Clinically-observed FOXA1 mutations upregulate SEMA3C through transcriptional derepression in prostate cancer.

FOXA1 is a pioneer transcription factor that is frequently mutated in prostate, breast, bladder, and salivary gland malignancies. Indeed, metastatic castration-resistant prostate cancer (mCRPC) commonly harbour FOXA1 mutations with a prevalence of 35%. However, despite the frequent recurrence of FOXA1 mutations in prostate cancer, the mechanisms by which FOXA1 variants drive its oncogenic effects are still unclear. Semaphorin 3C (SEMA3C) is a secreted autocrine growth factor that drives growth and treatment resistance of prostate and other cancers and is known to be regulated by both AR and FOXA1. In the present study, we characterize FOXA1 alterations with respect to its regulation of SEMA3C. Our findings reveal that FOXA1 alterations lead to elevated levels of SEMA3C both in prostate cancer specimens and in vitro. We further show that FOXA1 negatively regulates SEMA3C via intronic cis elements, and that mutations in FOXA1 forkhead domain attenuate its inhibitory function in reporter assays, presumably by disrupting DNA binding of FOXA1. Our findings underscore the key role of FOXA1 in prostate cancer progression and treatment resistance by regulating SEMA3C expression and suggest that SEMA3C may be a driver of growth and tumor vulnerability of mCRPC harboring FOXA1 alterations.

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.

SEMA3C induces androgen synthesis in prostatic stromal cells through paracrine signaling.

BACKGROUND: Castration resistant prostate cancer progression is associated with an acquired intratumoral androgen synthesis. Signaling pathways that can upregulate androgen production in prostate tumor microenvironment are not entirely known. In this study, we investigate the potential effect of a secreted signaling protein named semaphorin 3C (SEMA3C) on steroidogenic activities of prostatic stromal cells. METHODS: We treated human primary prostate stromal cells (PrSC) with 1uM recombinant SEMA3C protein and androgen precursor named dehydroepiandrosterone (DHEA) 1.7uM. Also, to test SEMA3C's effect on the conversion of DHEA to androgens, we exposed PrSCs to the conditioned media derived from LNCaP cells that were transduced with a lentiviral vector harboring full length SEMA3C gene or empty vector (CM-LNSEMA3C or CM-LNVector ). Then, liquid chromatography-mass spectrometry was performed on steroids isolated from PrSCs media. The messnger RNA expression of steroidogenic enzymes in PrSCs was quantified by quantitative polymerase chain reaction. RESULTS: Recombinant SEMA3C had no effect on steroidogenic activities in PrSCs. However, key steroidogenic enzymes expression and androgen synthesis were upregulated in PrSCs treated with CM-LNSEMA3C , compared to those treated with CM-LNVector . These results suggest that steroidogenic activities in PrSCs were upregulated in response to a signaling factor in CM-LNSEMA3C , other than SEMA3C. We hypothesized that SEMA3C overexpression in LNCaP cells affected androgen synthesis in PrSCs through sonic hedgehog (Shh) pathway activation in PrSCs. We verified this effect by blocking Shh signaling with smoothened antagonist. CONCLUSION: Based on known ability of Shh signaling pathway to activate steroidogenesis in stromal cells, we suggest that SEMA3C overexpression in LNCaP cells can upregulate Shh which in turn is able to stimulate steroidogenic activities in prostatic stromal cells.

SEMA3C drives cancer growth by transactivating multiple receptor tyrosine kinases via Plexin B1.

Growth factor receptor tyrosine kinase (RTK) pathway activation is a key mechanism for mediating cancer growth, survival, and treatment resistance. Cognate ligands play crucial roles in autocrine or paracrine stimulation of these RTK pathways. Here, we show SEMA3C drives activation of multiple RTKs including EGFR, ErbB2, and MET in a cognate ligand-independent manner via Plexin B1. SEMA3C expression levels increase in castration-resistant prostate cancer (CRPC), where it functions to promote cancer cell growth and resistance to androgen receptor pathway inhibition. SEMA3C inhibition delays CRPC and enzalutamide-resistant progression. Plexin B1 sema domain-containing:Fc fusion proteins suppress RTK signaling and cell growth and inhibit CRPC progression of LNCaP xenografts post-castration in vivo SEMA3C inhibition represents a novel therapeutic strategy for treatment of advanced prostate cancer.

Paracrine sonic hedgehog signaling contributes significantly to acquired steroidogenesis in the prostate tumor microenvironment.

Despite the substantial benefit of androgen deprivation therapy (ADT) for metastatic prostate cancer, patients often progress to castration-resistant disease (CRPC) that is more difficult to treat. CRPC is associated with renewed androgen receptor activity in tumor cells and restoration of tumor androgen levels through acquired intratumoral steroidogenesis (AIS). Although prostate cancer (PCa) cells have been shown to have steroidogenic capability in vitro, we previously found that benign prostate stromal cells (PrSCs) can also synthesize testosterone (T) from an adrenal precursor, DHEA, when stimulated with a hedgehog (Hh) pathway agonist, SAG. Here, we show exposure of PrSCs to a different Smoothened (Smo) agonist, Ag1.5, or to conditioned medium from sonic hedgehog overexpressing LNCaP cells induces steroidogenic enzyme expression in PrSCs and significantly increases production of T and its precursor steroids in a Smo-dependent manner from 22-OH-cholesterol substrate. Hh agonist-/ligand-treated PrSCs produced androgens at a rate similar to or greater than that of PCa cell lines. Likewise, primary bone marrow stromal cells became more steroidogenic and produced T under the influence of Smo agonist. Treatment of mice bearing LNCaP xenografts with a Smo antagonist, TAK-441, delayed the onset of CRPC after castration and substantially reduced androgen levels in residual tumors. These outcomes support the idea that stromal cells in ADT-treated primary or metastatic prostate tumors can contribute to AIS as a consequence of a paracrine Hh signaling microenvironment. As such, Smo antagonists may be useful for targeting prostate tumor stromal cell-derived AIS and delaying the onset of CRPC after ADT.

Androgen receptor transcriptionally regulates semaphorin 3C in a GATA2-dependent manner.

The androgen receptor (AR) is a member of the nuclear receptor superfamily of transcription factors and is central to prostate cancer (PCa) progression. Ligand-activated AR engages androgen response elements (AREs) at androgen-responsive genes to drive the expression of gene batteries involved in cell proliferation and cell fate. Understanding the transcriptional targets of the AR has become critical in apprehending the mechanisms driving treatment-resistant stages of PCa. Although AR transcription regulation has been extensively studied, the signaling networks downstream of AR are incompletely described. Semaphorin 3C (SEMA3C) is a secreted signaling protein with roles in nervous system and cardiac development but can also drive cellular growth and invasive characteristics in multiple cancers including PCa. Despite numerous findings that implicate SEMA3C in cancer progression, regulatory mechanisms governing its expression remain largely unknown. Here we identify and characterize an androgen response element within the SEMA3C locus. Using the AR-positive LNCaP PCa cell line, we show that SEMA3C expression is driven by AR through this element and that AR-mediated expression of SEMA3C is dependent on the transcription factor GATA2. SEMA3C has been shown to promote cellular growth in certain cell types so implicit to our findings is the discovery of direct regulation of a growth factor by AR. We also show that FOXA1 is a negative regulator of SEMA3C. These findings identify SEMA3C as a novel target of AR, GATA2, and FOXA1 and expand our understanding of semaphorin signaling and cancer biology.

Inhibition of phosphorylation of JNK suppresses Aß-induced ER stress and upregulates prosurvival mitochondrial proteins in rat hippocampus.

A growing body of evidence indicates that c-Jun N-terminal kinases (JNKs) is activated in Alzheimer's disease. Herein, we examine the effect of the JNK specific inhibitor, SP600125, on the level of functional proteins or transcription factors related to endoplasmic reticulum (ER) and oxidative stress induced by amyloid beta (Aß). Our results clearly showed the ability of SP600125 to decrease the levels of caspase 12 and calpain 2, two important enzymes involved in ER stress. Aß has been suggested to be able to decrease the phosphorylation level of cAMP response element-binding (CREB) through mitogen-activated protein kinase pathway. We observed that JNK inhibition in Aß-injected rats can restore the activation of CREB through increasing its phosphorylation level. This effect may explain the increase observed in c-fos level, as a CREB downstream factor under JNK inhibition in Aß-injected rats. Following Aß injection, the levels of pro-survival mitochondrial proteins including nuclear respiratory factor-1 (NRF-1), peroxisome proliferator-activated receptor gamma co-activator 1-alpha, and mitochondrial transcription factor A (TFAM) significantly decreased, which could be returned to control level with JNK inhibition. We suggest that the elevation in the level of PGC1-alpha and other mitochondrial proteins is the result of an increase in CREB activation as the upstream factor of PGC1-alpha. Also, we observed that pretreatment with SP600125 leads to a greater increase of nuclear related factor-2 (Nrf2) level compared with the Aß-injected group. Nrf2 has been shown to bind to CREB-binding factor leading to their contribution in Nrf2 target genes expression. Besides, NRF-1 and TFAM are reported as Nrf2 targets. Based on our data, we can conclude that JNK carry out partial destructive effects of Aß in rat brain.