Anti-cell growth and anti-cancer stem cell activities of the non-canonical hedgehog inhibitor GANT61 in triple-negative breast cancer cells

Background

Triple-negative breast cancer (TNBC) exhibits biologically aggressive behavior and has a poor prognosis. Novel molecular targeting agents are needed to control TNBC. Recent studies revealed that the non-canonical hedgehog (Hh) signaling pathway plays important roles in the regulation of cancer stem cells (CSCs) in breast cancer. Therefore, the anti-cell growth and anti-CSC effects of the non-canonical Hh inhibitor GANT61 were investigated in TNBC cells.

Methods

The effects of GANT61 on cell growth, cell cycle progression, apoptosis, and the proportion of CSCs were investigated in three TNBC cell lines. Four ER-positive breast cancer cell lines were also used for comparisons. The expression levels of effector molecules in the Hh pathway: glioma-associated oncogene (GLI) 1 and GLI2, were measured. The combined effects of GANT61 and paclitaxel on anti-cell growth and anti-CSC activities were also investigated.

Results

Basal expression levels of GLI1 and GLI2 were significantly higher in TNBC cells than in ER-positive breast cancer cells. GANT61 dose-dependently decreased cell growth in association with G1–S cell cycle retardation and increased apoptosis. GANT61 significantly decreased the CSC proportion in all TNBC cell lines. Paclitaxel decreased cell growth, but not the CSC proportion. Combined treatments of GANT61 and paclitaxel more than additively enhanced anti-cell growth and/or anti-CSC activities.

Conclusions

The non-canonical Hh inhibitor GANT61 decreased not only cell growth, but also the CSC population in TNBC cells. GANT61 enhanced the anti-cell growth activity of paclitaxel in these cells. These results suggest for the first time that GANT61 has potential as a therapeutic agent in the treatment of patients with TNBC.

     

Anti‐cancer stem cell activity of a hedgehog inhibitor GANT61 in estrogen receptor‐positive breast cancer cells

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.     

Targeting class I histone deacetylases by the novel small molecule inhibitor 4SC‐202 blocks oncogenic hedgehog‐GLI signaling and overcomes smoothened inhibitor resistance

Aberrant activation of Hedgehog (HH)/GLI signaling is causally involved in numerous human malignancies, including basal cell carcinoma (BCC) and medulloblastoma. HH pathway antagonists targeting smoothened (SMO), an essential effector of canonical HH/GLI signaling, show significant clinical success in BCC patients and have recently been approved for the treatment of advanced and metastatic BCC. However, rapid and frequent development of drug resistance to SMO inhibitors (SMOi) together with severe side effects caused by prolonged SMOi treatment call for alternative treatment strategies targeting HH/GLI signaling downstream of SMO. In this study, we report that 4SC‐202, a novel clinically validated inhibitor of class I histone deacetylases (HDACs), efficiently blocks HH/GLI signaling. Notably, 4SC‐202 treatment abrogates GLI activation and HH target gene expression in both SMOi‐sensitive and ‐resistant cells. Mechanistically, we propose that the inhibition of HDACs 1/2/3 is crucial for targeting oncogenic HH/GLI signaling, and that class I HDAC inhibitors either in combination with SMOi or as second‐line therapy may improve the treatment options for HH‐associated malignancies with SMOi resistance.     

Tumor shrinkage by cyclopamine tartrate through inhibiting hedgehog signaling

The link of hedgehog (Hh) signaling activation to human cancer and synthesis of a variety of Hh signaling inhibitors raise great expectation that inhibiting Hh signaling may be effective in human cancer treatment. Cyclopamine (Cyc), an alkaloid from the Veratrum plant, is a specific natural product inhibitor of the Hh pathway that acts by targeting smoothened (SMO) protein. However, its poor solubility, acid sensitivity, and weak potency relative to other Hh antagonists prevent the clinical development of C...     

Overexpression of Hedgehog signaling molecules and its involvement in triple-negative breast cancer

The purpose of this study was to investigate the activation of Hedgehog (Hh) signaling molecules and its involvement in triple-negative breast cancer (TNBC). A total of 123 cases of paraffin blocks, including 83?cases of primary breast carcinoma, 30 cases of mammary hyperplasia and 10?cases of normal breast tissue, were immunohistochemically analyzed for Sonic Hedgehog (SHH), Patched-1 (PTCH1), Smoothened (SMO) and glioma-associated oncogene homoglog?1 (GLI1) expression. The expression of SMO and GLI1 in TNBC was significantly increased in comparison to non-triple-negative breast cancer (nTNBC). GLI1 expression manifested an inverse association with the estrogen receptor. The levels of GLI1 expression were increased in lymph node-positive cases. The expression of SHH and SMO was increased in high histological grades. Furthermore, the expression of SMO and GLI1 was correlated with superior tumor stage. The expression of SHH, SMO and GLI1 was significantly increased in breast cancer and mammary hyperplasia. PTCH1 expression was significantly decreased in breast cancer compared to mammary hyperplasia and normal breast tissue. For the first time, clinical evidence has been provided in support of significant roles of Hh signaling in TNBC. Hh signaling is involved in breast ductal changes and malignant transformation. Measures to inhibit Hh activity may improve the prognosis of TNBC patients.     

Pharmacological inhibition of the Hedgehog pathway prevents human rhabdomyosarcoma cell growth

The Hedgehog pathway functions as an organizer in embryonic development. Recent studies have shown that mutation of the PTCH1 gene involved in the Hedgehog pathway affects rhabdomyosarcoma development. However, the expression of Hedgehog pathway molecules in human rhabdomyosarcoma cells has not been well clarified. In addition, the effect of pharmacological inhibition of the Hedgehog pathway is not known. We investigated the expression of the genes involved in the Hedgehog pathway using human rhabdomyosarcoma cell lines and biopsy specimens. Further, we evaluated the effect of pharmacological inhibition of the Hedgehog pathway using cyclopamine or GANT61 by WST assay, cell proliferation assay and cell death detection assay. Real-time PCR revealed that human rhabdomyosarcoma cell lines and biopsy specimens overexpressed the following genes: Sonic hedgehog, Indian hedgehog, Desert hedgehog, PTCH1, SMO, GLI1, GLI2 and ULK3. Immunohistochemistry revealed that rhabdomyosarcoma cell lines and biopsy specimens expressed SMO and GLI2. Inhibition of SMO by cyclopamine slowed the growth of human rhabdomyosarcoma cell lines. Similarly, inhibition of GLI by GANT61 slowed the growth of human rhabdomyosarcoma cell lines. Inhibition of cell proliferation and apoptotic cell death together prevented the growth of rhabdomyosarcoma cells by cyclopamine and GANT61 treatment. Our findings suggest that pharmacological inhibition of the Hedgehog pathway may be a useful approach for treating rhabdomyosarcoma patients.     

Hedgehog signaling pathway is inactive in colorectal cancer cell lines

The Hedgehog (Hh) signaling pathway plays an important role in human development. Abnormal activation of this pathway has been observed in several types of human cancers, such as the upper gastro-intestinal tract cancers. However, activation of the Hh pathway in colorectal cancers is controversial. We analyzed the expression of the main key members of the Hh pathway in 7 colon cancer cell lines in order to discover whether the pathway is constitutively active in these cells. We estimated the expression of SHH, IHH, PTCH, SMO, GLI1, GLI2, GLI3, SUFU and HHIP genes by RT-PCR. Moreover, Hh ligand, Gli3 and Sufu protein levels were quantified by western blotting. None of the cell lines expressed the complete set of Hh pathway members. The ligands were absent from Colo320 and HCT116 cells, Smo from Colo205, HT29 and WiDr. GLI1 gene was not expressed in SW480 cells nor were GLI2/GLI3 in Colo205 or Caco-2 cells. Furthermore the repressive form of Gli3, characteristic of an inactive pathway, was detected in SW480 and Colo320 cells. Finally treatment of colon cancer cells with cyclopamine, a specific inhibitor of the Hh pathway, did not downregulate PTCH and GLI1 genes expression in the colorectal cells, whereas it did so in PANC1 control cells. Taken together, these results indicate that the aberrant activation of the Hh signaling pathway is not common in colorectal cancer cell lines.     

The utility of hedgehog signaling pathway inhibition for cancer

The Hedgehog (Hh) signaling pathway has been implicated in tumor initiation and metastasis across different malignancies. Major mechanisms by which the Hh pathway is aberrantly activated can be attributed to mutations of members of Hh pathway or excessive/inappropriate expression of Hh pathway ligands. The Hh signaling pathway also affects the regulation of cancer stem cells, leading to their capabilities in tumor formation, disease progression, and metastasis. Preliminary results of early phase clinical trials of Hh inhibitors administered as monotherapy demonstrated promising results in patients with basal cell carcinoma and medulloblastoma, but clinically meaningful anticancer efficacy across other tumor types seems to be lacking. Additionally, cases of resistance have been already observed. Mutations of SMO, activation of Hh pathway components downstream to SMO, and upregulation of alternative signaling pathways are possible mechanisms of resistance development. Determination of effective Hh inhibitor-based combination regimens and development of correlative biomarkers relevant to this pathway should remain as clear priorities for future research.     

Hedgehog信号通路在食管癌中的异常活化

  目的   探讨Hedgehog（Hh）信号通路与食管鳞状细胞癌（ESCC）的关系。   方法   采用免疫组织化学随机分析30例ESCC及癌旁组织样本的Hh信号通路中主要组分Smo和Gli2及靶蛋白CyclinD1表达。构建分泌型配体ShhN的条件培养液,利用条件培养液及Hh信号通路激动剂Purmorphamine或Hh通路抑制剂环杷明及GANT61处理CaEs-17细胞后,MTT法检测细胞生存率的变化。   结果   ESCC组织中的Smo、Gli2和CyclinD1表达普遍高于癌旁组织。含有ShhN的条件培养液能有效激活Hh信号通路下游靶基因CCND1的表达并明显促进食管癌CaEs-17细胞的生存率,提示食管癌中Hh信号通路以配体依赖的方式激活。直接作用于Smo的Hh信号通路激动剂Purmorphamine促进食管癌CaEs-17细胞的生长;而Smo特异性抑制剂环杷明有效地抑制CaEs-17细胞生存率。Gli1/Gli2的抑制剂GANT61比环杷明更为有效地抑制CaEs-17细胞生存率。   结论   Hh信号通路在ESCC中异常活化,将可能成为新的治疗食管癌的药物靶点。      

Identification of a novel synthetic lethality of combined inhibition of hedgehog and PI3K signaling in rhabdomyosarcoma

We previously reported that aberrant HH pathway activation confers a poor prognosis in rhabdomyosarcoma (RMS). Searching for new treatment strategies we therefore targeted HH signaling. Here, we identify a novel synthetic lethality of concomitant inhibition of HH and PI3K/AKT/mTOR pathways in RMS by GLI1/2 inhibitor GANT61 and PI3K/mTOR inhibitor PI103. Synergistic drug interaction is confirmed by calculation of combination index (CI < 0.2). Similarly, genetic silencing of GLI1/2 significantly increases PI103-induced apoptosis. GANT61 and PI103 also synergize to induce apoptosis in cultured primary RMS cells emphasizing the clinical relevance of this combination. Importantly, GANT61/PI103 cotreatment suppresses clonogenic survival, three-dimensional sphere formation and tumor growth in an in vivo model of RMS. Mechanistic studies reveal that GANT61 and PI103 cooperate to trigger caspase-dependent apoptosis via the mitochondrial pathway, as demonstrated by several lines of evidence. First, GANT61/PI103 cotreatment increases mRNA and protein expression of NOXA and BMF, which is required for apoptosis, since knockdown of NOXA or BMF significantly reduces GANT61/PI103-induced apoptosis. Second, GANT61/PI103 cotreatment triggers BAK/BAX activation, which contributes to GANT61/PI103-mediated apoptosis, since knockdown of BAK provides protection. Third, ectopic expression of BCL-2 or non-degradable phospho-mutant MCL-1 significantly rescue GANT61/PI103-triggered apoptosis. Fourth, GANT61/PI103 cotreatment initiate activation of the caspase cascade via apoptosome-mediated cleavage of the initiator caspase-9, as indicated by changes in the cleavage pattern of caspases (e.g. accumulation of the caspase-9 p35 cleavage fragment) upon addition of the caspase inhibitor zVAD.fmk. Thus, combined GLI1/2 and PI3K/mTOR inhibition represents a promising novel approach for synergistic apoptosis induction and tumor growth reduction with implications for new treatment strategies in RMS.