Ovarian cancer (OC) is the most lethal female gynecological malignancy, mostly due to diagnosis in late stages when treatment options are limited. Hedgehog-GLI (HH-GLI) signaling is a major developmental pathway involved in organogenesis and stem cell maintenance, and is activated in OC. One of its targets is survivin (BIRC5), an inhibitor of apoptosis protein (IAP) that plays a role in multiple processes, including proliferation and cell survival. We wanted to investigate the role of different GLI proteins in the regulation of survivin isoform expression (WT, 2α, 2B, 3B, and Δex3) in the SKOV-3 OC cell line. We demonstrated that survivin isoforms are downregulated in GLI1 and GLI2 knock-out cell lines, but not in the GLI3 knock-out. Treatment of GLI1 knock-out cells with GANT-61 shows an additional inhibitory effect on several isoforms. Additionally, we examined the expression of survivin isoforms in OC samples and the potential role of BIRC5 polymorphisms in isoform expression. Clinical samples showed the same pattern of survivin isoform expression as in the cell line, and several BIRC5 polymorphisms showed the correlation with isoform expression. Our results showed that survivin isoforms are regulated both by different GLI proteins and BIRC5 polymorphisms in OC.

Several signaling pathways are aberrantly activated in head and neck squamous cell carcinoma (HNSCC), including the Hedgehog-Gli (HH-GLI), WNT, EGFR, and NOTCH pathways. The HH-GLI pathway has mostly been investigated in the context of canonical signal transduction and the inhibition of the membrane components of the pathway. In this work we investigated the role of downstream inhibitors GANT61 and lithium chloride (LiCl) on cell viability, wound closure, and colony forming ability of HNSCC cell lines. Five HNSCC cell lines were treated with HH-GLI pathway inhibitors affecting different levels of signal transduction. GANT61 and LiCl reduce the proliferation and colony formation capabilities of HNSCC cell lines, and LiCl has an additional effect on wound closure. The major effector of the HH-GLI signaling pathway in HNSCC is the GLI3 protein, which is expressed in its full-length form and is functionally regulated by GSK3β. LiCl treatment increases the inhibitory Ser9 phosphorylation of the GSK3β protein, leading to increased processing of GLI3 from full-length to repressor form, thus inhibiting HH-GLI pathway activity. Therefore, downstream inhibition of HH-GLI signaling may be a promising therapeutic strategy for HNSCC.

Prostate cancer is the second most frequent cancer diagnosed in men worldwide. Localized disease can be successfully treated, but advanced cases are more problematic. After initial effectiveness of androgen deprivation therapy, resistance quickly occurs. Therefore, we aimed to investigate the role of Hedgehog-GLI (HH-GLI) signaling in sustaining androgen-independent growth of prostate cancer cells. We found various modes of HH-GLI signaling activation in prostate cancer cells depending on androgen availability. When androgen was not deprived, we found evidence of non-canonical SMO signaling through the SRC kinase. After short-term androgen deprivation canonical HH-GLI signaling was activated, but we found little evidence of canonical HH-GLI signaling activity in androgen-independent prostate cancer cells. We show that in androgen-independent cells the pathway ligand, SHH-N, non-canonically binds to the androgen receptor through its cholesterol modification. Inhibition of this interaction leads to androgen receptor signaling downregulation. This implies that SHH-N activates the androgen receptor and sustains androgen-independence. Targeting this interaction might prove to be a valuable strategy for advanced prostate cancer treatment. Also, other non-canonical aspects of this signaling pathway should be investigated in more detail and considered when developing potential therapies.

Material and methods Immunohistochemical scoring for total-STAT3, pY705 and pS727 was done in 75 paired core-biopsy and post-NACT TNBC samples. Bioluminescence resonance energy transfer based sensor (PhosphoBRET) was developed by N-terminal fusion of STAT3 with Nanoluc (donor) and TurboFP635 (acceptor) and validated on multiple cancer cell lines (MCF7, A549, HT1080, PC3) or drug inhibitors (Niclosamide, Stattic). We also engineered 3'UTR STAT3shRNA cells overexpressing Nanoluc-STAT3 variants i.e. Wild type [wt], Y705F, S727A, K685R and performed biochemical as well as phenotypic assays to study biological role of STAT3 pathway. Results and discussions Over 90% TNBC cases showed positive staining for pS727 as compared to pY705 STAT3 indicating hyperactivation of non-canonical pathway. 3'UTR STAT3 shRNA cells over-expressing S727A PTM mutant, showed decreased pY705 expression along with abrogation of K685 acetylation. The level of downstream STAT3 targets like Myc, cyclin D1 etc. remained intact while some of the novel target genes (Her2, E-Cadherin and ERa) showed elevated expression in S727A and K685R mutants. Additionally, S727A or K685R mutant also exhibited 2-fold decrease in overall cell proliferation and survival potential. The designed STAT3-PhosphoBRET sensor demonstrated upto 3-fold gain in BRET ratio (p<0.01) using IL6 or EGF ligand. Further, PhosphoBRET sensor expressing either Y705F or S727A or K685R showed increased BRET ratio (p<0.001) implicating higher STAT3 dimerization and activation. Additionally, the PhosphoBRET platform was also extended to test various STAT3 inhibitors, of which Niclosamide was identified as a potent dual blocker (pS727 and pY705) of STAT3 activation for the first time. Conclusion Predominant expression of pS727 STAT3 in TNBC cases along with in vitro data implicate crucial role of non canonical (pS727 and acK685) pathway in controlling STAT3 dimerization and downstream function independent of pY705 activation. Hence, future drug design strategy should aim both arms of STAT3 pathway to completely abrogate its oncogenic function.