dactolisib and Prostatic-Neoplasms

Granulocytic myeloid-derived suppressor cells (Gr-MDSCs) promote immune evasion and resistance to immunotherapeutics in a variety of malignancies. Our previous study showed that dual PI3K/mTOR inhibitor Dactolisib impaired the viability and immunosuppressive function of Gr-MDSCs, and significantly synergized with immune checkpoint blockade (ICB) antibodies targeting PD1 and CTLA4 to eradicate metastatic castration-resistant prostate cancer (CRPC) in a preclinical transgenic mouse model. On the contrary, tyrosine kinase inhibitor Dasatinib diminished tumor-infiltrating T lymphocytes and showed no synergic activity with ICB. The understanding of the distinct effects of Dactolisib and Dasatinib on Gr-MDSCs, T cells and prostate neoplastic cells is inadequate, limiting the clinical translation of the combination immunotherapy. To address this question, we applied Reverse Phase Protein Array (RPPA) to profile 297 proteins and protein phosphorylation sites of Gr-MDSCs, T cells and prostate cancer cells isolated from the CRPC model. We found cell type-specific protein expression patterns and highly selective targets by the two drugs, including preferential inhibition of phospho-4E-BP1 in Gr-MDSCs by Dactolisib and preferential suppression of phospho-Src and phospho-p38 MAPK in T cells. Furthermore, transcriptomic profiling of Gr-MDSCs treated with the two inhibitors revealed downregulation of mitochondrial respiration pathways by Dactolisib but not Dasatinib. Overall, these results provide important mechanistic insight into the efficacious combination of Dactolisib and ICB as well as the detrimental effect of Dasatinib on anti-tumor immunity.

Topics: Animals; CTLA-4 Antigen; Dasatinib; Humans; Imidazoles; Immune Checkpoint Inhibitors; Immunotherapy; Lymphocytes, Tumor-Infiltrating; Male; Mice; Mice, Transgenic; Myeloid-Derived Suppressor Cells; Programmed Cell Death 1 Receptor; Prostate; Prostatic Neoplasms; Prostatic Neoplasms, Castration-Resistant; Protein Kinase Inhibitors; Quinolines; TOR Serine-Threonine Kinases

PIM and PI3K/mTOR pathways are often dysregulated in prostate cancer, and may lead to decreased survival, increased metastasis and invasion. The pathways are heavily interconnected and act on a variety of common effectors that can lead to the development of resistance to drug inhibitors. Most current treatments exhibit issues with toxicity and resistance. We investigated the novel multikinase PIM/PI3K/mTOR inhibitor, AUM302, versus a combination of the PIM inhibitor, AZD-1208, and the PI3K/mTOR inhibitor BEZ235 (Dactolisib) to determine their impact on mRNA and phosphoprotein expression, as well as their functional efficacy. We have determined that around 20% of prostate cancer patients overexpress the direct targets of these drugs, and this cohort are more likely to have a high Gleason grade tumour (≥ Gleason 8). A co-targeted inhibition approach offered broader inhibition of genes and phosphoproteins in the PI3K/mTOR pathway, when compared to single kinase inhibition. The preclinical inhibitor AUM302, used at a lower dose, elicited a comparable or superior functional outcome compared with combined AZD-1208 + BEZ235, which have been investigated in clinical trials, and could help to reduce treatment toxicity in future trials. We believe that a co-targeting approach is a viable therapeutic strategy that should be developed further in pre-clinical studies.

Topics: Antineoplastic Agents; Biphenyl Compounds; Cell Line, Tumor; Cell Proliferation; Cohort Studies; Drug Therapy, Combination; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Male; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Prostatic Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-pim-1; Pyridines; Pyrimidines; Quinolines; RNA, Messenger; Signal Transduction; Thiazolidines; Thiophenes; TOR Serine-Threonine Kinases

Use of drug combinations that target different pathways involved in the development and progression of prostate cancer (PCa) has emerged as an alternative to overcome the resistance caused by drug monotherapies. The antiandrogen abiraterone acetate and the PI3K/Akt inhibitor NVP-BEZ235 (BEZ235) may be suitable options for the prevention of drug resistance and the inhibition of PCa progression. The aim of the present study was to evaluate whether abiraterone acetate and BEZ235 achieve superior therapeutic effects to either drug administered as monotherapy, in the early stages of PCa in an androgen-dependent system. Our study showed that each drug might impair tumor growth by reducing proliferation and increasing cell death when administered as monotherapy. However, tumor growth continued to progress with each drug monotherapy and some important side effects were related to BEZ. Conversely, when used in combination, the drugs impaired the inflammatory response, decreased hyperplastic lesions, and blocked tumor progression from premalignant to a malignant stage. Our data showed that the strategy to block the androgenic and PI3K/AKT/mTOR pathway is an effective therapeutic option and should be investigated including distinct PI3K pathway inhibitors.

Topics: Abiraterone Acetate; Androgen Antagonists; Androgens; Animals; Antineoplastic Combined Chemotherapy Protocols; Carcinogenesis; Disease Models, Animal; Drug Synergism; Humans; Imidazoles; Male; Phosphoinositide-3 Kinase Inhibitors; Prostatic Neoplasms; Protein Kinase Inhibitors; Quinolines; Rats; Rats, Inbred F344; Signal Transduction; Tumor Cells, Cultured

To determine effects of BEZ235, an inhibitor of phosphoionsitol-3-kinase (PI3K)/mTOR, on the cell proliferation and migration in human prostate carcinoma lines including RWPE-1, PC3, and DU145 cells.
 Methods: Viability of RWPE-1, PC3, and DU145 cells was detected by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, while cell migration was analyzed by wound healing assay. Western blot and immunofluorescence were used to examine the changes of relevant protein expression. 
 Results: The proliferation of PC3 and DU145 cells was effectively inhibited by BEZ235 (P<0.01), whereas RWPE-1 was not obviously inhibited. Invasion and migration of PC3 and DU145 cells were attenuated by BEZ235 via EMT pathway.
 Conclusion: The PI3K/mTOR dual inhibitor BEZ235 shows substantial anti-tumor activity in human prostate carcinoma lines of PC3 and DU145 cells, which may be involved in the EMT pathway.. 目的：探讨PI3K/mTOR双重靶向抑制剂BEZ235对人前列腺癌细胞株的增殖、迁移能力的影响。方法：采用MTT实验分析BEZ235对正常前列腺上皮细胞RWPE-1和前列腺癌细胞PC3及DU145增殖能力的影响；划痕试验检测BEZ235对RWPE-1，PC3及DU145细胞迁移的影响；Western印迹及免疫荧光染色方法检测BEZ235对上皮-间质转化(epithelial-mesenchymal transition，EMT)相关标志物蛋白表达的影响。结果：BEZ235对PC3和DU145细胞的增殖具有明显的抑制作用(P<0.01)，而对RWPE-1作用不明显；BEZ235明显上调PC3和DU145细胞标志物E-cadherin的表达，下调间质标志物Vimentin的表达(P<0.01)。结论：BEZ235可体外抑制PC3和DU145细胞增殖和迁移，其部分机制可能是通过抑制EMT途径而实现。.

Topics: Cell Line, Tumor; Cell Proliferation; Humans; Imidazoles; Male; Phosphatidylinositol 3-Kinases; Prostatic Neoplasms; Quinolines; TOR Serine-Threonine Kinases

The intervention of advanced prostate cancer (PCa) in patients has been commonly depending on androgen deprivation therapy. Despite of tremendous research efforts, however, molecular mechanisms on AR regulation remain poorly understood, particularly for castration resistant prostate cancer (CRPC). Targeting AR and associated factors is considered an effective strategy in PCa treatment.. Human prostate cancer cells were used in this study. Manipulations of Skp2 expression were achieved by Skp2 shRNA/siRNA or overexpression of plasmids. Dual luciferase reporter assay was applied for AR activity assessment. Western blot, ubiquitination assay, immunoprecipitation, and immunofluorescence were applied to detect the proteins.. Our results demonstrated that Skp2 directly involves the regulation of AR expression through ubiquitination-mediated degradation. Skp2 interacted with AR protein in PCa cells, and enforced expression of Skp2 resulted in a decreased level and activity of AR. By contrast, Skp2 knockdown increased the protein accumulation and activity of AR. Importantly, changes of AR contributed by Skp2 led to subsequent alterations of PSA level in PCa cells. AR ubiquitination was significantly increased upon Skp2 overexpression but greatly reduced upon Skp2 knockdown. AR mutant at K847R abrogated Skp2-mediated ubiquitination of AR. NVP-BEZ235, a dual PI3K/mTOR inhibitor, remarkably inhibited Skp2 level with a striking elevation of AR.. The results indicate that Skp2 is an E3 ligase for proteasome-dependent AR degradation, and K847 on AR is the recognition site for Skp2-mediated ubiquitination. Our findings reveal an essential role of Skp2 in AR signaling.

Topics: Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Male; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Quinolines; Receptors, Androgen; S-Phase Kinase-Associated Proteins; Signal Transduction; TOR Serine-Threonine Kinases; Ubiquitin; Ubiquitination

Deregulation of the PI3K-AKT/mTOR pathway due to mutation of the tumor suppressor gene PTEN frequently occurs in human prostate cancer and is therefore considered to be an attractive therapeutic target. Here, we investigated how the PTEN genotype affected the antitumor effect of NVP-BEZ235 in human prostate cancer cells. In this setting, NVP-BEZ235 induced cell death in a PTEN-independent manner. NVP-BEZ235 selectively induced apoptotic cell death in the prostate cancer cell line DU145, which harbors wild-type PTEN; however, in the PC3 cell line, which is PTEN-null, treatment with NVP-BEZ235 resulted in autophagic cell death. Consistently, NVP-BEZ235 treatment did not result in the cleavage of caspase-3; instead, it resulted in the conversion of LC3-I to LC3-II, indicating autophagic cell death; these results suggest that an alternate mechanism of cell death is induced by NVP-BEZ235 in PTEN-null prostate cancer cells. Based on our findings, we conclude that the PTEN/PI3K/Akt pathway is critical for prostate cancer survival, and targeting PI3K signaling by NVP-BEZ235 may be beneficial in the treatment of prostate cancer, independent of the PTEN genotype.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Drug Resistance, Neoplasm; Genotype; Humans; Imidazoles; Male; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Prostatic Neoplasms; PTEN Phosphohydrolase; Quinolines; TOR Serine-Threonine Kinases

Aberrant activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway may account for development of radioadaptation and is not rare in prostate cancer. Neither PI3K nor mTOR blockade could completely inhibit the pathway owing to paradoxical feedback, so we anticipate dual PI3K/mTOR blockade by NVP-BEZ235 to radiosensitize prostate cancer cells.. We investigated into the radiosensitizing effect of NVP-BEZ235 on PC-3 cells, which are devoid of androgen receptors. Clonogenic survival and MTT assays were performed, and to pursue underlying cellular changes flow cytometric analysis of cell cycle and apoptosis as well as western blot were carried out.. Exposure to NVP-BEZ235 and irradiation caused a greater degree of survival inhibition than ionizing radiation (IR) or BEZ235 alone. Dual PI3K/mTOR blockade along with IR induced a G2/M arrest and enhanced proapoptotic effect. NVP-BEZ235 radiosensitized PC-3 cells through counteracting constitutive as well as IR-triggered activation of Akt/mTOR signaling.. Our study demonstrated that the dual PI3K/mTOR inhibitor NVP-BEZ235 prominently improved the radiosensitivity of PC-3 cells. It sensitized tumor cells to irradiation via interruption of cell cycle progression and augmentation of cell apoptosis, which was due to its constraint on constitutive and IR-elicited PI3K/Akt/mTOR signaling activation.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Survival; Combined Modality Therapy; Flow Cytometry; Humans; Imidazoles; Male; Phosphoinositide-3 Kinase Inhibitors; Prostatic Neoplasms; Quinolines; Radiation Tolerance; Radiation, Ionizing; Radiopharmaceuticals; Receptors, Androgen; Tetrazolium Salts; Thiazoles; TOR Serine-Threonine Kinases

Radioresistance is a major challenge in prostate cancer (CaP) radiotherapy (RT). In this study, we investigated the role and association of epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and the PI3K/Akt/mTOR signaling pathway in CaP radioresistance. We developed three novel CaP radioresistant (RR) cell lines (PC-3RR, DU145RR and LNCaPRR) by radiation treatment and confirmed their radioresistance using a clonogenic survival assay. Compared with untreated CaP-control cells, the CaP-RR cells had increased colony formation, invasion ability and spheroid formation capability (P<0.05). In addition, enhanced EMT/CSC phenotypes and activation of the checkpoint proteins (Chk1 and Chk2) and the PI3K/Akt/mTOR signaling pathway proteins were also found in CaP-RR cells using immunofluorescence, western blotting and quantitative real-time PCR (qRT-PCR). Furthermore, combination of a dual PI3K/mTOR inhibitor (BEZ235) with RT effectively increased radiosensitivity and induced more apoptosis in CaP-RR cells, concomitantly correlated with the reduced expression of EMT/CSC markers and the PI3K/Akt/mTOR signaling pathway proteins compared with RT alone. Our findings indicate that CaP radioresistance is associated with EMT and enhanced CSC phenotypes via activation of the PI3K/Akt/mTOR signaling pathway, and that the combination of BEZ235 with RT is a promising modality to overcome radioresistance in the treatment of CaP. This combination approach warrants future in vivo animal study and clinical trials.

Topics: Animals; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Enzyme Activation; Epithelial-Mesenchymal Transition; Humans; Imidazoles; Male; Models, Biological; Neoplasm Invasiveness; Neoplastic Stem Cells; Phenotype; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Quinolines; Radiation Tolerance; Reproducibility of Results; Signal Transduction; Spheroids, Cellular; TOR Serine-Threonine Kinases

Prostate cancer is an ideal target for chemoprevention. To date, chemoprevention clinical trials with 5α-reductase inhibitors have yielded encouraging yet ultimately confounding results. Using a preclinical mouse model of high-grade prostatic intraepithelial neoplasia (HG-PIN) induced by PTEN loss, we observed unprecedented deteriorating effects of androgen deprivation, in which surgical castration or MDV3100 treatment accelerated disease progression of the otherwise stable HG-PIN to invasive castration-resistant prostate cancer (CRPC). As an alternative, targeting the phosphoinositide 3-kinase (PI3K) signaling pathway via either genetic ablation of genes encoding PI3K components or pharmacologic inhibition of the PI3K pathway reversed the PTEN loss-induced HG-PIN phenotype. Finally, concurrent inhibition of the PI3K and mitogen-activated protein kinase (MAPK) pathways was effective in blocking the growth of PTEN-null CRPC. Together, these data have revealed the potential adverse effects of antiandrogen chemoprevention in certain genetic contexts (such as PTEN loss) while showing the promise of targeted therapy in the clinical management of this complex and prevalent disease.. Chemoprevention with antiandrogen therapies is attractive for prostate cancer, given its prevalence and established hormonally mediated pathogenesis. However, because PTEN loss has been found in 9% to 45% of HG-PIN in the clinic, the current findings suggest that patients with PTEN-deficient prostate tumors might be better treated with PI3K-targeted therapies.

Topics: Aminopyridines; Androgen Receptor Antagonists; Animals; Antineoplastic Agents; Benzamides; Benzimidazoles; Castration; Imidazoles; Male; MAP Kinase Kinase Kinases; Mice; Mice, Transgenic; Morpholines; Nitriles; Phenylthiohydantoin; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Prostatic Intraepithelial Neoplasia; Prostatic Neoplasms; Protein Kinase Inhibitors; PTEN Phosphohydrolase; Quinolines; Testosterone

Despite appropriate radiotherapy, high-risk prostate cancer patients often experience local relapse and progression to metastatic disease. Radioresistance may be due to tumor-hypoxia but also due to the PTEN mutation/deletion present in 70% prostate cancers. We investigated whether the novel PI3K/mTOR inhibitor BEZ235 might sensitize prostate cancer cells to radiation and reduce hypoxia-induced radioresistance.. The potential radiosensitizing properties of BEZ235 were investigated in vitro and in vivo using two prostate cancer cell lines, PC3 (PTEN(-/-)) and DU145 (PTEN(+/+)), under normoxic (21% O(2)) and hypoxic (0.5% O(2)) conditions.. BEZ235 rapidly inhibited PI3K and mTOR signaling in a dose dependent manner and limited tumor cell proliferation and clonogenic survival in both cell lines independently of PTEN status. In vivo, BEZ235 pretreatment enhanced the efficacy of radiation therapy on PC3 xenograft tumors in mice without inducing intestinal radiotoxicity. In culture, BEZ235 radiosensitized both cell lines in a comparable manner. Moreover, BEZ235 inhibited PI3K/mTOR activation and radiosensitized both cell lines under normoxia and hypoxia. BEZ235 radiosensitizing effects correlated with a decrease in γH2AX foci repair and increased G2/M cell cycle arrest.. BEZ235 is a potent radiosensitizer of normoxic and hypoxic prostate cancer cells.

Topics: Animals; Cell Cycle Checkpoints; Cell Hypoxia; Cell Line, Tumor; DNA Breaks; Humans; Imidazoles; Male; Mice; Phosphoinositide-3 Kinase Inhibitors; Prostatic Neoplasms; Quinolines; Radiation-Sensitizing Agents; TOR Serine-Threonine Kinases

The cancer stem cell hypothesis predicts that standard prostate cancer monotherapy eliminates bulk tumor cells but not a tumor-initiating cell population, eventually leading to relapse. Many studies have sought to determine the underlying differences between bulk tumor and cancer stem cells.. Our previous data suggest that the PTEN/PI3K/AKT pathway is critical for the in vitro maintenance of CD133(+)/CD44(+) prostate cancer progenitors and, consequently, that targeting PI3K signaling may be beneficial in treatment of prostate cancer.. Here, we show that inhibition of PI3K activity by the dual PI3K/mTOR inhibitor NVP-BEZ235 leads to a decrease in the population of CD133(+)/CD44(+) prostate cancer progenitor cells in vivo. Moreover, the combination of the PI3K/mTOR modulator NVP-BEZ235, which eliminates prostate cancer progenitor populations, and the chemotherapeutic drug Taxotere, which targets the bulk tumor, is significantly more effective in eradicating tumors in a prostate cancer xenograft model than monotherapy.. This combination treatment ultimately leads to the expansion of cancer progenitors with a PTEN E91D mutation, suggesting that the analysis of PTEN mutations could predict therapeutic response to the dual therapy.

Topics: AC133 Antigen; Animals; Antigens, CD; Antineoplastic Combined Chemotherapy Protocols; Carcinoma; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Delivery Systems; Glycoproteins; Humans; Hyaluronan Receptors; Imidazoles; Male; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, SCID; Neoplastic Stem Cells; Peptides; Prostatic Neoplasms; PTEN Phosphohydrolase; Quinolines; Xenograft Model Antitumor Assays