTD) of the combination of linsitinib (OSI-906), a dual inhibitor of IGFR and IR tyrosine kinase activity, and everolimus as treatment for patients with refractory metastatic colorectal cancer (mCRC).. Eligible adult patients with refractory mCRC, Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, and adequate end-organ function received escalating doses of OSI-906 and everolimus in a 3 + 3 design. Treatment continued until disease progression or unacceptable toxicity, with response evaluations every 8 weeks.. Eighteen patients with metastatic CRC were treated. There were no dose-limiting toxicities (DLTs) in the first dose level (DL, OSI-906 50 mg BID; everolimus 5 mg QD). At DL2 (OSI-906 100 mg BID; everolimus 10 mg QD, n =6), three patients had DLTs considered related to everolimus (grade 3 mucositis, 2; grade 3 thrombocytopenia, 1). An amendment introduced DL2a (OSI-906 100 mg BID; everolimus 5 mg QD, n =5); DLTs were seen in two patients (one patient each: grade 3 thrombocytopenia with bleeding; inability to receive 75 % of doses due to neutropenia/thrombocytopenia). DL1 was the MTD; a total of 7 patients were treated at this dose. Common adverse events across all DLs included grade 1/2 fatigue (50 %) and anorexia (50 %). There were no objective responses to treatment; median time of study treatment was 7.6 weeks (range: 3.9-53 weeks).. The MTD of OSI-906 and everolimus was 50 mg BID and 5 mg QD, respectively. No indications of clinical activity were observed in refractory mCRC patients.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Colorectal Neoplasms; Everolimus; Female; Humans; Imidazoles; Male; Maximum Tolerated Dose; Middle Aged; Protein Kinase Inhibitors; Pyrazines; Receptor, IGF Type 1; Sirolimus

To evaluate feasible doses of weekly everolimus and irinotecan given with cetuximab for previously treated metastatic colorectal cancer (mCRC).. Adults with mCRC that progressed after 5-fluorouracil or capecitabine-plus-oxaliplatin were treated using a sequential dose escalation scheme. Dosing decisions were based on the probability of experiencing a dose-limiting toxicity (DLT) during the first two 21-day treatment cycles.. Patients received everolimus 30 mg/week plus irinotecan 350 mg/m2 q3w (n=5; dose A1) or everolimus 30 mg/week plus irinotecan 250 mg/m2 q3w (n=14; dose B1). Among patients evaluable for the maximum tolerated dose, two out of four in A1 and one out of eight in B1 experienced four DLTs. The trial was terminated early based on changes in clinical practice and emerging data on everolimus dosing.. The feasible doses of everolimus and irinotecan administered with cetuximab as second-line therapy in mCRC were 30 mg/week and 250 mg/m2, respectively.

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Camptothecin; Cetuximab; Colorectal Neoplasms; Everolimus; Female; Humans; Irinotecan; Male; Middle Aged; Mutation; Neoplasm Metastasis; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Sirolimus

This phase I study investigated the safety, dose-limiting toxicity, and efficacy in three cohorts all treated with the mTOR inhibitor everolimus that was delivered (1) in combination with 5-fluorouracil with leucovorin (5-FU/LV), (2) with mFOLFOX6 (5-FU/LV + oxaliplatin), and (3) with mFOLFOX6 + panitumumab in patients with refractory solid tumors.. Patients were accrued using a 3-patient cohort design consisting of two sub-trials in which the maximum tolerated combination (MTC) and dose-limiting toxicity (DLT) of everolimus and 5-FU/LV was established in Sub-trial A and of everolimus in combination with mFOLFOX6 and mFOLFOX6 plus panitumumab in Sub-trial B.. Thirty-six patients were evaluable for toxicity, 21 on Sub-trial A and 15 on Sub-trial B. In Sub-trial A, DLT was observed in 1/6 patients enrolled on dose level 1A and 2/3 patients in level 6A. In Sub-trial B, 2/3 patients experienced DLT on level 1B and subsequent patients were enrolled on level 1B-1 without DLT. Three of six patients in cohort 2B-1 experienced grade 3 mucositis, and further study of the combination of everolimus, mFOLFOX6 and panitumumab was aborted. Among the 24 patients enrolled with refractory metastatic colorectal cancer, the median time on treatment was 2.7 months with 45 % of patients remaining on treatment with stable disease for at least 3 months.. While a regimen of everolimus in addition to 5-FU/LV and mFOLFOX6 appears safe and tolerable, the further addition of panitumumab resulted in an unacceptable level of toxicity that cannot be recommended for further study. Further investigation is warranted to better elucidate the role which mTOR inhibitors play in patients with refractory solid tumors, with a specific focus on mCRC as a potential for the combination of this targeted and cytotoxic therapy in future studies.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cohort Studies; Colorectal Neoplasms; Dose-Response Relationship, Drug; Drug Monitoring; Early Termination of Clinical Trials; Everolimus; Female; Fluorouracil; Humans; Leucovorin; Male; Middle Aged; Mucositis; Neoplasm Metastasis; Neoplasms; Organoplatinum Compounds; Oxaliplatin; Panitumumab; Protein Kinase Inhibitors; Severity of Illness Index; Sirolimus; TOR Serine-Threonine Kinases

Patients with chemotherapy refractory metastatic colorectal cancer and KRAS mutations have no effective treatment option. The present study evaluated the efficacy of temsirolimus in chemotherapy refractory mCRC with KRAS mutations. Furthermore, we wanted to investigate if resistance to temsirolimus could be reversed by the addition of irinotecan. Finally, we analyzed pre-treatment blood samples for KRAS mutations to investigate the association between quantitative measures of KRAS mutated alleles and clinical outcome.. Patients received weekly temsirolimus 25 mg until progression. Thereafter patients were treated with combination therapy comprising biweekly irinotecan 180 mg/m(2) and weekly temsirolimus. A polymerase chain reaction method was used to quantify the KRAS mutated alleles in plasma (pKRAS).. Sixty-four patients were included. Treatment was well tolerated. Thirty-eight percent achieved stable disease on monotherapy and 63% on combination therapy. Four and eight patients had a minimal response, respectively. Median overall survival was 160 days. Median time to progression was 45 and 84 days, respectively. The concordance between KRAS status in tumor and plasma was 82%. All patients with tumor reduction had low levels of pKRAS. Patients with high pKRAS had a 77% risk of early progression on monotherapy compared to 43% in patients with lower levels. Multivariate survival analysis confirmed that pKRAS was a strong prognostic factor.. Temsirolimus has limited efficacy in chemotherapy resistant KRAS mutant disease, but plasma KRAS quantification is a strong predictor of outcome.

Topics: Adenocarcinoma; Aged; Antineoplastic Combined Chemotherapy Protocols; Camptothecin; Colorectal Neoplasms; Disease Progression; Drug Resistance, Neoplasm; Female; Humans; Irinotecan; Male; Middle Aged; Mutation; Proportional Hazards Models; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Sirolimus; Treatment Outcome

Treatments that target the vascular endothelial growth factor (VEGF) pathway have efficacy in colorectal cancer. We evaluated tolerability and efficacy of tivozanib (an oral VEGF receptor-1, -2, -3 inhibitor) plus everolimus (an oral mammalian target of rapamycin inhibitor).. The phase Ib study followed a 3 + 3 dose-escalation design with three dose levels. The primary objective in the follow-on phase II study was improvement in 2-month progression-free survival (PFS) from 30% (historical benchmark) to 50% in patients with refractory, metastatic colorectal cancer.. Dose-limiting toxicities in the phase Ib study were grade 3 fatigue and dehydration. Oral tivozanib (1 mg daily for 3 of 4 weeks) and oral everolimus (10 mg daily continuously) were advanced to a 40-patient phase II study. The most common grade 3-4 adverse events were thrombocytopenia and hypophosphatemia. The 2-month PFS rate was 50%, with 20 of 40 patients having stable disease (SD). Seven (18%) patients were treated for ≥6 months. Median PFS and overall survival (OS) times were 3.0 months (95% confidence interval [CI]: 1.9-3.6 months) and 5.6 months (95% CI: 4.4-10.6 months), respectively. Patients who developed grade 1+ hypertension had increased SD rates (65.2% vs. 29.4%) and longer OS times (10.6 vs. 3.7 months).. The oral combination of tivozanib and everolimus was well tolerated, with stable disease achieved in 50% of patients with refractory, metastatic colorectal cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Colorectal Neoplasms; Disease-Free Survival; Drug-Related Side Effects and Adverse Reactions; Everolimus; Humans; Neoplasm Staging; Phenylurea Compounds; Quinolines; Sirolimus; Treatment Outcome

Dysregulation of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway is seen in 40% to 60% of patients with colorectal cancer. Everolimus, an oral inhibitor of mTOR, showed efficacy in patients with metastatic colorectal cancers in phase I studies.. In sequential phase II studies assessing two dosing schedules, patients with metastatic colorectal cancers refractory to bevacizumab-, fluoropyrimidine-, oxaliplatin-, and irinotecan-based regimens received everolimus 70 mg/wk (n = 99) or 10 mg/d (n = 100). Primary endpoints were disease control rate (DCR) and objective response rate; secondary endpoints included progression-free survival (PFS), overall survival (OS), and duration of response or stable disease (SD). Tumor tissue was collected from all patients for predefined exploratory biomarker analyses.. Seventy-one patients were included in the per-protocol set for each cohort. DCRs of 31.0% and 32.4% (all SD) were seen in the weekly and daily schedules, respectively. Median duration of SD was 3.9 months in each cohort. Median PFS and OS were 1.8 and 4.9 months and 1.8 and 5.9 months, respectively, for the weekly and daily schedules. Among patients receiving daily everolimus, those with a KRAS mutation experienced significantly shorter median OS (P = 0.008) and lower DCR (P = 0.035) compared with those with wild-type KRAS in exploratory biomarker analyses.. Everolimus 70 mg/wk or 10 mg/d was well tolerated but did not confer meaningful efficacy in heavily pretreated patients with metastatic colorectal cancers. Future studies may consider evaluating everolimus in combination with other agents or in patients with dysregulation of the PI3K/Akt/mTOR pathway.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Camptothecin; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; Disease-Free Survival; DNA Mutational Analysis; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Irinotecan; Kaplan-Meier Estimate; Male; Middle Aged; Organoplatinum Compounds; Oxaliplatin; Phosphatidylinositol 3-Kinases; Prospective Studies; Proto-Oncogene Proteins B-raf; Pyrimidines; Sirolimus; Treatment Outcome

Preclinical data suggest that combining the mTOR/hypoxia-inducible factor (HIF) inhibitor temsirolimus and the antiangiogenesis antibody bevacizumab may augment antitumor activity as well as resensitize cells to anthracyclines.. We initiated a phase I study of bevacizumab and temsirolimus plus liposomal doxorubicin in patients with advanced malignancies. Patients (N = 136) were enrolled according to a modified 3 + 3 design plus dose expansion in responsive tumor types.. The most common cancers were breast (n = 29), epithelial ovarian (n = 23), and colorectal cancer (n = 17). The median number of prior chemotherapy regimens was four (range: 0-16). Grade 3 or higher adverse events (> 5%) included pancytopenia, mucositis, hand-foot syndrome, hypertension, and fistula. This regimen led to a 21% (n = 28) stable disease (SD) ≥ 6 months and 21% (n = 29) rate of partial or complete remission [PR/CR; (total SD ≥ 6 months/PR/CR = 42% (n = 57)]. PR/CR was most common in parotid gland adenocarcinoma (4/6, 67%), metaplastic breast cancer (5/12, 42%), endometrial endometrioid carcinoma (6/15, 40%), and in patients with a PIK3CA mutation and/or a PTEN mutation/loss (11/28, 39%). The maximum tolerated dose was liposomal doxorubicin 30 mg/m(2) and bevacizumab 15 mg/kg every three weeks with temsirolimus 25 mg weekly.. Patients tolerated bevacizumab and temsirolimus together with liposomal doxorubicin. Further evaluation, especially in patients with parotid, metaplastic breast, and endometrial endometrioid cancer, and in patients with PIK3CA and/or PTEN aberrations is warranted.

Topics: Adolescent; Adult; Aged; Angiogenesis Inhibitors; Antibiotics, Antineoplastic; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Breast Neoplasms; Carcinoma, Ovarian Epithelial; Colorectal Neoplasms; Disease-Free Survival; Doxorubicin; Drug-Related Side Effects and Adverse Reactions; Female; Humans; Maximum Tolerated Dose; Middle Aged; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Protein Kinase Inhibitors; Sirolimus

We performed a single institution, phase I study of sirolimus and bevacizumab, in order to determine the dose limiting toxicity (DLT) and recommended phase II doses.. Eligible patients had previously treated advanced malignancies and were enrolled in three cohorts. Sirolimus 90 mg PO weekly (45 mg on days 1 and 2) was combined with bevacizumab 7.5mg/kg (cohort #1) or bevacizumab 15 mg/kg (cohort #2) IV q3weeks. Sirolimus 4 mg PO daily was combined with bevacizumab 15 mg/kg IV q3weeks (cohort #3).. Twenty-eight patients enrolled. The most common tumour types were colorectal (21%), head/neck (14%), and renal cell (11%). No DLTs were observed in cohorts #1 (4 patients) and #2 (12 patients), while two DLTs (grade 3 confusion and grade 3 fatigue) were observed in the first six patients in cohort #3 (12 patients). The most common grade 3 toxicities were fatigue (18%), hypertension (14%) and anorexia (11%). There were no responses, but one patient has had stable disease for 78 weeks.. The combination of sirolimus and bevacizumab at full doses is tolerable in the majority of patients. The availability and cost of sirolimus compared with other mTOR inhibitors make this an attractive agent to combine with bevacizumab.

Topics: Adult; Aged; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Carcinoma, Renal Cell; Cohort Studies; Colorectal Neoplasms; Female; Humans; Kidney Neoplasms; Male; Maximum Tolerated Dose; Middle Aged; Neoplasms; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome

For patients with metastatic colorectal cancer (mCRC), no standard therapy exists after progression on 5-fluorouracil, oxaliplatin, irinotecan, bevacizumab, and cetuximab or panitumumab. Preclinical data demonstrated that combined vascular endothelial growth factor and mammalian target of rapamycin inhibition has greater antiangiogenic and antitumor activity than either monotherapy. A phase I study of bevacizumab plus everolimus demonstrated that the combination is safe; activity was seen in several patients with refractory mCRC.. Fifty patients with refractory mCRC were enrolled and received bevacizumab at 10 mg/kg every 2 weeks and everolimus at 10 mg orally daily.. Of the 50 patients enrolled, the median age was 56 years and the median number of prior regimens was four. Forty-seven patients (96%) had prior bevacizumab exposure and 42 patients (84%) had documented progression on prior bevacizumab-based therapy. Forty-nine patients were evaluable for response; eight patients had minor responses (16%) and an additional 15 patients (30%) had stable disease (SD). No complete or partial responses were seen. The median progression-free survival interval was 2.3 months; however, 26% of patients achieved prolonged SD for ≥6 months, and three patients (6%) were on study for >1 year. The median overall survival duration was 8.1 months. The most common grade 1-2 toxicities were mucositis (68%) and hyperlipidemia (64%). Clinically significant grade ≥3 toxicities included hypertension (14%), fistula/abscess/perforation (8%), mucositis (6%), and hemorrhage (2%).. Bevacizumab plus everolimus is generally tolerable but may have risks related to mucosal damage and/or wound healing. Bevacizumab plus everolimus appears to have modest activity in refractory mCRC in patients.

Topics: Adult; Aged; Angiogenesis Inhibitors; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Colorectal Neoplasms; Disease-Free Survival; Everolimus; Humans; Immunosuppressive Agents; Middle Aged; Sirolimus; Treatment Outcome

To determine the pharmacokinetics and safety of RAD001 (everolimus) in Japanese patients with advanced solid tumors.. An open-label, non-randomized, dose-escalation Phase I study of RAD001 administered continuously once daily in a 28-day cycle was performed. The study had a '3 + 3' design, with three patients recruited to each of three successive cohorts treated with RAD001 at 2.5, 5.0 or 10.0 mg/day.. The pharmacokinetics of RAD001 in Japanese patients were similar to those previously determined in Caucasians. The drug safety profile was consistent with that of a mammalian target of rapamycin inhibitor. No dose-limiting toxicities were observed. One patient with esophageal cancer and one with gastric cancer treated with RAD001 at 10 mg/day showed marked tumor responses.. Treatment of Japanese cancer patients with RAD001 may be undertaken with the expectation that previously determined pharmacokinetic and safety profiles apply. The drug may hold promise for treatment of esophageal and gastric cancer.

Topics: Aged; Asian People; Colorectal Neoplasms; Dose-Response Relationship, Drug; Drug Administration Schedule; Esophageal Neoplasms; Everolimus; Female; Humans; Immunosuppressive Agents; Liver Neoplasms; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Neoplasms; Sirolimus; Stomach Neoplasms; Survival Analysis; Thyroid Neoplasms; Treatment Outcome

Temsirolimus (CCI-779) is a novel inhibitor of the mammalian target of rapamycin. This Phase 1 study was aimed at investigating the maximum-tolerated dose, toxicity, pharmacokinetics and antitumor activity in Japanese patients with advanced solid tumors.. Temsirolimus was given as a 30 min intravenous infusion once a week. Patients with solid tumors not amenable to standard forms of treatment were eligible. Dose escalation of temsirolimus was planned from 15, 45, 80 to 165 mg/m(2). The pharmacokinetics of temsirolimus and sirolimus in whole blood were examined for cycles 1, 2, 4 and 5 of treatment.. Ten patients (median age 60.5 years; range 41-69 years) with advanced solid tumors were enrolled. Their primary cancers were renal cell carcinoma (five patients), lung cancer (three patients) and colorectal cancer (two patients). The major toxicities were hypophosphatemia diarrhea, hyperglycemia, stomatitis, pyrexia, elevated aspartate aminotransferase, rash, reduced neutrophil count, elevated alanine aminotransferase, anorexia, hypertriglyceridemia and somnolence. Two of three patients who received temsirolimus 45 mg/m(2) developed dose-limiting toxicities of Grade 3 stomatitis (one patient) and Grade 3 diarrhea (two patients). The maximum-tolerated dose was 15 mg/m(2). The peak blood concentrations of temsirolimus and sirolimus, a major active metabolite, increased in a dose-dependent manner. The area under the concentration-versus-time curve of sirolimus, but not temsirolimus, increased in a dose-dependent manner.. The recommended dose for Phase 2 clinical studies of temsirolimus in Japanese patients with advanced solid tumors is 15 mg/m(2) intravenously once a week.

Topics: Adult; Aged; Anti-Allergic Agents; Antineoplastic Agents; Area Under Curve; Carcinoma, Renal Cell; Colorectal Neoplasms; Diarrhea; Diphenhydramine; Disorders of Excessive Somnolence; Drug Administration Schedule; Drug Eruptions; Fever; Humans; Hyperglycemia; Hypertriglyceridemia; Hypophosphatemia; Infusions, Intravenous; Intestinal Perforation; Kidney Neoplasms; Lung Neoplasms; Maximum Tolerated Dose; Middle Aged; Neoplasms; Neutropenia; Premedication; Sirolimus; Stomatitis; Treatment Outcome

The second-generation mammalian target of rapamycin (mTOR) inhibitor PP242 has demonstrated limited success in some rapamycin-insensitive tumours. We examined the therapeutic potential of combining PP242 with adenosine 50- monophosphate-activated protein kinase (AMPK) activator metformin, using a panel of colorectal carcinoma (CRC) cell lines. We found that the PP242 and metformin combination enhanced the suppression of CRC cell proliferation, colony formation, and cancer cell apoptosis induction. The effect of this combination was observed on AMPK phosphorylation. Western blotting showed that PP242 inhibited mTORC1 activation, as indicated by the reduced expression of its major substrate p-S6K1 and the partially reduced phosphorylation of eIF4E-binding protein 1 (4E-BP1). The inhibition of mTORC2-mediated AKT phosphorylation at Ser 473 (AKT Ser473) was transient and occurred in the first few hours of PP242 treatment; metformin exposure decreased the PP242 activity, counteracting AKT activation. We further demonstrated that this was related to direct AMPK-mediated phosphorylation of IRS-1 at Ser789. Thus, the combination of PP242 and metformin completely blocked the activity of both mTORC1 and mTORC2 kinase. This study suggests that this combination could be a more effective strategy for the treatment of CRC.

Topics: AMP-Activated Protein Kinases; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Humans; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Metformin; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The mammalian target of rapamycin (mTOR) is often activated in several cancers. We focused on two mTOR regulatory mechanisms: oxaliplatin-induced mTOR signaling and L-type amino acid transporter 1 (LAT1)-induced mTOR activation. High LAT1 expression in several cancers is associated with mTOR activation and resistance to chemotherapy. However, the significance of LAT1 has not yet been elucidated in colorectal cancer (CRC) patients treated with post-operative adjuvant chemotherapy. Immunohistochemistry was conducted to examine the significance of membrane LAT1 expression in 98 CRC patients who received adjuvant chemotherapy, including oxaliplatin. In vitro analysis was performed using CRC cell lines to determine the effects of LAT1 suppression on proliferation, oxaliplatin sensitivity, and mTOR signaling. LAT1 expression was associated with cancer aggressiveness and poor prognosis in 98 CRC patients treated with adjuvant chemotherapy. We found that positive LAT1 expression correlated with shorter survival in 43 patients treated with the capecitabine-plus-oxaliplatin (CAPOX) regimen. LAT1 suppression in CRC cells inhibited the proliferation potency and oxaliplatin-induced activation of mTOR signaling, and improved oxaliplatin sensitivity. LAT1 evaluation before adjuvant treatment may therefore be a sensitive marker for oxaliplatin-based regimens. Moreover, LAT1 may be a promising target for patients with refractory CRC.

Topics: Antineoplastic Combined Chemotherapy Protocols; Chemotherapy, Adjuvant; Colorectal Neoplasms; Fluorouracil; Humans; Oxaliplatin; Sirolimus; TOR Serine-Threonine Kinases

The purpose of this paper is to promote the medical treatment of colorectal cancer in our country and to save the lives of patients with colorectal cancer by studying mammalian target of rapamycin (mTOR) and the biologic information analysis of colorectal cancer.. We analyzed mTOR expression and survival differences using data from Coad & read from the TCGA public database and explored the coexpression regulatory network of mTOR. mTOR-regulated mirnas were screened using the Linked Omics database. In addition, we explored the association of mTOR with drug sensitivity, immune cell correlations, microsatellite deletions, tumor mutational burden, and mutational analysis.. The expression and survival of mTOR were significant different in colorectal cancer, and were related to the sensitivity of Bleomycin, Cisplatin and Gemcitabine. mTOR is associated with dendritic cell activation, NK cell dormancy, dendritic cell dormancy, and eosinophil granulocyte. mTOR is associated with microsatellite deletions, and tumor mutational load.. Based on these findings, we consumer mTOR as a biomarker for the diagnosis and prognosis of colorectal cancer.

Topics: Cell Line, Tumor; Colorectal Neoplasms; Humans; MicroRNAs; Prognosis; Sirolimus; TOR Serine-Threonine Kinases

Mucin 3A (MUC3A) is overexpressed in colorectal cancer (CRC) and associated with poor prognosis, but the related mechanism remains unclear. Our study found that MUC3A promotes the progression of CRC by activating the PI3K/Akt/mTOR signaling pathway. Knockout of MUC3A significantly inhibited the proliferation of CRC cells and induced G1 phase arrest by upregulating p21 protein, an important cell cycle regulator. Moreover, knockout of MUC3A significantly inhibited invasion ability and enhanced the sensitivity to the chemotherapeutic agent 5-FU. Furthermore, we found that knockout of MUC3A repressed the PI3K/Akt/mTOR pathway through RNA-seq. Treatment with the PI3K/Akt/mTOR pathway inhibitor rapamycin successfully eliminated the difference in proliferation, invasion and chemoresistance between MUC3A knockout cells and control cells. Our study suggests that MUC3A is a potential oncogene that promotes the proliferation, invasion, and chemotherapy resistance of CRC. Moreover, CRC patients with high expression of MUC3A may benefit from rapamycin treatment.

Topics: Cell Movement; Cell Proliferation; Colorectal Neoplasms; Humans; Mucin-3; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases

In digestive system, colorectal cancer (CRC) is a common malignant tumor. The phosphatidylinositol 3-kinase/protein kinase-B/mammalian target of the rapamycin (PI3K/AKT/mTOR) signaling pathway plays a central role in CRC, and the aberrant activation of this pathway is associated with tumorigenesis. We aimed to explore the role of Rho GTPase activating protein 9 (ARHGAP9) in the progression of CRC as well as its regulatory effects on the PI3K/AKT/mTOR pathway.. The expression of ARHGAP9 in CRC tumor tissues and cell lines were detected using reverse transcription-quantitative PCR (qRT-PCR). 5-ethynyl-2'-deoxyuridine (EdU) assay was applied to test the cell proliferation. Cell migration and invasion were both assessed through transwell assay. Xenograft mouse models were constructed to explore the effects of ARHGAP9 on CRC in vivo. The expressions of PI3K/AKT/mTOR-activating factors and epithelial-mesenchymal transition (EMT)-related factors were all determined using western blot. LY294002 was employed to block PI3K/AKT/mTOR pathway in CRC cells.. The expression of ARHGAP9 was down-regulated in CRC tumor tissues and cell lines when compared to normal tissues and cells. The over-expression of ARHGAP9 inhibited cell proliferation, invasion, migration and EMT in CRC cell lines while the knockdown of ARHGAP9 promoted them. In addition, ARHGAP9 up-regulation inhibited the activation of PI3K/AKT/mTOR signaling pathway in CRC cell lines while ARHGAP9 down-regulation led to an opposite effect. The over-expression of ARHGAP9 suppressed CRC tumor growth in vivo. When the PI3K/AKT/mTOR pathway was blocked in CRC cells, the effects of ARHGAP9 knockdown on cell proliferation, migration, invasion and EMT were all overturned.. ARHGAP9 inhibited the malignant phenotypes of CRC cells via interdicting PI3K/AKT/mTOR signaling pathway.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; GTPase-Activating Proteins; Humans; Mammals; Mice; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

This work firstly scrutinized the effect of KIF4 on the progression of CRC. KIF4 expression in CRC clinical tissues and cells was evaluated by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Cell counting kit-8 assay, Transwell invasion and migration assay were implemented to research the function of KIF4 on the proliferation, invasion and migration of CRC cells. The effect of KIF4 on the autophagy and the Hedgehog pathway activityavtivity in CRC cells was explored in the presence or absence of rapamycin and ring propylamine. The expression of autophagy-related proteins was scrutinized by qRT-PCR, Western blot and immunofluorescence. Autophagosomes in CRC cells was observed by transmission electron microscopy. In vivo xenograft experiment was executed. Immunohistochemistry of xenograft tumor tissues was executed to investigate the Hedgehog pathway activityactivtiy. KIF4 was abundantly expressed in CRC clinical tissues and cells. KIF4 enforced the proliferation, invasion, migration of CRC cells, repressed the autophagy and activated the Hedgehog pathway in CRC cells. Rapamycin and ring propylamine treatment reversed the inhibition of KIF4 on the autophagy and the promotion of KIF4 on the Hedgehog pathway activity in CRC cells. Ring propylamine treatment reversed the inhibition of KIF4 on the autophagy in CRC cells. KIF4 intensified the in vivo growth of CRC cells and activated the Hedgehog pathway in xenograft tumor tissues. KIF4 acted as an oncogene in CRC by inhibiting the autophagy via activating the Hedgehog pathway. It might be a potential target for CRC treatment.

Topics: Autophagy; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Hedgehog Proteins; Humans; Kinesins; Neoplasm Invasiveness; Propylamines; Sirolimus

As expected, Rapa/Lps and 5-FU significantly suppressed tumor formation, decreased the number of tumors, and tumor load both in two mouse models, and had no influence on mouse weight. Mechanically, the anti-tumor effect of the drug also was associated in inhibiting angiogenesis and proliferation. Furthermore, we found that Rapa/Lps obviously inhibited HUVECs tube formation and migration.. Altogether, we revealed the Rapa/Lps synergism with 5-FU decreased colon and small intestinal tumorigenesis in AOM/DSS-treated and APC

Topics: Animals; Azoxymethane; Colitis; Colorectal Neoplasms; Dextran Sulfate; Disease Models, Animal; Fluorouracil; Lipopolysaccharides; Liposomes; Mice; Mice, Inbred C57BL; Sirolimus

Long non‑coding RNA growth arrest specific 5 (GAS5) exerts inhibitory effects through the modulation of several target microRNAs (miRs) in cancer. However, its potential roles and underlying relationship during colorectal cancer (CRC) progression are unclear. Therefore, we explored the role of the negative feedback loop formed by the GAS5/miR‑34a axis and mammalian target of rapamycin/sirtuin 1 (mTOR/SIRT1) pathway on macroautophagy and apoptosis in CRC. Expression of GAS5, miR‑34a, SIRT1 and mTOR in CRC patients and cell lines was detected by quantitative reverse transcription polymerase chain reaction. Online bioinformatic analysis was used to predict the downstream miRs of GAS5. Luciferase assay and western blotting were performed to demonstrate miR‑34a as a downstream target gene of GAS5 in CRC cells. The effects of the GAS5/miR‑34a axis on apoptosis, macroautophagy, and the mTOR/SIRT1 pathway were assessed by flow cytometry, transmission electron microscopy and western blotting, respectively. Our results suggested that GAS5 was downregulated and acted as a molecular sponge of miR‑34a during CRC progression. miR‑34a participated in regulating GAS5‑suppressed CRC cell macroautophagy and induced apoptosis through the mTOR/SIRT1 pathway. GAS5‑mediated macroautophagy was maintained in an equilibrium state that might have a protective effect on CRC cell apoptosis. The mTOR signaling pathway suppressed GAS5 expression and formed a negative regulation feedback loop with miR‑34a in CRC cells. Our results suggested that the GAS5/miR‑34a/SIRT1/mTOR negative regulatory feedback loop mediated CRC cell macroautophagy, and maintained the cells in an autonomous equilibrium state, but not excessive activation state, which functions as a strong antiapoptotic phenotype during human CRC progression.

Topics: Aged; Animals; Azoxymethane; Cell Line, Tumor; Colon; Colorectal Neoplasms; Feedback, Physiological; Female; Gene Expression Regulation, Neoplastic; Humans; Macroautophagy; Male; MicroRNAs; Middle Aged; Neoplasms, Experimental; Rats; RNA, Long Noncoding; Signal Transduction; Sirolimus; Sirtuin 1; TOR Serine-Threonine Kinases

Rapamycin is a promising agent for treating tumors, but clinical applications of rapamycin are limited due to its poor water solubility and low bioavailability. This paper constructs a liposome delivery system for rapamycin to improve the effect in treating colorectal cancer.. We prepared the rapamycin liposomes using the ethanol injection method. The cellular uptake and biodistribution were detected by LC-MS and in vivo imaging system. MTT assay, transwell migration experiment, flow cytometry, and Western blot analysis evaluated the antitumor effect of rapamycin liposomes in vitro. Furthermore, HCT-116 tumor-bearing mice were used to assess the therapeutic efficacy of rapamycin liposomes in vivo.. The prepared rapamycin liposomes had a particle size of 100±5.5 nm and with a narrow size distribution. In vitro cellular uptake experiments showed that the uptake of rapamycin liposomes by colorectal cells was higher than that of free rapamycin. Subsequently, in vivo imaging experiments also demonstrated that rapamycin liposomes exhibited higher tumor accumulation. Therefore, the ability of rapamycin liposomes to inhibit tumor proliferation, migration and to induce tumor apoptosis is superior to that of free rapamycin. We also demonstrated in vivo good antitumor efficacy of the rapamycin liposomes in HCT-116 xenograft mice. In addition, rapamycin liposomes and 5-FU can synergistically improve the efficacy of colorectal cancer via the Akt/mTOR and P53 pathways.. Collectively, rapamycin liposomes are a potential treatment for colorectal cancer, as it not only improves rapamycin's antitumor effect but also synergistically enhances 5-FU's chemotherapy effect.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Drug Synergism; Fluorouracil; Humans; Inhibitory Concentration 50; Liposomes; Mice; Particle Size; Signal Transduction; Sirolimus; Tissue Distribution; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

The treatment and prognosis of advanced colorectal cancer (CRC) remain a challenging clinical research focus. Here, we describe a new CRC tumor suppressor and potential therapeutic target: thymocyte selection associated high mobility group box (TOX) protein. The expression of TOX was lower in CRC than para-CRC. With the increase of tumor stage, TOX expression decreased, indicating the presence of TOX relates to better overall survival (OS). TOX suppressed the mechanistic target of rapamycin kinase (mTOR) signaling to inhibit cell proliferation, migration, invasion, and change the epithelial-mesenchymal transition (EMT) process. In addition, TOX promoted apoptosis. As tumor mutation burden and tumor microenvironment play vital roles in the occurrence and development of tumors, we analyzed the TOX expression in the immune microenvironment of CRC. The high TOX expression was negatively correlated with TumorPurity. Moreover, it was positively related to ImmuneScore, StromalScore, microsatellite instability (MSI) status, and Consensus Molecular Subtypes (CMS) 3 typing. Based on gene set enrichment analysis (GSEA), the reduced expression of TOX activated mTOR. We found rapamycin, a mTOR inhibitor, partly inhibited cell proliferation, invasion, and migration in shTOX HCT116 cells. Lastly, TOX suppressed tumorigenesis and lung metastasis of CRC

Topics: Aged; Animals; Apoptosis; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Genes, Tumor Suppressor; HCT116 Cells; High Mobility Group Proteins; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Microsatellite Instability; Middle Aged; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transfection; Tumor Burden; Tumor Microenvironment; Xenograft Model Antitumor Assays

Zotarolimus is a semi-synthetic derivative of rapamycin and an inhibitor of mammalian target of rapamycin (mTOR) signaling. Currently, zotarolimus is used to prolong the survival time of organ grafts, but it is also a novel immunosuppressive agent with potent anti-proliferative activity. Here, we examine the anti-tumor effect of zotarolimus, alone and in combination with 5-fluorouracil, on HCT-116 colorectal adenocarcinoma cells implanted in BALB/c nude mice. Compared with the control mice, mice treated with zotarolimus or zotarolimus combined with 5-FU showed retarded tumor growth; increased tumor apoptosis through the enhanced expression of cleaved caspase 3 and extracellular signal-regulated kinase (ERK) phosphorylation; reduced inflammation-related factors such as IL-1β, TNF-α, and cyclooxygenase-2 (COX-2) protein; and inhibited metastasis-related factors such as CD44, epidermal growth factor receptor (EGFR), transforming growth factor β (TGF-β), and vascular endothelial growth factor (VEGF). Notably, mice treated with a combination of zotarolimus and 5-FU showed significantly retarded tumor growth, reduced tumor size, and increased tumor inhibition compared with mice treated with 5-FU or zotarolimus alone, indicating a strong synergistic effect. This in vivo study confirms that zotarolimus or zotarolimus combined with 5-FU can be used to retard colorectal adenocarcinoma growth and inhibit tumorigenesis. Our results suggest that zotarolimus may increase the chemo-sensitization of tumor cells. Therefore, zotarolimus alone and zotarolimus combined with 5-FU may be potential anti-tumor agents in the treatment of human colon adenocarcinoma. Future research on zotarolimus may lead to the development of new therapeutic strategies.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Proliferation; Colorectal Neoplasms; Fluorouracil; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Sirolimus; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Mammalian target of rapamycin complex 1 (mTORC1) is evolutionally conserved and frequently activated in various tumors, including colorectal cancer (CRC). It has been reported that the ribosome assembly factor Urb1 acts downstream of mTORC1/raptor signaling and contributes to digestive organ development in zebrafish. Previously, we highlighted that URB1 was overexpressed in CRC. Here, we assessed the mTORC1/regulatory associated protein with mTOR (RAPTOR)-URB1 axis in CRC tumorigenesis. We found that RAPTOR was overexpressed in CRC tissues and cell lines, was a favorable predictor in patients with CRC, and positively correlated with URB1. Silencing of RAPTOR suppressed CRC cell proliferation and migration and induced cell cycle arrest and apoptosis in vitro and inhibited xenograft growth in vivo. Moreover, ectopic overexpression of RAPTOR exerted an inverse biological phenotype. Knockdown of RAPTOR quenched mTORC1 activity and reduced the expression of URB1 and cyclinA2 (CCNA2). In contrast, overexpression of RAPTOR activated mTORC1 and upregulated URB1 and CCNA2. Furthermore, URB1 and CCNA2 expression were also impeded by rapamycin, which is a specific inhibitor of mTORC1. Thus, RAPTOR promoted CRC proliferation, migration, and cell cycle progression by inducing mTORC1 signaling and transcriptional activation of both URB1 and CCNA2. Taken together, we concluded that RAPTOR has the potential to serve as a novel biomarker and therapeutic target for CRC.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Colon; Colorectal Neoplasms; Cyclin A2; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Kaplan-Meier Estimate; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Middle Aged; Nuclear Proteins; Rectum; Regulatory-Associated Protein of mTOR; Signal Transduction; Sirolimus; Up-Regulation; Xenograft Model Antitumor Assays

Radiotherapy plays an important role in the treatment of colorectal cancer (CRC). However, some patients benefit minimally from radiotherapy because of radioresistance. This study investigated the effects of andrographolide on radiosensitivity in HCT116 CRC cells and examined its mechanism of action.. Cell survival, proliferation, apoptosis, and migration were evaluated using MTT, colony formation, flow cytometry, and Transwell cell invasion assays, respectively. Glycolysis-related indicators were measured to examine cell glycolytic activity. The expression of related proteins was detected by western blotting.. After andrographolide treatment, the expression of phosphoinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway-related proteins, glycolytic activity, and cell survival and invasion rates were decreased in HCT116 cells. Andrographolide plus irradiation increased apoptosis and decreased survival, invasion, and colony formation compared with the effects of irradiation alone.. Andrographolide enhanced radiosensitivity by downregulating glycolysis via inhibition of the PI3K-Akt-mTOR signaling pathway in HCT116 cells.

Topics: Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Diterpenes; Glycolysis; HCT116 Cells; Humans; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Radiation Tolerance; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Germ-free (GF). GF. Human clinical isolate

Topics: Animals; Bacterial Toxins; Campylobacter jejuni; Carcinogenesis; Colorectal Neoplasms; DNA Damage; DNA, Neoplasm; Feces; Gastrointestinal Microbiome; Gene Expression; Humans; Mice; RNA, Neoplasm; Sirolimus; Transcriptome

Mechanistic target of rapamycin mTOR complex 1 (mTORC1) plays a key role in the integration of various environmental signals to regulate cell growth and metabolism. mTORC1 is recruited to the lysosome where it is activated by its interaction with GTP-bound Rheb GTPase. However, the regulatory mechanism of Rheb activity remains largely unknown. Here, we show that ubiquitination governs the nucleotide-bound status of Rheb. Lysosome-anchored E3 ligase RNF152 catalyzes Rheb ubiquitination and promotes its binding to the TSC complex. EGF enhances the deubiquitination of Rheb through AKT-dependent USP4 phosphorylation, leading to the release of Rheb from the TSC complex. Functionally, ubiquitination of Rheb is linked to mTORC1-mediated signaling and  consequently regulates tumor growth. Thus, we propose a mechanistic model whereby Rheb-mediated mTORC1 activation is dictated by a dynamic opposing act between Rheb ubiquitination and deubiquitination that are catalyzed by RNF152 and USP4 respectively.

Topics: Animals; Colorectal Neoplasms; Gene Knockout Techniques; HCT116 Cells; HEK293 Cells; Humans; Intercellular Signaling Peptides and Proteins; Lysosomes; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Mice, Nude; Ras Homolog Enriched in Brain Protein; Sirolimus; Transfection; Tumor Burden; Ubiquitin-Protein Ligases; Ubiquitin-Specific Proteases; Ubiquitination; Xenograft Model Antitumor Assays

Identification of new therapeutic targets may improve the survival rate of patients with colorectal cancer (CRC). Recent studies have suggested that the level of phosphodiesterase 4B (PDE4B) is elevated in fatal/refractory diffuse large B-cell lymphoma (DLBCL), and therapeutic efficacy of a PDE4 inhibitor in B-cell lymphoma has been successfully tested in clinical settings. Here, we show that PDE4B is a potential therapeutic target in CRC. Treatment with forskolin, an activator of adenylyl cyclase (AC), increased intracellular cyclic AMP (cAMP) levels in PDE4B-low, but not PDE4B-high cells, indicating that PDE4B was a major regulator of cAMP levels in CRC cells. Furthermore, cAMP modulated the activities of AKT and AMPK in a PDE4B-dependent manner, which was associated with a marked decrease in mTOR-Myc signals and oncogenic properties of CRC cells such as anchorage-independent growth and colony formation. We found that the Myc proto-oncogene was a crucial downstream target of the AKT/mTOR and AMPK/mTOR signals that mediated cAMP-induced anti-tumor effect. A natural polyphenol resveratrol that was reported to have PDE4 inhibitory effects also showed tumor suppressive effects by inhibiting the mTOR-Myc axis. Intriguingly, we identified Myc as a transcriptional activator of PDE4B in CRC cells, which maintains the intracellular cAMP levels low and promotes cell survival. These data suggest that cAMP/PDE4B signals play a significant role in regulating the malignant phenotype of CRC cells and targeting of PDE4B should be actively pursued.

Topics: AMP-Activated Protein Kinases; Antineoplastic Agents; Azepines; Carcinogenesis; Cell Line, Tumor; Colorectal Neoplasms; Cyclic AMP; Cyclic Nucleotide Phosphodiesterases, Type 4; Humans; Phosphodiesterase 4 Inhibitors; Proto-Oncogene Mas; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Resveratrol; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Triazoles

The PI3K/AKT/mTOR pathway and autophagy are known to play important roles in cancer radioresistance. The aim of the present study was to investigate whether the combination of temsirolimus (TEM), an mTOR inhibitor, and chloroquine (CQ), an autophagy inhibitor, can increase radiosensitivity in colorectal cancer (CRC) cells. The efficacies of TEM and/or CQ as radiosensitizers were examined using clonogenic assays in CRC cell lines SW480 and HT‑29. The expression levels of the phosphorylated isoforms of S6 and 4E‑BP1, downstream proteins of mTOR, as well as the expression levels of p62 and LC3, autophagy‑related proteins, were assessed by western blot analysis. The formation of acidic organelles was detected in acridine orange‑stained cells. Apoptosis and caspase activity were assessed using flow cytometry. The results revealed that ionizing radiation (IR) activated the downstream proteins of mTOR and induced autophagy. In the clonogenic assays, neither TEM nor CQ influenced the efficacy of IR, whereas their combination significantly increased the dose‑dependent efficacy of IR. TEM inhibited phosphorylation of the downstream proteins of mTOR and induced autophagy. CQ inhibited autophagy in the late phase and did not influence the downstream proteins of mTOR. TEM and CQ inhibited both the phosphorylation of downstream proteins of mTOR and autophagy. Cell death analysis revealed that the combination of TEM and CQ strongly induced apoptosis in cells exposed to IR. In conclusion, the combination of TEM and CQ increased radiosensitivity in CRC cells through co‑inhibition of mTOR and autophagy.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Combined Chemotherapy Protocols; Autophagy; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chloroquine; Colorectal Neoplasms; HT29 Cells; Humans; Microtubule-Associated Proteins; Phosphorylation; Radiation-Sensitizing Agents; Ribosomal Protein S6 Kinases; RNA-Binding Proteins; Sirolimus

Objective To investigate the molecular mechanisms underlying the effect of homoharringtonine (HHT) on the proliferation and apoptosis in HT29 human colorectal tumor cells. Methods HT29 cells were treated by 0, 0.007812, 0.015625, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8 μg/mL HHT for 24, 48 and 72 hours. CCK-8 assay was used to assess the cell viability. Colony formation assay was performed to detect the cell proliferation ability. Flow cytometry was used to analyze cell apoptosis. Hoechst33258 fluorescent staining was used to observe the morphology of the cell nuclei. The real-time quantitative PCR was used to detect the mRNA expressions of BAX, Bcl2, caspase-3, caspase-9, mammalian target of rapamycin (mTOR), PI3K, pyruvate dehydrogenase kinase-1 (PDK1), protein kinase B (AKT), raptor, rictor. The protein levels of Bax, Bcl2, pro-caspase-3, cleaved caspase 3 (c-caspase-3), pro-caspase-9, cleaved caspase-9 (c-caspase- 9), poly(ADP-ribose)polymerase (PARP), cleaved PARP (c-PARP), mTOR, raptor, rictor, PI3K, PDK1, AKT, p-AKT were detected by Western blotting. Results Compared with the control group, the proliferation of HT29 cells was inhibited when treated with HHT. Meanwhile, the nuclear fragmentation, chromatin condensation, and apoptotic body of the cells could be observed. Treatment of HHT could increase the mRNA expressions of BAX/Bcl2, caspase-3, caspase-9 and raptor, and decrease PI3K, AKT and rictor in the HT29 cells. The protein levels of pro-caspase-3, pro-caspase-9, PARP, PI3K, PDK1, AKT, mTOR, and rictor were down-regulated, and the c-caspase-3, c-caspase-9, c-PARP, BAX and raptor were up-regulated. Conclusion HHT has the function of inhibiting the HT29 cell proliferation and inducing its apoptosis by blockage of mTOR signaling pathway.

Topics: Apoptosis; Cell Proliferation; Colorectal Neoplasms; Homoharringtonine; HT29 Cells; Humans; Phosphatidylinositol 3-Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Previous studies by our group have demonstrated that extract of clove exhibits potent anticancer effects in vitro and in vivo. In the present study, the effect of an extracted and isolated active fraction of clove (AFC) on induction of cellular apoptosis in human colorectal cancer HCT-116 cells was investigated by morphological observation, flow cytometry, and western blotting analysis. The results revealed that AFC induced apoptosis of HCT-116 cells. AFC also induced autophagy, demonstrated by increased punctuate microtubule-associated protein 1A/1B-light chain 3 (LC3) staining, and LC3-II and Beclin-1 protein expression levels. Furthermore, the autophagy inhibitors 3-MA and baflomycin A1 potentiated the pro-apoptotic activity of AFC in HCT-116 cells. AFC also inhibited the phosphorylation of the phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin signaling pathway. The present study may improve the existing understanding of the anticancer mechanisms of clove and provide a scientific rationale for AFC to be further developed as a promising novel anticancer agent for the treatment of colorectal cancer.

Topics: Adenine; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Drug Screening Assays, Antitumor; HCT116 Cells; Humans; Macrolides; Phosphatidylinositol 3-Kinases; Plant Extracts; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Syzygium; TOR Serine-Threonine Kinases

The roles of autophagy performed in chemotherapy-induced cell death or proliferation inhibition were still in debate. In this study, we aimed to disclose the function of autophagy in chemotherapy of HCT-116 colon cells.. Pharmacological and genetic methods were applied to induce and inhibit autophagy and elucidate the roles of autophagy performed in chemotherapy-induced proliferation inhibition and apoptosis. Autophagy was assessed by microtubule-associated protein light chain 3 (LC3) expression and monodansylcadaverine (MDC) staining.. After treatment with 5-fluorouracil (5-FU), HCT-116 cells showed typical autophagy as stained by MDC. Autophagy inhibitor (3-methyladenine [3-MA]) or inducer (rapamycin) was applied in combination with 5-FU, respectively. As evidenced by our data, 3-MA inhibited while rapamycin facilitated 5-FU-induced apoptosis and proliferation inhibition of HCT-116 cells. Consistently, 3-MA inhibited, while rapamycin facilitated 5-FU-induced expressions of Beclin1 and LC3B. Moreover, 3-MA inhibited while rapamycin facilitated 5-FU-induced p53 protein expression. Using genetic method, Beclin1 overexpression increased while Beclin1 knockdown decreased 5-FU-induced cell proliferation inhibition and apoptosis. Especially, Beclin1 overexpression increased while Beclin1 knockdown decreased 5-FU-induced p53 expression.. Our study provides both of pharmacological and genetic evidence to support that autophagy facilitates anticancer effect of the chemotherapeutic agent. The associated application of autophagy inducer with 5-FU would be beneficial for the chemotherapy in HCT-116 cancer cells.

Topics: Adenine; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Beclin-1; Cell Proliferation; Colorectal Neoplasms; Drug Screening Assays, Antitumor; Drug Synergism; Fluorouracil; HCT116 Cells; Humans; Microtubule-Associated Proteins; RNA, Small Interfering; Sirolimus

SNORD44 is a C/D box small nucleolar RNA, and exhibits low expression in breast cancer and head and neck squamous cell carcinoma tissues. Its host gene is growth arrest specific transcript 5 (GAS5), which is a long noncoding RNA. GAS5 is downregulated in colorectal cancer (CRC), and overexpression of GAS5 suppresses cell proliferation. However, the function of SNORD44 in CRC remains largely unknown, and the application of SNORD44 combined with GAS5 in CRC treatment has not been reported. In this study, the expression levels of SNORD44 and GAS5 were measured in CRC tissues by quantitative RT-PCR. The correlation between SNORD44 and GAS5 was evaluated by Pearson correlation analysis. An oncolytic adenovirus expressing SNORD44 and GAS5 (SPDD-UG) was constructed. The biological effects of SPDD-UG were investigated in CRC cell line SW620 and LS174T in vitro and in xenografts. The synergistic effect of rapamycin and SPDD-UG was explored in SW620 and LS174T cells and tumors. We demonstrated that SNORD44 expression level was markedly decreased in CRC tissues and positively correlated with GAS5 expression. SPDD-UG significantly inhibited SW620 and LS174T cell growth and induced cell apoptosis. Intratumoral injection of SPDD-UG significantly suppressed xenografts growth in nude mice. Moreover, the mechanistic target of rapamycin (mTOR) inhibitor, rapamycin, enhanced the antitumor effect through antagonizing the PI3K/Akt pathway activated by SPDD-UG. These results suggest that overexpression of SNORD44 and GAS5 by oncolytic adenovirus provides a promising method for CRC therapy.

Topics: Adenoviridae; Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Disease Models, Animal; Gene Expression; Gene Order; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Lentivirus; Mice; Oncolytic Viruses; RNA, Long Noncoding; RNA, Small Nucleolar; Signal Transduction; Sirolimus; Tumor Burden; Xenograft Model Antitumor Assays

SAV1 is a human homologue of Salvador that contains two protein-protein interaction modules known as WW domains and acts as a scaffolding protein. SAV1 participates in the development of diverse types of cancer. We aimed to investigate the role of SAV1 in human colorectal cancer.. Human colorectal cancer samples were used to study the expression of SAV1 and YAP. Loss-of-function and gain-of-function strategies were used to study the effects of SAV1 on colorectal cancer cell growth. Rapamycin was used to treat cells and mice to investigate the effect of mTOR signalling.. SAV1 represses the development of colorectal cancer by inhibiting the Akt-mTOR signalling in a YAP-dependent manner. The mRNA and protein levels of SAV1 are down-regulated in human colorectal cancer tissues compared with adjacent non-cancer tissues. SAV1 knockdown promotes the growth of colorectal cancer cells in vitro and in vivo, whereas SAV1 overexpression leads to opposing results. SAV1 represses the activation of the Akt-mTOR signalling, and rapamycin treatment blunts the effects of SAV1 on in vitro and in vivo growth of colorectal cancer cells. Finally, we show that SAV1 promotes the phosphorylation and inactivation of YAP, which contributes to the effect of SAV1 on Akt-mTOR signalling pathway.. SAV1 is a repressor during the development of human colorectal cancer by inhibiting the YAP-Akt-mTOR signalling pathway.

Topics: Adult; Aged; Animals; Cell Cycle Proteins; Colorectal Neoplasms; Down-Regulation; Female; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Nuclear Proteins; Phosphorylation; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Transplantation, Heterologous

With the goal of modeling human disease of the large intestine, we sought to develop an effective protocol for deriving colonic organoids (COs) from differentiated human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs). Extensive gene and immunohistochemical profiling confirmed that the derived COs represent colon rather than small intestine, containing stem cells, transit-amplifying cells, and the expected spectrum of differentiated cells, including goblet and endocrine cells. We applied this strategy to iPSCs derived from patients with familial adenomatous polyposis (FAP-iPSCs) harboring germline mutations in the WNT-signaling-pathway-regulator gene encoding APC, and we generated COs that exhibit enhanced WNT activity and increased epithelial cell proliferation, which we used as a platform for drug testing. Two potential compounds, XAV939 and rapamycin, decreased proliferation in FAP-COs, but also affected cell proliferation in wild-type COs, which thus limits their therapeutic application. By contrast, we found that geneticin, a ribosome-binding antibiotic with translational 'read-through' activity, efficiently targeted abnormal WNT activity and restored normal proliferation specifically in APC-mutant FAP-COs. These studies provide an efficient strategy for deriving human COs, which can be used in disease modeling and drug discovery for colorectal disease.

Topics: Adenoma; Adenomatous Polyposis Coli; Adenomatous Polyposis Coli Protein; Antibiotics, Antineoplastic; Blotting, Western; Cell Differentiation; Cell Proliferation; Colon; Colorectal Neoplasms; Drug Screening Assays, Antitumor; Enteroendocrine Cells; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression Profiling; Gentamicins; Germ-Line Mutation; Goblet Cells; Heterocyclic Compounds, 3-Ring; Human Embryonic Stem Cells; Humans; Immunohistochemistry; Induced Pluripotent Stem Cells; Microscopy, Confocal; Mutation; Organoids; Real-Time Polymerase Chain Reaction; Sirolimus; Wnt Signaling Pathway

The mTor-inhibitor temsirolimus (TEM) has potent anti-tumor activities on extrahepatic colorectal metastases. Treatment of patients with advanced disease may require portal branch ligation (PBL). While PBL can induce intrahepatic tumor growth, the effect of PBL on extrahepatic metastases under TEM treatment is unknown. Therefore, we analyzed the effects of TEM treatment on extrahepatic metastases during PBL-associated liver regeneration. GFP-transfected CT26.WT colorectal cancer cells were implanted into the dorsal skinfold chamber of BALB/c-mice. Mice were randomized to four groups (n = 8). One was treated daily with TEM (1.5 mg/kg), PBS-treated animals served as controls. Another group underwent PBL of the left liver lobe and received daily TEM treatment. Animals with PBL and PBS treatment served as controls. Tumor vascularization and growth as well as tumor cell migration, proliferation and apoptosis were studied over 14 days. In non-PBL animals TEM treatment inhibited tumor cell proliferation as well as vascularization and growth of the extrahepatic metastases. PBL did not influence tumor cell engraftment, vascularization and metastatic growth. Of interest, TEM treatment significantly reduced tumor cell engraftment, neovascularization and metastatic groth also after PBL. PBL does not counteract the inhibiting effect of TEM on extrahepatic colorectal metastatic growth.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Female; Ligation; Liver Neoplasms; Liver Regeneration; Mice, Inbred BALB C; Neoplasm Invasiveness; Neovascularization, Pathologic; Portal Vein; Sirolimus; Tumor Burden; Xenograft Model Antitumor Assays

Tumour cells can use strategies that make them resistant to nutrient deprivation to outcompete their neighbours. A key integrator of the cell's responses to starvation and other stresses is amino-acid-dependent mechanistic target of rapamycin complex 1 (mTORC1). Activation of mTORC1 on late endosomes and lysosomes is facilitated by amino-acid transporters within the solute-linked carrier 36 (SLC36) and SLC38 families. Here, we analyse the functions of SLC36 family member, SLC36A4, otherwise known as proton-assisted amino-acid transporter 4 (PAT4), in colorectal cancer. We show that independent of other major pathological factors, high PAT4 expression is associated with reduced relapse-free survival after colorectal cancer surgery. Consistent with this, PAT4 promotes HCT116 human colorectal cancer cell proliferation in culture and tumour growth in xenograft models. Inducible knockdown in HCT116 cells reveals that PAT4 regulates a form of mTORC1 with two distinct properties: first, it preferentially targets eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and second, it is resistant to rapamycin treatment. Furthermore, in HCT116 cells two non-essential amino acids, glutamine and serine, which are often rapidly metabolised by tumour cells, regulate rapamycin-resistant mTORC1 in a PAT4-dependent manner. Overexpressed PAT4 is also able to promote rapamycin resistance in human embryonic kidney-293 cells. PAT4 is predominantly associated with the Golgi apparatus in a range of cell types, and in situ proximity ligation analysis shows that PAT4 interacts with both mTORC1 and its regulator Rab1A on the Golgi. These findings, together with other studies, suggest that differentially localised intracellular amino-acid transporters contribute to the activation of alternate forms of mTORC1. Furthermore, our data predict that colorectal cancer cells with high PAT4 expression will be more resistant to depletion of serine and glutamine, allowing them to survive and outgrow neighbouring normal and tumorigenic cells, and potentially providing a new route for pharmacological intervention.

Topics: Amino Acid Transport Systems; Amino Acids; Animals; Colorectal Neoplasms; Drug Resistance, Neoplasm; Female; Golgi Apparatus; HCT116 Cells; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred BALB C; Mice, Nude; Mice, SCID; Multiprotein Complexes; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome

To study the correlation between the phosphorylation of keratin 18 (K18) and the autophagy and apoptosis of HCT116 cells under the effect of oxaliplatin (OXA) and investigate its possible mechanism.. HCT116 cells were transfected with empty plasmid, wild-type K18 expression plasmid and 33, 52 phosphorylation site mutated K18 (Ser33/52A) expression plasmid separately, and all cells were then treated with 60 μmol/L OXA, followed by supplementation of autophagy inhibitor 3-methyladenine (3-MA) or autophagy inducer rapamycin. FITC-conjugated annexin V and propidium iodide (PI) double staining combined with flow cytometry, calcein-AM/PI staining were used to analyze the effects of K18 and its mutants on cell apoptosis; Western blotting was performed to detect the expressions of K18 phosphorylation, autophagy related proteins microtubule associated protein 1 light chain 3 (LC3) and beclin-1.. Transfection of Ser33/52A plasmid significantly reduced the level of K18 phosphorylation. After treated with OXA, the apoptosis rate of K18 plasmid transfected group was significantly higher than that of empty plasmid transfected group, while the apoptosis rate of Ser33/52A plasmid transfected HCT116 cells was significantly lower than that of empty plasmid or K18 plasmid transfected group. Compared with empty plasmid group, the autophagy of K18 plasmid transfected group was significantly promoted, while the autophagy in Ser33/52A plasmid transfected group was significantly inhibited.. K18 overexpression enhanced the autophagy in HCT116 cells and increased its sensitivity to OXA. The decrease of K18 ser33 and ser52 phosphorylation inhibited autophagy and decreased apoptosis of HCT116 cells.

Topics: Adenine; Antineoplastic Agents; Apoptosis; Autophagy; Blotting, Western; Colorectal Neoplasms; Flow Cytometry; HCT116 Cells; Humans; Keratin-18; Microscopy, Fluorescence; Mutation; Organoplatinum Compounds; Oxaliplatin; Phosphorylation; Serine; Sirolimus; Transfection

We investigated the effects of TORC1/2 kinase inhibitors on colorectal cancer (CRC) cell lines.. Using selective TORC1/2 inhibitors, rapamycin and PP242, we assessed their effect on the growth of CRC cells in vitro and tumor growth in vivo.. Rapamycin and PP242 inhibit proliferation and induce apoptosis of CRC cells. They also enhance proapoptotic effect of conventional chemo drug doxorubicin in CRC cells in vitro. When combined with doxorubicin, rapamycin and PP242 almost completely inhibit tumor growth in vivo. Rapamycin and PP242 inhibit phosphorylation of Akt, ribosomal S6 kinase, 4EBP1 and mTOR.. Our study suggests rapamycin and PP242 may be a useful therapeutic agent and inhibiting mTOR signaling pathway represents a new targeted therapy for CRC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Disease Models, Animal; Doxorubicin; Humans; Indoles; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Multiprotein Complexes; Protein Kinase Inhibitors; Purines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

Cancer stem cells (CSCs) have tumor initiation, self-renewal, metastasis and chemo-resistance properties in various tumors including colorectal cancer. Targeting of CSCs may be essential to prevent relapse of tumors after chemotherapy. Phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) signals are central regulators of cell growth, proliferation, differentiation, and apoptosis. These pathways are related to colorectal tumorigenesis. This study focused on PI3K and mTOR pathways by inhibition which initiate differentiation of SW620 derived CSCs and investigated its effect on tumor progression. By using rapamycin, LY294002, and NVP-BEZ235, respectively, PI3K and mTOR signals were blocked independently or dually in colorectal CSCs. Colorectal CSCs gained their differentiation property and lost their stemness properties most significantly in dual-blocked CSCs. After treated with anti-cancer drug (paclitaxel) on the differentiated CSCs cell viability, self-renewal ability and differentiation status were analyzed. As a result dual-blocking group has most enhanced sensitivity for anti-cancer drug. Xenograft tumorigenesis assay by using immunodeficiency mice also shows that dual-inhibited group more effectively increased drug sensitivity and suppressed tumor growth compared to single-inhibited groups. Therefore it could have potent anti-cancer effects that dual-blocking of PI3K and mTOR induces differentiation and improves chemotherapeutic effects on SW620 human colorectal CSCs.

Topics: AC133 Antigen; Animals; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cell Survival; Chromones; Colorectal Neoplasms; Humans; Imidazoles; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Morpholines; Neoplastic Stem Cells; Paclitaxel; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Signal Transduction; Sirolimus; SOXB1 Transcription Factors; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

The inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) by chemical inhibitors, such as rapamycin, has demonstrated anti-cancer activity in preclinical and clinical trials. Their efficacy is, however, limited and tumors eventually relapse through resistance formation. In this study, using two different cancer mouse models, we identify tumor hypoxia as a novel mechanism of resistance of cancer cells against mTORC1 inhibitors. Indeed, we show that the activity of mTORC1 is mainly restricted to the non-hypoxic tumor compartment, as evidenced by a mutually exclusive staining pattern of the mTORC1 activity marker pS6 and the hypoxia marker pimonidazole. Consequently, whereas rapamycin reduces cancer cell proliferation in non-hypoxic regions, it has no effect in hypoxic areas, suggesting that cancer cells proliferate independently of mTORC1 under hypoxia. Targeting the hypoxic tumor compartment by knockdown of carbonic anhydrase IX (CAIX) using short hairpin RNA or by chemical inhibition of CAIX with acetazolamide potentiates the anti-cancer activity of rapamycin. Taken together, these data emphasize that hypoxia impairs the anti-cancer efficacy of rapalogs. Therapeutic strategies targeting the hypoxic tumor compartment, such as the inhibition of CAIX, potentiate the efficacy of rapamycin and warrant further clinical evaluation.

Topics: Acetazolamide; Animals; Antibiotics, Antineoplastic; Carbonic Anhydrase Inhibitors; Carbonic Anhydrase IX; Cell Line, Tumor; Colorectal Neoplasms; Drug Synergism; Female; HT29 Cells; Humans; Hypoxia; Mice, Inbred C57BL; Mice, Nude; Neoplasms, Experimental; RNA Interference; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome; Xenograft Model Antitumor Assays

The cancer stem cell (CSC) hypothesis proposes a hierarchical organization of tumors, in which stem-like cells sustain tumors and drive metastasis. The molecular mechanisms underlying the acquisition of CSCs and metastatic traits are not well understood. SOX9 is a transcription factor linked to stem cell maintenance and commonly overexpressed in solid cancers including colorectal cancer. In this study, we show that SOX9 levels are higher in metastatic (SW620) than in primary colorectal cancer cells (SW480) derived from the same patient. This elevated expression correlated with enhanced self-renewal activity. By gain and loss-of-function studies in SW480 and SW620 cells respectively, we reveal that SOX9 levels modulate tumorsphere formation and self-renewal ability in vitro and tumor initiation in vivo. Moreover, SOX9 regulates migration and invasion and triggers the transition between epithelial and mesenchymal states. These activities are partially dependent on SOX9 post-transcriptional modifications. Importantly, treatment with rapamycin inhibits self-renewal and tumor growth in a SOX9-dependent manner. These results identify a functional role for SOX9 in regulating colorectal cancer cell plasticity and metastasis, and provide a strong rationale for a rapamycin-based therapeutic strategy.

Topics: Cell Line, Tumor; Cell Movement; Cell Plasticity; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplastic Stem Cells; Signal Transduction; Sirolimus; SOX9 Transcription Factor

Blocking the mechanistic target of rapamycin complex-1 (mTORC1) with chemical inhibitors such as rapamycin has shown limited clinical efficacy in cancer. The tumor microenvironment is characterized by an acidic pH which interferes with cancer therapies. The consequences of acidity on the anti-cancer efficacy of mTORC1 inhibitors have not been characterized and are thus the focus of our study.. Cancer cell lines were treated with rapamycin in acidic or physiological conditions and cell proliferation was investigated. The effect of acidity on mTORC1 activity was determined by Western blot. The anticancer efficacy of rapamycin in combination with sodium bicarbonate to increase the intratumoral pH was tested in two different mouse models and compared to rapamycin treatment alone. Histological analysis was performed on tumor samples to evaluate proliferation, apoptosis and necrosis.. Exposing cancer cells to acidic pH in vitro significantly reduced the anti-proliferative effect of rapamycin. At the molecular level, acidity significantly decreased mTORC1 activity, suggesting that cancer cell proliferation is independent of mTORC1 in acidic conditions. In contrast, the activation of mitogen-activated protein kinase (MAPK) or AKT were not affected by acidity, and blocking MAPK or AKT with a chemical inhibitor maintained an anti-proliferative effect at low pH. In tumor mouse models, the use of sodium bicarbonate increased mTORC1 activity in cancer cells and potentiated the anti-cancer efficacy of rapamycin. Combining sodium bicarbonate with rapamycin resulted in increased tumor necrosis, increased cancer cell apoptosis and decreased cancer cell proliferation as compared to single treatment.. Taken together, these results emphasize the inefficacy of mTORC1 inhibitors in acidic conditions. They further highlight the potential of combining sodium bicarbonate with mTORC1 inhibitors to improve their anti-tumoral efficacy.

Topics: Acids; Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Drug Therapy, Combination; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Sirolimus; Sodium Bicarbonate; TOR Serine-Threonine Kinases; Tumor Microenvironment; Xenograft Model Antitumor Assays

Tumor growth and metastasis are not determined by cancer cells alone but also by a variety of stromal cells, and platelet-derived growth factor receptors (PDGF-Rs) are overexpressed by various stromal cell populations. Activation of PI3K-AKT-mTOR signaling is frequently observed in many cancer types. We investigated whether the mTOR inhibitor everolimus, alone or in combination with the PDGF-R tyrosine kinase inhibitor nilotinib, can inhibit growth and metastasis of human colon cancer. The effects of nilotinib and everolimus on tumor growth and metastasis were examined in an orthotopic mouse model of human colon cancer and a model of liver metastasis. After treatment with nilotinib (versus distilled water), the stromal reaction of xenografts growing in the cecal wall and liver was significantly decreased. After treatment with everolimus, the stromal reaction did not decrease, but tumor cell proliferation and microvessel density decreased. With the two drugs in combination, both stromal reaction and tumor cell proliferation decreased and apoptosis of tumor cells increased, resulting in remarkable inhibition of tumor growth at both the orthotopic and the metastatic site. Concurrent inhibition of tumor cells and activated stromal cells by a PDGF-R tyrosine kinase inhibitor and an mTOR inhibitor used in combination may represent a novel therapeutic approach for colorectal cancer.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Colorectal Neoplasms; Everolimus; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Platelet-Derived Growth Factor; Pyrimidines; Receptors, Platelet-Derived Growth Factor; Sirolimus; TOR Serine-Threonine Kinases; Tumor Microenvironment; Xenograft Model Antitumor Assays

Many powerful drugs have limited clinical utility because of poor water solubility and high systemic toxicity. Here, we formulated a targeted nanomedicine, rapamycin encapsulated in pegylated octadecyl lithocholate micelles labeled with a new ligand for colorectal neoplasia, LTTHYKL peptide. CPC;Apc mice that spontaneously develop colonic adenomas were treated with free rapamycin, plain rapamycin micelles, and peptide-labeled rapamycin micelles via intraperitoneal injection for 35days. Endoscopy was performed to monitor adenoma regression in vivo. We observed complete adenoma regression at the end of therapy. The mean regression rate for peptide-labeled rapamycin micelles was significantly greater than that for plain rapamycin micelles, P<0.01. On immunohistochemistry, we observed a significant reduction in phospho-S6 but not β-catenin expression and reduced tumor cell proliferation, suggesting greater inhibition of downstream mTOR signaling. We observed significantly reduced renal toxicity for peptide-labeled rapamycin micelles compared to that of free drug, and no other toxicities were found on chemistries. Together, this unique targeted micelle represents a potential therapeutic for colorectal neoplasia with comparable therapeutic efficacy to rapamycin free drug and significantly less systemic toxicity.

Topics: Adenoma; Animals; Antibiotics, Antineoplastic; beta Catenin; Binding, Competitive; Cell Line, Tumor; Chemistry, Pharmaceutical; Colorectal Neoplasms; Drug Delivery Systems; Humans; Lithocholic Acid; Mice; Micelles; Peptides; Polyethylene Glycols; Sirolimus

Portal branch ligation (PBL) can be performed before major hepatic resection of colorectal liver metastases (mCRC) to increase the remnant liver mass. However, PBL may also stimulate mCRC growth through hepatic arterial hyperperfusion and growth factor release. Herein, we studied whether hepatic arterial infusion (HAI) of the mTOR-inhibitor temsirolimus (Tem) is capable of inhibiting the growth of colorectal liver metastases after PBL. WAG/Rij rats were randomized to four groups (n=6 each) and underwent subcapsular implantation of 5×10(5) CC531 cells into the left liver lobe. The animals of two groups underwent simultaneous PBL of the tumour bearing liver lobe. Ten days later animals underwent a HAI either of temsirolimus (Tem and PBL Tem) or saline solution (Sham and PBL Sham). Tumour size was analyzed at days 10 and 13 using three-dimensional ultrasound. In Sham controls tumour volume increased by 43%. After PBL Sham tumour volume increased by 52%. In contrast, in animals undergoing HAI of temsirolimus the tumour growth was not only completely inhibited, but tumour volume was found decreased, irrespective of PBL. After HAI of temsirolimus immunohistochemistry revealed an increased cleaved caspase-3 activity, indicating stimulation of apoptotic cell death. In parallel temsirolimus treatment was associated with a significant reduction of PECAM-1 positive cells within the tumour tissue, implying a reduced tumour vascularisation. HAI of temsirolimus is capable of inhibiting the growth of CC531 colorectal rat liver metastases also after PBL.

Topics: Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Infusions, Intra-Arterial; Ligation; Liver; Liver Neoplasms; Male; Neoplasm Transplantation; Neovascularization, Pathologic; Platelet Endothelial Cell Adhesion Molecule-1; Portal Vein; Random Allocation; Rats; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden

Many epithelial cancers, particularly gastrointestinal tract cancers, remain poor prognosis diseases, due to resistance to cytotoxic therapy and local or metastatic recurrence. We have previously shown that apoptosis incompetent esophageal cancer cells induce autophagy in response to chemotherapeutic agents and this can facilitate their recovery. However, known pharmacological inhibitors of autophagy could not enhance cytotoxicity. In this study, we have examined two well known, clinically approved autophagy inducers, rapamycin and lithium, for their effects on chemosensitivity in apoptosis incompetent cancer cells. Both lithium and rapamycin were shown to induce autophagosomes in esophageal and colorectal cancer cells by western blot analysis of LC3 isoforms, morphology and FACS quantitation of Cyto-ID or mCherry-GFP-LC3. Analysis of autophagic flux indicates inefficient autophagosome processing in lithium treated cells, whereas rapamycin treated cells showed efficient flux. Viability and recovery was assessed by clonogenic assays. When combined with the chemotherapeutic agent 5-fluorouracil, rapamycin was protective. In contrast, lithium showed strong enhancement of non-apoptotic cell death. The combination of lithium with 5-fluorouracil or oxaliplatin was then tested in the syngenic mouse (balb/c) colorectal cancer model--CT26. When either chemotherapeutic agent was combined with lithium a significant reduction in tumor volume was achieved. In addition, survival was dramatically increased in the combination group (p < 0.0001), with > 50% of animals achieving long term cure without re-occurrence (> 1 year tumor free). Thus, combination treatment with lithium can substantially improve the efficacy of chemotherapeutic agents in apoptosis deficient cancer cells. Induction of compromised autophagy may contribute to this cytotoxicity.

Topics: Animals; Antineoplastic Agents; Autophagy; Cell Line, Tumor; Colorectal Neoplasms; Drug Synergism; Esophageal Neoplasms; Female; Fluorouracil; Genes, Reporter; Humans; Lithium Chloride; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Organoplatinum Compounds; Oxaliplatin; Sirolimus; Transplantation, Heterologous

To determine the role of the mammalian target of rapamycin (mTOR) signaling in sustaining cancer stem-like cells and its clinical values in colorectal cancer (CRC).. mTOR expression in CRC patients was analyzed by immunohistochemistry and survival analysis was used to confirm the clinical value of mTOR. Colorectal cell lines were treated by mTOR inhibitors rapamycin and PP242, and sphere formation assay and aldehyde dehydrogenase (ALDH) assay were utilized to determine the impact of mTOR inhibition in CRC stem-like cells, combined or not combined with chemotherapeutic drug (fluorouracil and oxaliplatin).. mTOR expression was associated with outcomes of CRC patients and predicted poor prognosis in stage II CRC patients. mTOR signaling was activated in stem-like colorectal cancer cells, and mTOR inhibitors (rapamycin and PP242) decreased the capacity of sphere formation as well as ALDH activity. Furthermore, mTOR inhibitors also were demonstrated to suppress the stimulation of stem-like cells by chemotherapy.. mTOR shared predictive significance in stage II CRC patients' outcomes and played a vital role in the maintenance of colorectal cancer stem-like cells. mTOR inhibitors might hold the potential to become a therapeutic target against CRC stem cells.

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Antineoplastic Combined Chemotherapy Protocols; Cell Proliferation; Colorectal Neoplasms; Female; Fluorouracil; Follow-Up Studies; Humans; Male; Middle Aged; Neoplasm Staging; Neoplastic Stem Cells; Organoplatinum Compounds; Oxaliplatin; Prognosis; Sirolimus; Survival Rate; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Temsirolimus (TEM) is a novel, water-soluble mammalian target of rapamycin (mTOR) inhibitor that has shown activity against a wide range of cancers in preclinical models, but its efficacy against colorectal cancer (CRC) has not been fully explored.. We evaluated the antitumor effect of TEM in CRC cell lines (CaR-1, HT-29, Colon26) in vitro and in vivo. In vitro, cell growth inhibition was assessed using a MTS assay. Apoptosis induction and cell cycle effects were measured using flow cytometry. Modulation of mTOR signaling was measured using immunoblotting. Antitumor activity as a single agent was evaluated in a mouse subcutaneous tumor model of CRC. The effects of adding chloroquine, an autophagy inhibitor, to TEM were evaluated in vitro and in vivo.. In vitro, TEM was effective in inhibiting the growth of two CRC cell lines with highly activated AKT, possibly through the induction of G1 cell cycle arrest via a reduction in cyclin D1 expression, whereas TEM reduced HIF-1α and VEGF in all three cell lines. In a mouse subcutaneous tumor model, TEM inhibited the growth of tumors in all cell lines, not only through direct growth inhibition but also via an anti-angiogenic effect. We also explored the effects of adding chloroquine, an autophagy inhibitor, to TEM. Chloroquine significantly potentiated the antitumor activity of TEM in vitro and in vivo. Moreover, the combination therapy triggered enhanced apoptosis, which corresponded to an increased Bax/Bcl-2 ratio.. Based on these data, we propose TEM with or without chloroquine as a new treatment option for CRC.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Chloroquine; Colorectal Neoplasms; Cyclin D1; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Mice; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Liver kinase B1 (LKB1) loss by gene mutation, loss of heterozygosity, and promoter methylation rarely occurs in colorectal cancer. We wondered whether LKB1 loss could be deregulated at the transcriptional level to promote tumor progression and poor outcome in colorectal cancer.. Mechanistic studies were performed in two each of p53 wild-type (HCT116, LoVo) and p53-mutated (SW480, HT29) colon cancer cells to explore whether LKB1 loss could be deregulated by NKX2-1-mediated p53 pathway. LKB1 and NK2 homeobox 1 (NKX2-1) expressions in colorectal tumors were determined by immunohistochemistry, and the prognostic value of both molecules was assessed by Kaplan-Meier test and Cox regression model.. Mechanistically, LKB1 loss at the transcriptional level due to alteration of the NKX2-1-mediated p53 pathway promotes invasiveness in colon cancer cells. The cell invasiveness induced by LKB1 loss was nearly suppressed by mammalian target of rapamycin (mTOR) inhibitor (rapamycin and everolimus) and mTOR/AKT dual inhibitor Palomid 529 (P529). Among patients, low LKB1 tumors exhibited shorter overall survival (OS) and relapse-free survival periods than high LKB1 tumors. The highest hazard ratio value for OS and relapse-free survival was observed in wild-type p53 with low LKB1/low NKX2-1 tumors and in mutated p53 with low LKB1/high NKX2-1 tumors when wild-type p53 with high LKB1/high NKX2-1 and mutated p53 with high LKB1/low NKX2-1 tumors were used as references.. LKB1 loss at the transcriptional level via alteration of the NKX2-1/p53 axis promotes cell invasion, consequently resulting in poor outcome in colorectal cancer patients.

Topics: Aged; AMP-Activated Protein Kinase Kinases; Antineoplastic Agents; Benzopyrans; Colorectal Neoplasms; Disease Progression; Disease-Free Survival; Everolimus; Gene Knockdown Techniques; HCT116 Cells; HT29 Cells; Humans; Kaplan-Meier Estimate; Middle Aged; Neoplasm Invasiveness; Nuclear Proteins; Proportional Hazards Models; Protein Serine-Threonine Kinases; Signal Transduction; Sirolimus; Survival Rate; Thyroid Nuclear Factor 1; Transcription Factors; Transcription, Genetic; Tumor Suppressor Protein p53

The aim of this study was to investigate the antitumor effect of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) signaling, combined with 5-fluorouracil treatment on CT-26 colorectal adenocarcinoma cells implanted into BALB/c mice.. Two experiments were carried out: treatment from day 1 after CT-26 cell implantation; and treatment from day 7 after CT-26 cell implantation after the detection of a tumor mass. There were four groups in each experiment: control; treatment with 5-fluorouracil; with rapamycin; and with rapamycin with 5-fluorouracil.. Rapamycin combined with 5-fluorouracil significantly reduced tumor size, suppressed expression of B-cell lymphoma 2, increased tumor apoptosis, and inhibited mTOR signaling activity by de-phosphorylation of S6K.. The results strongly suggest that rapamycin might increase the chemosensitization of tumor cells. Rapamycin combined with 5-fluorouracil treatment had a synergistic tumor-inhibition effect. Future research on rapamycin is required to develop new therapeutic strategies.

Topics: Adenocarcinoma; Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Growth Processes; Cell Line, Tumor; Colorectal Neoplasms; Drug Synergism; Fluorouracil; In Situ Nick-End Labeling; Male; Mice; Mice, Inbred BALB C; Phosphorylation; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Random Allocation; Ribosomal Protein S6 Kinases; Sirolimus; Vascular Endothelial Growth Factor A

Chronic inflammation is an underlying risk factor for colorectal cancer. No direct evidence has proven that inflammation in the colon promotes carcinogenesis. STAT3 plays an important role in the development of colitis-associated colorectal cancer (CAC). There is crosstalk between the mammalian target of rapamycin complex 1 (mTORC1) and the STAT3 pathways. The aim of the present study was to confirm that colitis promotes CAC and if so, to explore the function of the STAT3 and mTORC1 pathways in CAC. C57BL/6 mice were treated with axozymethane (AOM) and dextran sulfate sodium (DSS) to induce CAC. By varying the concentration of DSS (0, 1 and 2% respectively), we mimicked the CAC model with different degrees of inflammation and determined the risk of carcinogenesis. Expression of the STAT3 and mTORC1 pathways was detected. Finally, rapamycin, an mTORC1 inhibitor, was used to treat the CAC model. Tumor load, protein and gene expression of chemokines were determined. The multiplicity and tumor load of the high inflammation group were higher than those of the low inflammation group. Immunohistochemical staining and western blot analysis revealed that activation of the STAT3 and mTORC1 pathways increased gradually in the inflammation tissues and tumors. When we treated the mice with rapamycin, the tumor incidence, multiplicity and tumor load decreased. In addition, rapamycin widely suppressed the expression of pro‑inflammatory and anti-inflammatory chemokines in the tissues, including tumor necrosis factor-α, interferon-γ, IL-6, IL-10 and IL-12α. In conclusion, inflammation promotes the development of CAC via the STAT3 and mTORC1 pathways, which may be a viable treatment strategy for the chemoprevention of CAC.

Topics: Animals; Antineoplastic Agents; Azoxymethane; Cell Transformation, Neoplastic; Colitis; Colorectal Neoplasms; Cytokines; Dextran Sulfate; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Multiprotein Complexes; Signal Transduction; Sirolimus; STAT3 Transcription Factor; TOR Serine-Threonine Kinases

Amino acid (AA) is a potent mitogen that controls growth and metabolism. Here we describe the identification of Rab1 as a conserved regulator of AA signaling to mTORC1. AA stimulates Rab1A GTP binding and interaction with mTORC1 and Rheb-mTORC1 interaction in the Golgi. Rab1A overexpression promotes mTORC1 signaling and oncogenic growth in an AA- and mTORC1-dependent manner. Conversely, Rab1A knockdown selectively attenuates oncogenic growth of Rab1-overexpressing cancer cells. Moreover, Rab1A is overexpressed in colorectal cancer (CRC), which is correlated with elevated mTORC1 signaling, tumor invasion, progression, and poor prognosis. Our results demonstrate that Rab1 is an mTORC1 activator and an oncogene and that hyperactive AA signaling through Rab1A overexpression drives oncogenesis and renders cancer cells prone to mTORC1-targeted therapy.

Topics: Amino Acids; Animals; Antibiotics, Antineoplastic; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Female; Gene Expression; HEK293 Cells; Humans; MAP Kinase Kinase Kinases; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred BALB C; Mice, Nude; Multiprotein Complexes; Neoplasm Invasiveness; NIH 3T3 Cells; Oncogenes; Phosphatidylinositol 3-Kinases; rab GTP-Binding Proteins; rab1 GTP-Binding Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Tumor Burden; Xenograft Model Antitumor Assays

The aim of the present study was to determine the role of hcrcn81 in the regulation of the mammalian target of rapamycin (mTOR) pathway in human colorectal adenocarcinoma cells. The effect of rapamycin treatment on hcrcn81 expression was evaluated by examining the mRNA and protein expression of hcrcn81 in rapamycin‑treated human colon carcinoma cell lines, SW480 and LoVo, using real‑time PCR and western blot analysis, respectively. The results demonstrated that mRNA and protein levels of hcrcn81 were elevated following rapamycin treatment in these cell lines, indicating that hcrcn81 expression is upregulated by rapamycin treatment in human colorectal adenocarcinoma cells. Observations of the current study indicate that hcrcn81 may play a role in tumorigenesis by regulating the mTOR signaling pathway.

Topics: Adenocarcinoma; Carcinogenesis; Cell Line, Tumor; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Colorectal cancer is the second leading cause of cancer-related deaths. Drug resistance and/or off-target toxicity against normal cells limit the effectiveness of current chemotherapies for the treatment of colorectal cancer. In the current study, we studied the potential cytotoxic effects of short-chain and cell-permeable C6 ceramide in cultured colorectal cancer HT-29 cells and focused on the underlying mechanisms. We observed that C6 ceramide-induced HT-29 cell death and growth inhibition in a dose- and time-dependent manner. However, no significant apoptosis was observed in C6 ceramide-treated HT-29 cells. Our data support that autophagy contributed to C6 ceramide-induced cytotoxic effects, as autophagy inhibitors, 3-methyladenine (3-MA) and hydroxychloroquine, inhibited C6 ceramide's effect; however, autophagy activators, everolimus (RAD001) and temsirolimus, mimicked C6 ceramide effects and induced HT-29 cell death. Further, we indentified that AMP-activated protein kinase (AMPK)/Ulk1 signaling was required for autophagy induction by C6 ceramide, and AMPK silencing by a specific short hairpin RNA suppressed C6 ceramide-induced autophagy and cytotoxic effects. Reversely, forced activation of AMPK by its activator AICAR or by genetic manipulation caused autophagic death in HT-29 cells, which was inhibited by 3-MA. Our results suggest that autophagy, but not apoptosis, is a major contributor for C6 ceramide-induced cytotoxic effects in HT-29 cells, and activation of AMPK/Ulk1 is required for the process.

Topics: Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Antineoplastic Agents; Apoptosis; Autophagy; Autophagy-Related Protein-1 Homolog; Cell Survival; Ceramides; Colorectal Neoplasms; Enzyme Activation; Everolimus; HCT116 Cells; HT29 Cells; Humans; Intracellular Signaling Peptides and Proteins; Protein Serine-Threonine Kinases; Ribonucleotides; Signal Transduction; Sirolimus

Increased cell migration and invasion lead to cancer metastasis and are crucial to cancer prognosis. In this study, we explore whether FBXW7 plays any role in metastatic process. We show that depletion of FBXW7 induces epithelial-mesenchymal transition (EMT) in human colon cancer cells along with the increase in cell migration and invasion. Moreover, FBXW7 deficiency promotes the generation of colon cancer stem-like cells in tumor-sphere culture. mTOR inhibition by rapamycin suppresses FBXW7 loss-driven EMT, invasion and stemness. Our results define the FBXW7/mTOR axis as a novel EMT pathway that mediates cancer invasion.

Topics: Blotting, Western; Cell Cycle Proteins; Cell Movement; Cell Shape; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; F-Box Proteins; F-Box-WD Repeat-Containing Protein 7; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Neoplasm Invasiveness; Neoplastic Stem Cells; Sirolimus; TOR Serine-Threonine Kinases; Ubiquitin-Protein Ligases

Hepatic arterial infusion (HAI) of specific anti-tumor drugs can be more effective compared with systemic drug application. Herein, we studied whether HAI of temsirolimus is effective to inhibit tumor growth of colorectal liver metastases after liver resection.. Twenty-four Wistar Albino Glaxo from Rijswijk (WAG/Rij) rats were randomized to four groups and underwent subcapsular implantation of CC531 colorectal cancer cells in the left liver lobe. In two groups, a 70% liver resection (Phx) was performed simultaneously. After 10 d, animals received either a HAI of temsirolimus (CCI-779) or saline solution (controls). Tumor growth was determined on d 10 and 13 using three-dimensional ultrasound. On d 13, tumor tissue was removed for histologic and immunohistochemical analysis.. Sham controls revealed a tumor growth of ∼40% from d 10 to d 13. HAI of temsirolimus completely inhibited this tumor growth. Controls with Phx showed a tumor growth of >60%. In contrast, HAI of temsirolimus in Phx animals did not only inhibit tumor growth but was even capable of decreasing the tumor size by ∼8%. Immunohistochemical analysis of the tumors showed a decreased proliferation rate and an increased cleaved caspase-3 activity, which was associated with a significant reduction of platelet endothelial cell adhesion molecule (PECAM)-1-positive cells after HAI of temsirolimus.. HAI of temsirolimus inhibits tumor growth of CC531 colorectal liver metastases even if a growth-stimulating procedure like Phx is performed. Inhibition of tumor growth is provided by a decrease of tumor vascularization associated with an inhibition of tumor cell proliferation and an induction of tumor cell apoptosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Disease Models, Animal; Hepatic Artery; Liver; Liver Neoplasms, Experimental; Liver Regeneration; Male; Neovascularization, Pathologic; Random Allocation; Rats; Rats, Wistar; Sirolimus

KRAS mutation is a negative predictive prognostic factor during metastatic colorectal cancer treatment with antiepidermal growth factor receptor antibodies. For affected patients, new therapeutics must be explored. Our objective was to study efficacy of two drugs with different mechanisms of action, everolimus (mTOR inhibitor) and lapatinib (tyrosine kinase inhibitor), in a mouse xenograft model. We chose a model obtained after engraftment of a tumor originating from a human tumor collection. The patient was affected by a metastasis colorectal carcinoma resistant to cetuximab with KRAS mutation. From a previous study in mice, we know that everolimus is a P-glycoprotein (P-gp) substrate and that a lapatinib pretreatment increases significantly (2.6-fold) everolimus AUC by inhibiting its intestinal P-gp efflux. We hence tested the effect of these drugs alone or combined. Mice bearing the xenografts were divided in four groups: control, lapatinib, everolimus, and L/E group (L/E: 2 days of lapatinib 200 mg/kg and then 3 days of everolimus 1 mg/kg). Tumor volumes and treatment toxicities were evaluated. Sixteen days after treatment initiation, the group L/E was the first one in which tumor volume average was significantly lower than the one of control group (193 ± 90 vs. 395 ± 171 mm(3) ; P = 0.0025). After 4 weeks of treatment, inhibition of tumor growth in lapatinib, everolimus, and L/E groups reached, respectively, 49, 53, and 57%. Each drug showed significant antitumor activity. Only moderate hematologic toxicity signs were observed. These results lead to new perspectives for new oral drugs in metastatic KRAS-mutated colorectal cancer resistant to standard chemotherapy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; ATP Binding Cassette Transporter, Subfamily B; Colorectal Neoplasms; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Lapatinib; Mice; Mice, Nude; Mutation; Quinazolines; ras Proteins; Sirolimus; Xenograft Model Antitumor Assays

The incidence of colorectal neuroendocrine tumors (NETs) is increasing, and patients with this disease have particularly poor prognoses. Treatment options are limited, and survival times have not improved in the past decade.. A post hoc analysis of the efficacy and tolerability of everolimus plus octreotide long-acting repeatable (LAR) was conducted in patients with colorectal NETs enrolled in the phase III RAD001 in Advanced Neuroendocrine Tumors, Second Trial (RADIANT-2) study. The primary endpoint (progression-free survival [PFS]), secondary endpoints (including objective response rate), and safety were assessed.. Patients with colorectal NETs receiving everolimus plus octreotide LAR had a significantly longer median PFS (29.9 months; n = 19) than did those receiving placebo plus octreotide LAR (6.6 months; n = 20). Everolimus plus octreotide LAR treatment also significantly reduced the risk for disease progression (hazard ratio: 0.34; 95% confidence interval: 0.13-0.89; p = .011). Although no objective responses were observed, tumor shrinkage was more frequently noted in the everolimus plus octreotide LAR arm than in the placebo plus octreotide LAR arm (67% vs. 37%, respectively). The combination of everolimus plus octreotide LAR was generally well tolerated by patients with colorectal NETs; rash and stomatitis were the most commonly reported adverse events.. Everolimus plus octreotide LAR treatment had significant benefits and improved outcomes for patients with advanced colorectal NETs compared with placebo plus octreotide LAR treatment. Results of this exploratory analysis are consistent with those reported from the RADIANT-2 primary analysis. These findings support additional investigations of everolimus plus octreotide LAR in patients with colorectal NETs.

Topics: Adult; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Clinical Trials, Phase III as Topic; Colorectal Neoplasms; Disease-Free Survival; Everolimus; Female; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Neoplasm Staging; Neuroendocrine Tumors; Octreotide; Sirolimus

Epidemiological studies have indicated that obesity is associated with colorectal cancer. The obesity hormone leptin is considered as a key mediator for cancer development and progression. The present study aims to investigate regulatory effects of leptin on colorectal carcinoma. The expression of leptin and its receptor Ob-R was examined by immunohistochemistry in 108 Chinese patients with colorectal carcinoma. The results showed that leptin/Ob-R expression was significantly associated with T stage, TNM stage, lymph node metastasis, distant metastasis, differentiation and expression of p-mTOR, p-70S6 kinase, and p-Akt. Furthermore, the effects of leptin on proliferation and apoptosis of HCT-116 colon carcinoma cells were determined. The results showed that leptin could stimulate the proliferation and inhibit the apoptosis of HCT-116 colon cells through the PI3K/Akt/mTOR pathway. Ly294002 (a PI3K inhibitor) and rapamycin (an mTOR inhibitor) could prevent the regulatory effects of leptin on the proliferation and apoptosis of HCT-116 cells via abrogating leptin-mediated PI3K/Akt/mTOR pathway. All these results indicated that leptin could regulate proliferation and apoptosis of colorectal carcinoma through the PI3K/Akt/ mTOR signalling pathway.

Topics: Apoptosis; Asian People; Cell Proliferation; Chromones; Colorectal Neoplasms; Humans; Immunohistochemistry; Leptin; Morpholines; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptors, Leptin; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Epidermal growth factor receptor (EGFR) is highly expressed in colorectal carcinomas and, as a result, it leads to the activation of downstream mammalian target of rapamycin (mTOR) kinase pathways for cancer growth and progression. Clinical and preclinical studies, however, have shown that inhibition of epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) alone is not sufficient to treat colorectal carcinomas. In search of effective combination therapies, we show here that simultaneous targeting of EGFR with its inhibitor, erlotinib and mTOR with its inhibitor, rapamycin inhibits the phosphorylation and activation of downstream phosphatidylinositol 3-kinase (PI3K), Akt, mTOR and extracellular-signal-regulated kinase 1/2 (Erk1/2) pathways, resulting in the inhibition of cell cycle progression and the growth of both KRAS wild-type and mutated colorectal carcinoma cells. This study has demonstrated the principle that the combination of erlotinib and rapamycin may provide an effective therapy for colorectal carcinomas.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Drug Synergism; ErbB Receptors; Erlotinib Hydrochloride; G1 Phase Cell Cycle Checkpoints; Humans; MAP Kinase Signaling System; Mutation; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Quinazolines; ras Proteins; Sirolimus; TOR Serine-Threonine Kinases

Activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling is associated with tumorigenesis and metastasis of colorectal cancer (CRC). The mammalian target of rapamycin (mTOR) kinase, a downstream effector of PI3K/Akt signaling, regulates tumorigenesis and metastasis of CRCs, indicating that mTOR inhibition may have therapeutic potential. Notwithstanding, many cancers, including CRC, demonstrate resistance to the antitumorigenic effects of rapamycin. In this study, we show that inhibition of mTORC1 with rapamycin leads to feedback activation of PI3K/Akt and Ras-MAPK signaling, resulting in cell survival and possible contribution to rapamycin resistance. Combination with the multikinase inhibitor, sorafenib, abrogates rapamycin-induced activation of PI3K/Akt and Ras-MAPK signaling pathways. Combination of rapamycin with sorafenib synergistically inhibits proliferation of CRC cells. CRCs harboring coexistent KRAS and PIK3CA mutations are partially sensitive to either rapamycin or sorafenib monotherapy, but highly sensitive to combination treatment with rapamycin and sorafenib. Combination with sorafenib enhances therapeutic efficacy of rapamycin on induction of apoptosis and inhibition of cell-cycle progression, migration and invasion of CRCs. We demonstrate efficacy and safety of concomitant treatment with rapamycin and sorafenib at inhibiting growth of xenografts from CRC cells with coexistent mutations in KRAS and PIK3CA. The efficacy and tolerability of combined treatment with rapamycin and sorafenib provides rationale for use in treating CRC patients, particularly those with tumors harboring coexistent KRAS and PIK3CA mutations.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzenesulfonates; Cell Cycle; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; Drug Synergism; Humans; Male; MAP Kinase Signaling System; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Mutation; Niacinamide; Phenylurea Compounds; Phosphatidylinositol 3-Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Pyridines; ras Proteins; Sirolimus; Sorafenib; TOR Serine-Threonine Kinases

The mammalian target of rapamycin (mTOR) is a downstream integrator of essential pathways. mTOR signaling is frequently dysregulated in a variety of human cancers, and in silico analysis has revealed two miR-144 binding sites in the mTOR 3' untranslated region. We investigated the clinicopathologic magnitude of the mTOR pathway regulating microRNA, miR-144 in colorectal cancer (CRC) cases. The regulation of mTOR by miR-144 was examined with inhibitor miR-144-transfected cells. We also investigated changes in sensitivity to the mTOR inhibitor, rapamycin, in inhibitor miR-144-transfected cells. Quantitative RT-PCR was used to evaluate the clinicopathologic significance of miR-144 expression in 137 CRC. Furthermore, we assessed the correlation between CRC prognosis and the expression of 16 genes in the Akt/mTOR pathway. In vitro assays showed that mTOR is a direct target of miR-144, and downregulation of miR-144 facilitated proliferation of CRC cell line, HT29. In addition, the viability of HT29 cells with downregulated miR-144 expression was significantly reduced with rapamycin treatment. Low expression levels of miR-144 were associated with enhanced malignant potential such as venous invasion (P = 0.0013), liver metastasis (P = 0.08), liver recurrence (P = 0.0058) and poor prognosis (P = 0.0041). Multivariate analysis indicated that low miR-144 expression was an independent prognostic factor for survival. Among many genes consisting of the mTOR pathway, only high expression of Rictor was associated with poor prognosis of CRC. miR-144 is a meaningful prognostic marker. Downregulation of miR-144 leads to poor prognosis of CRC patients via activation of the mTOR signaling pathway.

Topics: Cell Proliferation; Colorectal Neoplasms; Disease Progression; Down-Regulation; HT29 Cells; Humans; MicroRNAs; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Thymidine phosphorylase (TPase) is also known as the platelet-derived endothelial cell growth factor (PD-ECGF) and plays a role in angiogenesis. Deoxyribose (dR; a downstream TPase-product) addition to endothelial cells may stimulate FAK and p70/S6k signaling, which can be inhibited by rapamycin. Rapamycin is a specific mammalian target of the rapamycin (mTOR) inhibitor, a kinase that lies directly upstream of p70/S6k. This suggests a role for TPase in the mTOR/p70/S6k pathway. In order to study this in more detail, we exposed cells with and without TPase expression to dR and rapamycin and determined the effect on cell growth. We observed protection in cytotoxicity in Colo320 cells, but not Colo320 TP1 cells. This was in part mediated by activation of p70/S6k and inhibition of autophagy. Further studies are recommended to elucidate the mechanism behind the protective effect of dR.

Topics: Autophagy; Blotting, Western; Cell Death; Cell Line, Tumor; Colorectal Neoplasms; Cytoprotection; Deoxyribose; Fluorescent Antibody Technique; Humans; Inhibitory Concentration 50; Intracellular Space; Microtubule-Associated Proteins; Phosphorylation; Sirolimus; TOR Serine-Threonine Kinases

APC mutations cause activation of Wnt/beta-catenin signaling, which invariably leads to colorectal cancer. Similarly, overexpressed Dvl proteins are potent activators of beta-catenin signaling. Screening a large tissue microarray of different staged colorectal tumors by immunohistochemistry, we found that Dvl2 has a strong tendency to be overexpressed in colorectal adenomas and carcinomas, in parallel to nuclear beta-catenin and Axin2 (a universal transcriptional target of Wnt/beta-catenin signaling). Furthermore, deletion of Dvl2 reduced the intestinal tumor numbers in a dose-dependent way in the Apc(Min) model for colorectal cancer. Interestingly, the small intestines of Dvl2 mutants are shortened, reflecting in part a reduction of their crypt diameter and cell size. Consistent with this, mammalian target of rapamycin (mTOR) signaling is highly active in normal intestinal crypts in which Wnt/beta-catenin signaling is active, and activated mTOR signaling (as revealed by staining for phosphorylated 4E-BP1) serves as a diagnostic marker of Apc(Min) mutant adenomas. Inhibition of mTOR signaling in Apc(Min) mutant mice by RAD001 (everolimus) reduces their intestinal tumor load, similarly to Dvl2 deletion. mTOR signaling is also consistently active in human hyperplastic polyps and has a significant tendency for being active in adenomas and carcinomas. Our results implicate Dvl2 and mTOR in the progression of colorectal neoplasia and highlight their potential as therapeutic targets in colorectal cancer.

Topics: Adaptor Proteins, Signal Transducing; Animals; Carrier Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cell Transformation, Neoplastic; Colorectal Neoplasms; Dishevelled Proteins; Eukaryotic Initiation Factors; Everolimus; Female; Humans; Inbreeding; Intestine, Small; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Phosphoproteins; Phosphorylation; Precancerous Conditions; Protein Serine-Threonine Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The PP2A serine/threonine protein phosphatase serves as a critical cellular regulator of cell growth, proliferation, and survival. However, how this pathway is altered in human cancer to confer growth advantage is largely unknown. Here, we show that PPP2R2B, encoding the B55β regulatory subunit of the PP2A complex, is epigenetically inactivated by DNA hypermethylation in colorectal cancer. B55β-associated PP2A interacts with PDK1 and modulates its activity toward Myc phosphorylation. On loss of PPP2R2B, mTORC1 inhibitor rapamycin triggers a compensatory Myc phosphorylation in PDK1-dependent, but PI3K and AKT-independent manner, resulting in resistance. Reexpression of PPP2R2B, genetic ablation of PDK1 or pharmacologic inhibition of PDK1 abrogates the rapamycin-induced Myc phosphorylation, leading to rapamycin sensitization. Thus, PP2A-B55β antagonizes PDK1-Myc signaling and modulates rapamycin sensitivity.

Topics: Animals; Antibiotics, Antineoplastic; Cell Proliferation; Cell Transformation, Neoplastic; Cellular Senescence; Class I Phosphatidylinositol 3-Kinases; Cluster Analysis; Colorectal Neoplasms; DNA Methylation; Drug Resistance, Neoplasm; Epigenesis, Genetic; Humans; Mice; Nerve Tissue Proteins; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Phosphatase 2; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Signal Transduction; Sirolimus; Transplantation, Heterologous

The role of the mTOR signal pathway in colorectal cancer (CRC) pathogenesis remains unclear, and the combination effect of PD98059 (an inhibitor for MEK) and rapamycin (an inhibitor for mTOR) on CRC is still unknown. Here, we found that combination treatment with PD98059 and rapamycin suppressed the proliferation of CRC cells, induced apoptosis, arrested cell cycle, and reduced the incidence and volume of CRC in mice, as well as inhibited phosphorylation of mTOR and the MEK signal pathway components, of which the effects were more significant than single-drug treatments. These findings indicate that PD98059 combined with rapamycin appears to be a promising strategy for inhibiting the initiation, and progression of CRC, which may provide a novel strategy for CRC prevention.

Topics: Animals; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Disease Progression; Female; Flavonoids; Humans; Mice; Mice, Inbred ICR; Mitogen-Activated Protein Kinase Kinases; Protein Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Although the anticancer effects of rapamycin (RPM) and 5-aza-deoxycytidine (AZA) have been studied extensively, the combined effect of these two drugs on colorectal cancer (CRC) is still unknown. This study addresses the effect of AZA and RPM combination therapy on CRC and its influence on the mammalian target of rapamycin (mTOR) and its signal transduction pathway.. Human CRC cell line HCT116 was treated with AZA alone, RPM alone, or concurrently with a combination of both drugs. Cell viability, apoptosis, and cell cycle distribution were analyzed. CRC was initiated in S-ICR mice, which were then treated with the drugs mentioned above, and tumor incidence and volume were measured. The activity of the mTOR signal transduction pathway was detected by Western blot analysis or immunohistochemistry.. Combination treatment with AZA and RPM inhibited the growth of HCT116 cells, induced apoptosis, arrested the cell cycle, and reduced the incidence and tumor volume of CRC in mice, as well as inhibited the phosphorylation of components of the mTOR signal transduction pathway. These effects were more significant than those of single-drug treatments.. Combination treatment with AZA and RPM inhibits the formation and growth of CRC. These findings may provide a novel strategy for CRC treatment.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Azacitidine; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Colorectal Neoplasms; DNA (Cytosine-5-)-Methyltransferases; Drug Synergism; HCT116 Cells; Humans; Immunohistochemistry; Mice; Phosphoproteins; Phosphorylation; Protein Kinases; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden

High-frequency microsatellite-instable (MSI-H) tumors account for approximately 15% of colorectal cancers. Therapeutic decisions for colorectal cancer are empirically based and currently do not emphasize molecular subclassification despite an increasing collection of gene expression information. Our objective was to identify low molecular weight compounds with preferential activity against MSI colorectal cancers using combined gene expression data sets.. Three expression/query signatures (discovery data set) characterizing MSI-H colorectal cancer were matched with information derived from changes induced in cell lines by 164 compounds using the systems biology tool "Connectivity Map." A series of sequential filtering and ranking algorithms were used to select the candidate compounds. Compounds were validated using two additional expression/query signatures (validation data set). Cytotoxic, cell cycle, and apoptosis effects of validated compounds were evaluated in a panel of cell lines.. Fourteen of the 164 compounds were validated as targeting MSI-H cell lines using the bioinformatics approach; rapamycin, LY-294002, 17-(allylamino)-17-demethoxygeldanamycin, and trichostatin A were the most robust candidate compounds. In vitro results showed that MSI-H cell lines due to hypermethylation of MLH1 are preferentially targeted by rapamycin (18.3 versus 4.4 mumol/L; P = 0.0824) and LY-294002 (15.02 versus 10.37 mumol/L; P = 0.0385) when compared with microsatellite-stable cells. Preferential activity was also observed in MSH2 and MSH6 mutant cells.. Our study shows that the phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathway is of special relevance in mismatch repair-deficient colorectal cancer. In addition, we show that amalgamation of gene expression information across studies provides a robust approach for selection of potential therapies corresponding to specific groups of patients.

Topics: Algorithms; Antineoplastic Agents; Benzoquinones; Cell Cycle; Cell Line, Tumor; Chromones; Colorectal Neoplasms; Computational Biology; DNA Mismatch Repair; Drug Evaluation, Preclinical; Enzyme Inhibitors; Gene Expression Profiling; Humans; Hydroxamic Acids; Immunosuppressive Agents; Lactams, Macrocyclic; Microsatellite Instability; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Sirolimus

Under conditions of nutrient stress, cells switch to a survival mode catabolizing cellular and tissue constituents for energy. Proline metabolism is especially important in nutrient stress because proline is readily available from the breakdown of extracellular matrix (ECM), and the degradation of proline through the proline cycle initiated by proline oxidase (POX), a mitochondrial inner membrane enzyme, can generate ATP. This degradative pathway generates glutamate and alpha-ketoglutarate, products that can play an anaplerotic role for the TCA cycle. In addition the proline cycle is in a metabolic interlock with the pentose phosphate pathway providing another bioenergetic mechanism. Herein we have investigated the role of proline metabolism in conditions of nutrient stress in the RKO colorectal cancer cell line. The induction of stress either by glucose withdrawal or by treatment with rapamycin, stimulated degradation of proline and increased POX catalytic activity. Under these conditions POX was responsible, at least in part, for maintenance of ATP levels. Activation of AMP-activated protein kinase (AMPK), the cellular energy sensor, by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), also markedly upregulated POX and increased POX-dependent ATP levels, further supporting its role during stress. Glucose deprivation increased intracellular proline levels, and expression of POX activated the pentose phosphate pathway. Together, these results suggest that the induction of proline cycle under conditions of nutrient stress may be a mechanism by which cells switch to a catabolic mode for maintaining cellular energy levels.

Topics: Adenosine Triphosphate; Cell Line, Tumor; Colorectal Neoplasms; Glucose; Humans; Malnutrition; Proline; Proline Oxidase; Sirolimus; Stress, Physiological; Up-Regulation

Liver regeneration after hepatectomy stimulates metastatic tumor growth through the release of potent growth factors. In the signaling cascades of several growth factors, mTOR is a downstream mediator, triggering cell survival and cell cycle progression. The mTOR inhibitor rapamycin (RAPA) has been shown to exhibit potent antitumor activities. However, it is unknown whether RAPA is capable of exerting these effects under the conditions of hepatectomy-associated liver regeneration. We therefore analyzed the effect of RAPA and cyclosporine A (CyA) on tumor growth characteristics after major hepatectomy using a mouse model of colorectal metastasis.. Tumor growth was studied by using GFP-transfected CT26.WT colorectal cancer cells, which were implanted into the dorsal skinfold chambers of BALB/c-mice after 70% hepatectomy. The animals were treated daily with RAPA (1.5 mg/kg) or CyA (10 mg/kg). Tumors were analyzed for angiogenesis, microvascular blood perfusion, cell proliferation, apoptotic cell death, and tumor growth.. RAPA significantly inhibited tumor growth compared with CyA and sham treatment. This was associated with a decreased microvascular density within the tumors and a markedly reduced microvascular blood perfusion. CyA only slightly reduced angiogenesis and tumor growth. The effects of RAPA were associated with a significant reduction of tumor cell proliferation, whereas manifestation of apoptotic cell death was not affected by the immunosuppressive treatment regimen.. RAPA is capable of inhibiting angiogenesis, microvascular blood perfusion, and tumor growth of colorectal metastasis during hepatectomy-associated liver regeneration. Thus, targeting mTOR might represent an interesting strategy to prevent tumor recurrence after hepatectomy for colorectal metastasis.

Topics: Adenocarcinoma; Angiogenesis Inhibitors; Animals; Apoptosis; Cell Cycle; Cell Proliferation; Colorectal Neoplasms; Cyclosporine; Female; Hepatectomy; Humans; Immunoenzyme Techniques; Immunosuppressive Agents; In Situ Nick-End Labeling; Liver Regeneration; Mice; Mice, Inbred BALB C; Mice, Nude; Microcirculation; Neovascularization, Pathologic; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The mTOR-inhibitor rapamycin has shown antitumor activity in various tumors. Bedside observations have suggested that rapamycin may be effective as a treatment for colorectal carcinomatosis.. We established an orthotopic syngenic model by transplanting CT26 peritoneal tumors in Balb/C mice and an orthotopic xenograft model by transplanting SW620 peritoneal tumors in nu/nu mice. Expression levels of tissue inhibitor of matrix-metalloproteinases 1 (TIMP-1) in the tumor and serum was determined by enzyme-linked immunosorbent assay.. Rapamycin significantly suppressed growth of syngenic and xenografted peritoneal tumors. The effect was similar with intraperitoneal or oral rapamycin administration. Tumor suppression was further enhanced when rapamycin was combined with 5-fluorouracil and/or oxaliplatin. The combination treatment showed no acute toxicity. TIMP-1 serum levels correlated well (CC = 0.75; P < 0.01) with rapamycin treatment.. Rapamycin suppressed advanced stage colorectal cancer, even with oral administration. Combining rapamycin with current chemotherapy regimens significantly increased antitumor efficacy without apparent toxicity. The treatment efficacy correlated with serum TIMP-1 levels, suggesting its potential as a surrogate marker in future clinical trials.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Colorectal Neoplasms; Fluorouracil; Humans; Mice; Organoplatinum Compounds; Oxaliplatin; Peritoneal Neoplasms; Sirolimus; Tissue Inhibitor of Metalloproteinase-1; Treatment Outcome; Xenograft Model Antitumor Assays

The underlying mechanism regulating the expression of the cancer stem cell/tumor-initiating cell marker CD133/prominin-1 in cancer cells remains largely unclear, although knowledge of this mechanism would likely provide important biological information regarding cancer stem cells. Here, we found that the inhibition of mTOR signaling up-regulated CD133 expression at both the mRNA and protein levels in a CD133-overexpressing cancer cell line. This effect was canceled by a rapamycin-competitor, tacrolimus, and was not modified by conventional cytotoxic drugs. We hypothesized that hypoxia-inducible factor-1 alpha (HIF-1 alpha), a downstream molecule in the mTOR signaling pathway, might regulate CD133 expression; we therefore investigated the relation between CD133 and HIF-1 alpha. Hypoxic conditions up-regulated HIF-1 alpha expression and inversely down-regulated CD133 expression at both the mRNA and protein levels. Similarly, the HIF-1 alpha activator deferoxamine mesylate dose-dependently down-regulated CD133 expression, consistent with the effects of hypoxic conditions. Finally, the correlations between CD133 and the expressions of HIF-1 alpha and HIF-1 beta were examined using clinical gastric cancer samples. A strong inverse correlation (r = -0.68) was observed between CD133 and HIF-1 alpha, but not between CD133 and HIF-1 beta. In conclusion, these results indicate that HIF-1 alpha down-regulates CD133 expression and suggest that mTOR signaling is involved in the expression of CD133 in cancer cells. Our findings provide a novel insight into the regulatory mechanisms of CD133 expression via mTOR signaling and HIF-1 alpha in cancer cells and might lead to insights into the involvement of the mTOR signal and oxygen-sensitive intracellular pathways in the maintenance of stemness in cancer stem cells.

Topics: AC133 Antigen; Antigens, CD; Cell Line, Tumor; Chromones; Colorectal Neoplasms; Down-Regulation; Glycoproteins; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Lung Neoplasms; Morpholines; Neoplasms; Peptides; Protein Kinases; RNA, Messenger; Signal Transduction; Sirolimus; Stomach Neoplasms; Tacrolimus; TOR Serine-Threonine Kinases; Transcription, Genetic; Up-Regulation

To evaluate the efficacy and the safety of combined 5-Fluorouracil, irinotecan, bevacizumab and sirolimus in refractory advanced colorectal carcinoma.. We initiated a regimen with at day 1 an injection (iv) of bevacizumab at 5 mg/kg, followed by 180 mg/m(2) irinotecan, followed by Leucovorin 400 mg/m(2), followed by a 5-Fluorouracil bolus 400 mg/m(2); and a 46-h infusion 2400 mg/m(2). Sirolimus was given orally as continuous administration of 2 mg twice a day every days. This treatment was repeated every 14 d.. A total of 12 patients were enrolled. All patients presented with metastatic disease that had failed at least three lines of chemotherapy that contained oxaliplatin, irinotecan and bevacizumab. Cetuximab failure was also observed in all K-Ras wild-type patients. The median number of cycles was 8.5 (range 2-20) and clinical benefit was observed in eight patients. The median time to progression was 5 mo and the median survival was 8 mo. Grade 3 neutropenia developed in four patients, and grade 3 diarrhea and stomatitis in two.. The combination regimen of 5-Fluorouracil, irinotecan, bevacizumab and sirolimus in advanced colorectal carcinoma after failure of classical treatment is feasible and promising. Further evaluation of this combination is required.

Topics: Aged; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Camptothecin; Carcinoma; Colorectal Neoplasms; Female; Fluorouracil; Humans; Irinotecan; Male; Middle Aged; Pilot Projects; Sirolimus; Treatment Outcome

We investigated the Akt-mTOR pathway and effects of rapamycin using human colorectal cancer cell lines. LoVo and CaRI reduced proliferative activity in response to rapamycin in a dose-dependent manner. The phosphorylation of Akt and p70 S6K was prominent in these cells. Rapamycin quickly downregulated phospho-S6K but not phospho-Akt. Therefore, phospho-S6K is considered a good indicator of the activated Akt-mTOR pathway as well as rapamycin sensitivity in colorectal cancer cells. By immunohistochemical study, nearly 40% of adenomas and carcinomas of the colorectum exhibited either partial or whole positive staining for phospho-S6K, suggestive of rapamycin-sensitive lesions.

Topics: Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Immunohistochemistry; Ki-67 Antigen; Phosphorylation; Protein Kinases; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

To investigate the synergistic effect of rapamycin (RPM) and PD98059 on human colorectal cancer cells and its potential mechanisms.. Three human colorectal cancer cell lines SW480, HCT116 and HT29 were treated with RPM 10 nmol/L, PD98059 (10 micromol/L, 20 micromol/L, 40 micromol/L, 50 micromol/L), or RPM plus PD98059, respectively, and the sensitivity was analyzed by MTT assay. The cell cycle progression was evaluated by flow cytometry. Western blotting analysis was performed to examine the total and phosphorylated levels of mammalian target of rapamycin (mTOR) and its downstream translational signaling intermediates, 70 kDa ribosomal protein S6 kinase (p70s6k) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1).. Both RPM and PD98059 could inhibit viability of the three cell lines. The anti-proliferative effect of PD98059 exhibited a time/dose dependent manner and was strengthen by RPM. All the treatment with RPM, PD98059, and RPM + PD98059 induced arrest of cell cycle, although the arrest was confined at different cell cycle phases. In addition to their effect on proliferation and cell cycle, both inhibitors also reduced phosphorylation levels of mTOR, p70s6k, and 4E-BP1, as well as total 4E-BP1 levels in SW480 and HCT116 cells. That effect was reinforced when cells were treated with RPM plus PD98059 simultaneously, whereas total protein levels of mTOR and p70s6k remained unchanged.. RPM and PD98059 inhibit proliferation of colorectal cancer cells synergistically, and induce cell cycle arrest. The modulation of mammalian target of rapamycin signaling pathway is involved in its potential mechanisms.

Topics: Adaptor Proteins, Signal Transducing; Antibiotics, Antineoplastic; Calcium-Calmodulin-Dependent Protein Kinases; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Colorectal Neoplasms; Drug Synergism; Flavonoids; HCT116 Cells; HT29 Cells; Humans; Intracellular Signaling Peptides and Proteins; Phosphoproteins; Phosphorylation; Protein Serine-Threonine Kinases; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Immunosuppressive therapies associated with organ transplantation produce an increased risk of cancer development. Malignancies are increased in transplant recipients because of the impaired immune system. Moreover, experimental data point to a tumor-promoting activity of various immunosuppressive agents. In this study, we compared the effects of 4 immunosuppressive agents with different mechanisms of action (cyclosporine, rapamycin, mycophenolic acid, and leflunomide) on the in vitro growth of various tumor cell lines and umbilical vein endothelial cells. To varying degrees rapamycin (10 ng/mL), mycophenolic acid (300 nmol/L), and leflunomide (30 micromol/L) highly inhibited the growth of human rhabdomyosarcoma, hepatocellular carcinoma, colorectal carcinoma, and endothelial cells. In contrast, cyclosporine (100 ng/mL) did not affect their growth. Our data suggest that regimens containing rapamycin, mycophenolic acid, or leflunomide, which have both immunosuppressive and antitumor activities, should be preferred in transplant recipients to minimize the risk of tumors.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Cyclosporine; Humans; Immunosuppression Therapy; Immunosuppressive Agents; Isoxazoles; Jurkat Cells; Leflunomide; Liver Neoplasms; Mycophenolic Acid; Rhabdomyosarcoma; Sirolimus

We aimed to use cell-based carriers to direct vector production to target sites for systemic therapy. We used T cells engineered to express a chimeric T cell receptor that can specifically recognize target cells expressing the tumor-associated carcinoembryonic antigen (CEA). These T cells were modified to produce a retrovirus under tight pharmacological control using the rapamycin-inducible transcriptional regulatory system. The retroviral vectors produced were transcriptionally targeted to CEA-expressing target cells. We found that vector production and transgene expression from these T cells in vitro was dependent on pharmacological induction and expression of CEA in target cells, respectively. Mice bearing metastatic tumors that received cell carriers delivering the HSVtk gene demonstrated a significant increase in survival, but only in response to pharmacological induction of vector production. Interestingly, the therapeutic effect required the presence of the tumor-specific chimeric receptor on T cells. Further studies demonstrated that systemic delivery of tumor-specific T cells to mice bearing metastatic tumors caused recruitment of nonspecific T cells to the tumor site. We hypothesize that this enhanced targeting to tumor sites is responsible for the efficiency of T cell-mediated retroviral gene transfer and that this principle can be used to enhance systemic therapies using immune-cell carriers.

Topics: Adenocarcinoma; Animals; Antigens, Neoplasm; Carcinoembryonic Antigen; Colorectal Neoplasms; Drug Delivery Systems; Fluorescent Dyes; Gene Expression Regulation, Viral; Genes, Synthetic; Genetic Therapy; Genetic Vectors; Humans; Jurkat Cells; Liver Neoplasms, Experimental; Melanoma; Mice; Moloney murine leukemia virus; Organ Specificity; Promoter Regions, Genetic; Recombinant Fusion Proteins; Simplexvirus; Sirolimus; Terminal Repeat Sequences; Thymidine Kinase; Transcription, Genetic; Transduction, Genetic; Transfection; Viral Proteins; Virus Replication; Xenograft Model Antitumor Assays