Semaphorin 3C promotes de novo steroidogenesis in prostate cancer cells.

Intratumoral androgen biosynthesis contributes to castration-resistant prostate cancer progression in patients treated with androgen deprivation therapy. The molecular mechanisms by which castration-resistant prostate cancer acquires the capacity for androgen biosynthesis to bypass androgen deprivation therapy are not entirely known. Here, we show that semaphorin 3C, a secreted signaling protein that is highly expressed in castration-resistant prostate cancer, can promote steroidogenesis by altering the expression profile of key steroidogenic enzymes. Semaphorin 3C not only upregulates enzymes required for androgen synthesis from dehydroepiandrosterone or de novo from cholesterol but also simultaneously downregulates enzymes involved in the androgen inactivation pathway. These changes in gene expression correlate with increased production of androgens induced by semaphorin 3C in prostate cancer model cells. Moreover, semaphorin 3C upregulates androgen synthesis in LNCaP cell-derived xenograft tumors, likely contributing to the enhanced in vivo tumor growth rate post castration. Furthermore, semaphorin 3C activates sterol regulatory element-binding protein, a transcription factor that upregulates enzymes involved in the synthesis of cholesterol, a sole precursor for de novo steroidogenesis. The ability of semaphorin 3C to promote intratumoral androgen synthesis may be a key mechanism contributing to the reactivation of the androgen receptor pathway in castration-resistant prostate cancer, conferring continued growth under androgen deprivation therapy. These findings identify semaphorin 3C as a potential therapeutic target for suppressing intratumoral steroidogenesis.

Dependency of Tamoxifen Sensitive and Resistant ER+ Breast Cancer Cells on Semaphorin 3C (SEMA3C) for Growth.

Estrogen receptor positive (ER+) breast cancer (BCa) accounts for the highest proportion of breast cancer-related deaths. While endocrine therapy is highly effective for this subpopulation, endocrine resistance remains a major challenge and the identification of novel targets is urgently needed. Previously, we have shown that Semaphorin 3C (SEMA3C) is an autocrine growth factor that drives the growth and treatment resistance of various cancers, but its role in breast cancer progression and endocrine resistance is poorly understood. Here, we report that SEMA3C plays a role in maintaining the growth of ER+ BCa cells and is a novel, tractable therapeutic target for the treatment of ER+ BCa patients. Analyses of publicly available clinical datasets indicate that ER+ BCa patients express significantly higher levels of SEMA3C mRNA than other subtypes. Furthermore, SEMA3C mRNA expression was positively correlated with ESR1 mRNA expression. ER+ BCa cell lines (MCF7 and T47D) expressed higher levels of SEMA3C mRNA and protein than a normal mammary epithelial MCF10A cell line. ER siRNA knockdown was suppressed, while dose-dependent beta-estradiol treatment induced SEMA3C expression in both MCF7 and T47D cells, suggesting that SEMA3C is an ER-regulated gene. The stimulation of ER+ BCa cells with recombinant SEMA3C activated MAPK and AKT signaling in a dose-dependent manner. Conversely, SEMA3C silencing inhibited Estrogen Receptor (ER) expression, MAPK and AKT signaling pathways while simultaneously inducing apoptosis, as monitored by flow cytometry and Western blot analyses. SEMA3C silencing significantly inhibited the growth of ER+ BCa cells, implicating a growth dependency of ER+ BCa cells on SEMA3C. Moreover, the analysis of tamoxifen resistant (TamR) cell models (TamC3 and TamR3) showed that SEMA3C levels remain high despite treatment with tamoxifen. Tamoxifen-resistant cells remained dependent on SEMA3C for growth and survival. Treatment with B1SP Fc fusion protein, a SEMA3C pathway inhibitor, attenuated SEMA3C-induced signaling and growth across a panel of tamoxifen sensitive and resistant ER+ breast cancer cells. Furthermore, SEMA3C silencing and B1SP treatment were associated with decreased EGFR signaling in TamR cells. Here, our study implicates SEMA3C in a functional role in ER+ breast cancer signaling and growth that suggests ER+ BCa patients may benefit from SEMA3C-targeted therapy.

8-Hydroxyquinoline derivatives suppress GLI1-mediated transcription through multiple mechanisms.

Aberrant activation of the Hedgehog (Hh) signaling pathway has been observed in various human malignancies. Glioma-associated oncogene transcription factor 1 (GLI1) is the ultimate effector of the canonical Hh pathway and has also been identified as a common regulator of several tumorigenic pathways prevalent in Hh-independent cancers. The anti-cancer potential of GLI1 antagonism with small molecule inhibitors has demonstrated initial promise; however, the continued development of GLI1 inhibitors is still needed. We previously identified a scaffold containing an 8-hydroxyquinoline as a promising lead GLI1 inhibitor (compound 1). To further develop this scaffold, we performed a systematic structure-activity relationship study to map the structural requirements of GLI1 inhibition by this chemotype. A series of biophysical and cellular experiments identified compound 39 as an enhanced GLI1 inhibitor with improved activity. In addition, our studies on this scaffold suggest a potential role for SRC family kinases in regulating oncogenic GLI1 transcriptional activity.

DNA Sequence Specificity Reveals a Role of the HLTF HIRAN Domain in the Recognition of Trinucleotide Repeats.

DNA damage tolerance (DDT) pathways enable cells to cope with a variety of replication blocks that threaten their ability to complete DNA replication. Helicase-like transcription factor (HLTF) plays a central role in the error-free DDT pathway, template switching (TS), by serving as a ubiquitin ligase to polyubiquitinate the DNA sliding clamp PCNA, which promotes TS initiation. HLTF also serves as an ATP-dependent DNA translocase facilitating replication fork remodeling. The HIP116, Rad5p N-terminal (HIRAN) domain of HLTF specifically recognizes the unmodified 3'-end of single-stranded DNA (ssDNA) at stalled replication forks to promote fork regression. Several crystal structures of the HIRAN domain in complex with ssDNA have been reported; however, optimal ssDNA sequences for high-affinity binding with the domain have not been described. Here we elucidated DNA sequence preferences of HLTF HIRAN through systematic studies of its binding to ssDNA substrates using fluorescence polarization assays and a computational analysis of the ssDNA:HIRAN interaction. These studies reveal that the HLTF HIRAN domain preferentially recognizes a (T/C)TG sequence at the 3'-hydroxyl ssDNA end, which occurs in the CTG trinucleotide repeat (TNR) regions that are susceptible to expansion and deletion mutations identified in neuromuscular and neurodegenerative disorders. These findings support a role for HLTF in maintaining the stability of difficult to replicate TNR microsatellite regions.

Targeting the GLI family of transcription factors for the development of anti-cancer drugs.

INTRODUCTION: GLI1 is a transcription factor that has been identified as a downstream effector for multiple tumorigenic signaling pathways. These include the Hedgehog, RAS-RAF-MEK-ERK, and PI3K-AKT-mTOR pathways, which have all been separately validated as individual anti-cancer drug targets. The identification of GLI1 as a key transcriptional regulator for each of these pathways highlights its promise as a therapeutic target. Small molecule GLI1 inhibitors are potentially efficacious against human malignancies arising from multiple oncogenic mechanisms. AREAS COVERED: This review provides an overview of the key oncogenic cellular pathways that regulate GLI1 transcriptional activity. It also provides a detailed account of small molecule GLI1 inhibitors that are currently under development as potential anti-cancer chemotherapeutics. EXPERT OPINION: Interest in developing inhibitors of GLI1-mediated transcription has significantly increased as its role in multiple oncogenic signaling pathways has been elucidated. To date, it has proven difficult to directly target GLI1 with small molecules, and the majority of compounds that inhibit GLI1 activity function through indirect mechanisms. To date, no direct-acting GLI1 inhibitor has entered clinical trials. The identification and development of new scaffolds that can bind and directly inhibit GLI1 are essential to further advance this class of chemotherapeutics.