gant-61 and Breast-Neoplasms

Current conventional mono and combination therapeutic strategies often fail to target breast cancer tissue effectively due to tumor heterogeneity comprising cancer stem cells (CSCs) and bulk tumor cells. This is further associated with drug toxicity and resistivity in the long run. A nanomedicine platform incorporating combination anti-cancer treatment might overcome these challenges and generate synergistic anti-cancer effects and also reduce drug toxicity. GANT61 and curcumin were co-delivered via polymeric nanoparticles (NPs) for the first time to elicit enhanced anti-tumor activity against heterogeneous breast cancer cell line MCF-7. We adopted the single-emulsion-solvent evaporation method for the preparation of the therapeutic NPs. The GANT61-curcumin PLGA NPs were characterized for their size, shape and chemical properties, and anti-cancer cell studies were undertaken for the plausible explanation of our hypothesis. The synthesized GANT61-curcumin PLGA NPs had a spherical, smooth surface morphology, and an average size of 347.4 d. nm. The NPs induced cytotoxic effects in breast cancer cells at a mid-minimal dosage followed by cell death via autophagy and apoptosis, reduction in their target protein expression along with compromising the self-renewal property of CSCs as revealed by their in vitro cell studies. The dual-drug NPs thus provide a novel perspective on aiding existing anti-cancer nanomedicine therapies to target a heterogeneous tumor mass effectively.

Topics: Adenocarcinoma; Animals; Apoptosis; Autophagy; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Curcumin; Drug Liberation; Endocytosis; Female; Humans; MCF-7 Cells; Mice; Nanoparticles; Particle Size; Phosphatidylinositol 3-Kinases; Photoelectron Spectroscopy; Polylactic Acid-Polyglycolic Acid Copolymer; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidines; Spectroscopy, Fourier Transform Infrared; Spheroids, Cellular; Static Electricity; Zinc Finger Protein GLI1

Cholesterol is a risk factor for breast cancer. However, it is still unclear whether the cholesterol biosynthesis pathway plays any significant role in breast carcinogenesis. 24-Dehydrocholesterol reductase (DHCR24) is a key enzyme in the cholesterol synthesis pathway. Although DHCR24 is reported to have different functions in different cancers, it is not clear whether DHCR24 is involved in breast cancer. In this study, we found that DHCR24 expression was higher in breast cancer especially in luminal and HER2 positive breast cancer tissues compared with normal breast. Changes in DHCR24 expression altered cellular cholesterol content without affecting the adherent growth of breast cancer cells. However, DHCR24 knockdown reduced whereas DHCR24 overexpression enhanced breast cancer stem-like cell populations such as mammosphere and aldehyde dehydrogenase positive cell numbers. In addition, DHCR24 overexpression increased the expression of the Hedgehog pathway-regulated genes. Treating DHCR24 overexpressing breast cancer cell lines with the Hedgehog pathway inhibitor GANT61 blocked DHCR24-induced mammosphere growth and increased mRNA levels of the Hedgehog regulated genes. Furthermore, expression of a constitutively activated mutant of Smoothened, a key hedgehog signal transducer, rescued the decreases in mammosphere growth and Hedgehog regulated gene expression induced by knockdown of DHCR24. These results indicate that DHCR24 promotes the growth of breast cancer stem-like cells in part through enhancing the Hedgehog signaling pathway. Our data suggest that cholesterol contribute to breast carcinogenesis by enhancing Hedgehog signaling and cancer stem-like cell populations. Enzymes including DHCR24 involved in cholesterol biosynthesis should be considered as potential treatment targets for breast cancer.

Topics: Antineoplastic Agents; Breast; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Proliferation; Cholesterol; Female; Gene Expression; Hedgehog Proteins; HEK293 Cells; Humans; MCF-7 Cells; Neoplastic Stem Cells; Nerve Tissue Proteins; Oxidoreductases Acting on CH-CH Group Donors; Pyridines; Pyrimidines; RNA, Messenger; Signal Transduction

The use of carbon ion therapy for cancer treatment is becoming more widespread due to the advantages of carbon ions compared with X‑rays. Breast cancer patients may benefit from these advantages, as the surrounding healthy tissues receive a lower dose, and the increased biological effectiveness of carbon ions can better control radioresistant cancer cells. Accumulating evidence indicates that the Hedgehog (Hh) pathway is linked to the development and progression of breast cancer, as well as to resistance to X‑irradiation and the migratory capacity of cancer cells. Hence, there is an increasing interest in targeting the Hh pathway in combination with radiotherapy. Several studies have already investigated this treatment strategy with conventional radiotherapy. However, to the best of our knowledge, the combination of Hh inhibitors with particle therapy has not yet been explored. The aim of the present study was to investigate the potential of the Hh inhibitor GANT61 as an effective modulator of radiosensitivity and migration potential in MCF‑7 breast cancer cells, and compare potential differences between carbon ion irradiation and X‑ray exposure. Although Hh targeting was not able to radiosensitise cells to any radiation type used, the combination of GANT61 with X‑rays or carbon ions (energy: 95 MeV/n; linear energy transfer: 73 keV/µm) was more effective in decreasing MCF‑7 cell migration compared with either radiation type alone. Gene expression of the Hh pathway was affected to different degrees in response to X‑ray and carbon ion irradiation, as well as in response to the combination of GANT61 with irradiation. In conclusion, combining Hh inhibition with radiation (X‑rays or carbon ions) more effectively decreased breast cancer cell migration compared with radiation treatment alone.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Chemoradiotherapy; Gene Expression Profiling; Heavy Ion Radiotherapy; Hedgehog Proteins; Humans; MCF-7 Cells; Pyridines; Pyrimidines; Radiation Tolerance; Signal Transduction; X-Ray Therapy

Sonic Hedgehog signaling is critical for breast morphogenesis and cancer. The present study was conducted to explore the influence of SHH/GLI1 axis on epithelial mesenchymal transition and invasion in breast cancer cells. SHH/GLI1 positive samples demonstrated high expression of Snail and Vimentin with relatively low expression of E-cadherin. Overexpression of Vimentin and Snail in SHH/GLI1 positive patients was also associated with poor overall survival. Interestingly, GANT61 (GLI1 inhibitor) exposure significantly reduced cell viability and induced apoptosis at 10 µM. Suppression of Hedgehog pathway either by CRISPR mediated SHH knock out or GANT61 altered regulation of EMT markers in breast cancer cells. Moreover, in-activation of SHH/GLI1 axis also significantly restricted cell migration and invasiveness. These findings suggest that targeting SHH/GLI1 axis alters expression of EMT markers and abrogates neoplastic invasion in breast cancer cells.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Breast Neoplasms; Cadherins; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Humans; Male; Middle Aged; Pyridines; Pyrimidines; Signal Transduction; Snail Family Transcription Factors; Vimentin; Young Adult; Zinc Finger Protein GLI1

Dysregulation of hedgehog pathway is observed in numerous cancers. Relevance of hedgehog pathway genes in cancer cohort and inhibition of its downstream effector (GLI1) towards metastasis in cell lines are explored in the study.. One hundred fifty fresh tumours of breast cancer patients were collected for the study. Based on differential expression, panel of 6 key regulators of the pathway (SHH, DHH, IHH, PTCH1, SMO and GLI1) in microarray datasets were identified. Expressional profiles of aforementioned genes were later correlated with clinico-pathological parameters in Pakistani breast cancer cohort at transcript and protein levels. In addition, GLI1 over expressing breast cancer cell lines (MDA-MB-231 and MCF-7) were treated with GANT61 to explore its probable effects on metastasis.. SHH, DHH, PTCH1 and GLI1 were significantly over-expressed in tumours as compared with respective normal mammary tissues. A significant correlation of SHH, DHH and GLI1 expression with advanced tumour size, stages, grades, nodal involvement and distant metastasis was observed (p < 0.05). Over-expression of SHH, DHH and GLI1 was significantly related with patients having early onset and pre-menopausal status. Of note, hedgehog pathway was frequently up regulated in luminal B and triple negative breast cancer affected women. In addition, positive correlations were observed among aforementioned members of pathway and Ki67 (r-value: 0.63-0.78) emphasizing their role towards disease progression. Exposure of GANT61 (inhibitor for GLI1) significantly restricted cell proliferation, reduced cell motility and invasion.. Role of activated hedgehog pathway in breast cancer metastasis provides a novel target for cancer therapy against aggressive cancer subtypes.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Hedgehog Proteins; Humans; MCF-7 Cells; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; Patched-1 Receptor; Proportional Hazards Models; Pyridines; Pyrimidines; Signal Transduction; Zinc Finger Protein GLI1

Recent fate-mapping studies concluded that EMT is not required for metastasis of carcinomas. Here we challenge this conclusion by showing that these studies failed to account for possible crosstalk between EMT and non-EMT cells that promotes dissemination of non-EMT cells. In breast cancer models, EMT cells induce increased metastasis of weakly metastatic, non-EMT tumour cells in a paracrine manner, in part by non-cell autonomous activation of the GLI transcription factor. Treatment with GANT61, a GLI1/2 inhibitor, but not with IPI 926, a Smoothened inhibitor, blocks this effect and inhibits growth in PDX models. In human breast tumours, the EMT-transcription factors strongly correlate with activated Hedgehog/GLI signalling but not with the Hh ligands. Our findings indicate that EMT contributes to metastasis via non-cell autonomous effects that activate the Hh pathway. Although all Hh inhibitors may act against tumours with canonical Hh/GLI signalling, only GLI inhibitors would act against non-canonical EMT-induced GLI activation.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Heterografts; Humans; Mice, Inbred NOD; Mice, SCID; Neoplasm Metastasis; Paracrine Communication; Pyridines; Pyrimidines; Tumor Microenvironment; Veratrum Alkaloids; Zinc Finger Protein GLI1

Itraconazole is a common antifungal which may have promise for treating various human cancers. We report that itraconazole was cytotoxic to MCF-7 and SKBR-3 breast cancer cell lines via apoptosis by altering mitochondria membrane potential, reducing BCL-2 expression and elevating caspase-3 activity. Itraconazole also induced autophagic cell death via LC3-II expression upregulation, P62/SQSTM1 degradation, autophagosome formation and increases in autophagic puncta. Itraconazole treatment inhibited hedgehog pathway key molecular expression, such as SHH and Gli1, resulting in promotion of apoptosis and autophagy. The anti-proliferation effect of itraconazole-induced apoptosis and autophagy via hedgehog pathway inhibition was confirmed with Gli1 inhibitor GANT61 and SHH siRNA, GANT61 and SHH siRNA synergistically enhanced cytotoxicity induced by itraconazole. A human xenograft nude mouse model corroborated the anti-breast cancer activity as evidenced by reduced tumor size, and increased tumor tissue apoptosis and autophagy. Thus, itraconazole has a potent anti-breast cancer activity that may be improved when combined with hedgehog pathway inhibitors.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Breast Neoplasms; Cell Cycle Checkpoints; Cell Proliferation; Dose-Response Relationship, Drug; Drug Synergism; Female; Hedgehog Proteins; Humans; Itraconazole; MCF-7 Cells; Mice, Nude; Pyridines; Pyrimidines; Signal Transduction; Time Factors; Transfection; Tumor Burden; Xenograft Model Antitumor Assays; Zinc Finger Protein GLI1

Estradiol (E2) increases not only the cell growth but also the cancer stem cell (CSC) proportion in estrogen receptor (ER)-positive breast cancer cells. It has been suggested that the non-canonical hedgehog (Hh) pathway activated by E2 plays an important role in the regulation of CSC proportion in ER-positive breast cancer cells. We studied anti-CSC activity of a non-canonical Hh inhibitor GANT61 in ER-positive breast cancer cells. Effects of GANT61 on the cell growth, cell cycle progression, apoptosis and CSC proportion were investigated in four ER-positive breast cancer cell lines. CSC proportion was measured using either the mammosphere assay or CD44/CD24 assay. Expression levels of pivotal molecules in the Hh pathway were measured. Combined effects of GANT61 with antiestrogens on the anti-cell growth and anti-CSC activities were investigated. E2 significantly increased the cell growth and CSC proportion in all ER-positive cell lines. E2 increased the expression levels of glioma-associated oncogene (GLI) 1 and/or GLI2. GANT61 decreased the cell growth in association with a G1-S cell cycle retardation and increased apoptosis. GANT61 decreased the E2-induced CSC proportion measured by the mammosphere assay in all cell lines. Antiestrogens also decreased the E2-induced cell growth and CSC proportion. Combined treatments of GANT61 with antiestrogens additively enhanced anti-cell growth and/or anti-CSC activities in some ER-positive cell lines. In conclusion, the non-canonical Hh inhibitor GANT61 inhibited not only the cell growth but also the CSC proportion increased by E2 in ER-positive breast cancer cells. GANT61 enhanced anti-cell growth and/or anti-CSC activities of antiestrogens in ER-positive cell lines.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Estradiol; Estrogen Antagonists; Female; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; MCF-7 Cells; Neoplastic Stem Cells; Pyridines; Pyrimidines; Receptors, Estrogen; Signal Transduction

Aberrant Hedgehog (Hh)/glioma-associated oncogene (GLI) signaling has been implicated in cancer progression. Here, we analyzed GLI1, Sonic Hedgehog (Shh) and NF-κB expression in 51 breast cancer (ductal carcinoma) tissues using immunohistochemistry. We found a positive correlation between nuclear GLI1 expression and tumor grade in ductal carcinoma cases. Cytoplasmic Shh staining significantly correlated with a lower tumor grade. Next, the in vitro effects of two Hh signaling pathway inhibitors on breast cancer cell lines were evaluated using the Smoothened (SMO) antagonist GDC-0449 and the direct GLI1 inhibitor GANT-61. GDC-0449 and GANT-61 exhibited the following effects: a) inhibited breast cancer cell survival; b) induced apoptosis; c) inhibited Hh pathway activity by decreasing the mRNA expression levels of GLI1 and Ptch and inhibiting the nuclear translocation of GLI1; d) increased/decreased EGFR and ErbB2 protein expression, reduced p21-Ras and ERK1/ERK2 MAPK activities and inhibited AKT activation; and e) decreased the nuclear translocation of NF-κB. However, GANT-61 exerted these effects more effectively than GDC-0449. The in vivo antitumor activities of GDC-0449 and GANT-61 were analyzed in BALB/c mice that were subcutaneously inoculated with mouse breast cancer (TUBO) cells. GDC-0449 and GANT-61 suppressed tumor growth of TUBO cells in BALB/c mice to different extents. These findings suggest that targeting the Hh pathway using antagonists that act downstream of SMO is a more efficient strategy than using antagonists that act upstream of SMO for interrupting Hh signaling in breast cancer.

Topics: Active Transport, Cell Nucleus; Anilides; Animals; Apoptosis; Breast Neoplasms; Carcinoma, Ductal; Cell Line, Tumor; Cell Proliferation; Cell Survival; Enzyme Activation; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Mammary Neoplasms, Experimental; MCF-7 Cells; Mice; Mice, Inbred BALB C; NF-kappa B; Patched-1 Receptor; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Pyridines; Pyrimidines; Receptor, ErbB-2; RNA, Messenger; Signal Transduction; Smoothened Receptor; Zinc Finger Protein GLI1

Anti-estrogen treatment, exemplified by tamoxifen, is a well-established adjuvant therapy for estrogen receptor alpha (ERα)-positive breast cancer. However, the effectiveness of this drug is limited due to the development of resistance. The Hedgehog (HH) signaling pathway is critical in embryonic development, and aberrant activation of this transduction cascade is linked to various malignancies. However, it remains unclear whether HH signaling is activated in human breast cancer and related to tamoxifen resistance. Deciphering how this pathway may be involved in breast cancer is a crucial step towards the establishment of targeted combinatorial treatments for this disease. Here, we show that the expression of the HH signaling effector protein GLI1 is higher in tamoxifen resistant compared to sensitive cells. Tamoxifen resistant cells have stronger ERα transcriptional activity relative to sensitive cells, even though the ERα expression is similar in both cell types. Knockdown of GLI1 attenuates cell proliferation and reduces ERα transcriptional activity in both sensitive and resistant cells, irrespective of estrogen stimulation. Combinatorial treatment of tamoxifen and the GLI antagonist GANT61 further suppresses the growth of sensitive and resistant cells relative to administration of only tamoxifen, and this was irrespective of estrogen stimulation. Moreover, a positive correlation between GLI1 and ERα expression was identified in breast cancer samples. Additionally, high GLI1 expression predicted worse distant metastasis-free survival in breast cancer patients. These data suggest that the HH pathway may be a new candidate for therapeutic targeting and prognosis in ERα-positive breast cancer.

Topics: Breast Neoplasms; Cell Proliferation; Down-Regulation; Drug Resistance, Neoplasm; Estrogen Receptor alpha; Estrogens; Female; Hedgehog Proteins; Humans; MCF-7 Cells; Pyridines; Pyrimidines; Response Elements; Signal Transduction; Tamoxifen; Zinc Finger Protein GLI1