Greater utility of molecular subtype rather than epithelial-to-mesenchymal transition (EMT) markers for prognosis in high-risk non-muscle-invasive (HGT1) bladder cancer.

Approximately 75% of bladder cancers are non-muscle invasive (NMIBC). Of these, up to 53% of cases progress to life-threatening muscle-invasive bladder cancer (MIBC). Patients with high-grade stage T1 (HGT1) NMIBC frequently undergo radical cystectomy (RC), although this represents overtreatment for many. Identification of progressors versus non-progressors could spare unnecessary treatment. Recent studies have confirmed that urothelial carcinoma is composed of two main molecular subtypes, basal and luminal, with 12% of basal tumours exhibiting epithelial-to-mesenchymal transition (EMT). Levels of immune cell infiltration have been shown to be subtype-specific. Here, we performed immunohistochemistry (IHC) for 11 antibodies relating to molecular subtypes or EMT in 26 cases of HGT1 urothelial carcinoma cases with 6 matched samples subsequently obtained at cystectomy (n = 6; 1 muscle-invasive, 5 non-muscle-invasive; 3 = pTis, 1 = pT1, 1 = pTa) and at recurrence (n = 2, pT2). RNAScope was also conducted in a subset. Expression patterns in HGT1 specimens versus MIBC (pT2+) were examined, and correlated with disease-specific survival (DSS). Levels of stromal tumour-infiltrating lymphocytes (sTILs) were assessed manually to determine whether lymphocyte infiltration was associated with DSS and whether differences existed between HGT1 and MIBC. Molecular subtype markers demonstrated increased prognostic potential compared to the EMT markers assessed. Increased expression of the luminal markers FOXA1 and SCUBE2, were found to be significantly associated with better DFS. No EMT markers were significantly associated with DFS. In areas of non-invasive papillary urothelial carcinoma, but not invasive carcinoma, sTIL levels were found to be significantly associated with DFS. While differences were observed between HGT1 cases that progressed versus those that did not, a larger cohort study is required for validation of these findings. Taken together, an emphasis on molecular subtype markers, rather than EMT markers, may be preferable when studying biomarkers of HGT1 urothelial carcinoma in the future.

Over-Expression of Activin-ßC Is Associated with Murine and Human Prostate Disease.

Activins are members of the TGF-ß superfamily and have been linked to prostate cancer. There are four mammalian activin subunits (ßA, ßB, ßC, and ßE) that dimerize to form functional proteins. The role of activin-A (ßA-ßA) has been relatively well characterized and has been shown to generally inhibit growth in the prostate. In contrast, little is known about the biological function of the ßC and ßE subunits. Previous work indicated activin-C (ßC-ßC) to be an antagonist of activin-A. This is important because resistance to activin-A growth inhibition occurs during prostate cancer progression. This paradox is not currently well understood. Hence, we hypothesize that local expression of the activin-ßC subunit antagonizes activin-A-dependent growth inhibition and represents a key factor contributing to acquired insensitivity to activin-A observed in prostate cancer progression. To test our hypothesis, we characterized the ventral prostate lobes of 9-month-old transgenic mice over-expressing activin-ßC and examined the expression of activin-ßA, activin-ßC, and the activin intracellular signaling factor, Smad-2, in human prostate diseases. Prostate epithelial cell hyperplasia, low-grade prostatic intraepithelial neoplasia (PIN) lesions, alterations in cell proliferation, and reduced Smad-2 nuclear localization were evident in mice over-expressing activin-ßC. Increased activin-ßA and -ßC subunit immunoreactive scores and decreased Smad-2 nuclear localization were also evident in human prostate cancer. This study suggests that over-expression of activin-ßC is associated with murine and human prostate pathologies. We conclude that the activin-ßC subunit may have therapeutic and/or diagnostic implications in human prostate disease.

Activin A regulates microRNAs and gene expression in LNCaP cells.

BACKGROUND: Prostate cancer (PCa) is an increasing health issue worldwide. For patients with advanced castration-resistant PCa (CRPC) treatment options are limited and overall survival is relatively short. Paired with this, non-invasive diagnostic options are yet to be established. Activins are members of the TGF-ß superfamily and have been linked to prostate physiology. For instance, activin A is an inhibitor of growth in the prostate. A novel class of non-coding RNA, microRNAs (miRNAs) have been intrinsically linked to a range of cellular processes and carcinogenesis. No studies have investigated the impact of activin A on miRNA expression in PCa cell lines. Hence, the objective of this study was to determine the effect of activin A on miRNA expression and downstream target genes in PCa. METHODS: Activin-sensitive (LNCaP) and insensitive (PC3) prostate cells were treated with 50 ng/ml of activin A for 72 hr. To examine miRNA expression following treatment, SYBR RT-qPCR miRNA arrays were used in conjunction with TaqMan RT-qPCR. MiRPath-TarBase analysis was conducted using the miRNAs that were significantly altered following activin A treatment of LNCaP cells to highlight enriched target genes within biological pathways. Highlighted target genes were assessed using pathway-focused TGF-ß and cell cycle SYBR RT-qPCR arrays. RESULTS: Activin A treatment altered nine miRNAs in LNCaP cells: miR-222-3p, miR-15b-5p, miR-93-5p, miR-18a-5p, and let-7i-5p were significantly decreased, while miR-30a/30d-5p, let-7c, and miR-196b-5p were significantly increased versus media control. In PC3 cells five miRNAs were altered: miR-130a-3p, miR-7-5p, and miR-140-3p were significantly decreased while miR-191-5p and miR-26a-5p were significantly increased versus media control. MiRPath-TarBase analysis highlighted that the miRNAs significantly altered in LNCaP cells targeted genes contained in activin A-related KEGG pathways. Furthermore, when LNCaP cells were treated with activin A the expression of the targeted genes was the inverse of the expression of activin A-mediated miRNAs. CONCLUSIONS: This study demonstrated the ability of activin A to modulate miRNA expression in PCa cell lines and suggests a correlative relationship between miRNA expression and downstream target genes in LNCaP cells. This study provides impetus for further studies into activin A and miRNAs in PCa. Prostate 76:951-963, 2016. © 2016 Wiley Periodicals, Inc.

microRNA and non-canonical TGF-ß signalling: implications for prostate cancer therapy.

The incidence of prostate cancer is increasing worldwide and marks a significant health issue. Paired with this, current therapeutic options for advanced prostate cancer, notably androgen deprivation therapy (ADT), fail to provide a consistent level of efficacy throughout the treatment period, highlighting the need for new robust therapies. Growth factors, such as Transforming Growth Factor-beta (TGF-ß), possess the ability to impede cancer development in the early stages, via alterations in either apoptosis, cell proliferation, or the promotion of cellular senescence. However, later in the pathogenesis, advanced prostate cancer cells become insensitive to the previously beneficial effects of TGF-ß. The molecular mechanisms behind this acquired insensitivity are not well understood. Thus, the aim of this review is to examine the effects of a class of small non-coding RNA, microRNA (miRNA), on TGF-ß signalling. The impact of miRNA on the canonical TGF-ß Smad signalling pathway has been well investigated, hence, in this review, we will examine whether miRNA targeting members of non-canonical TGF-ß signalling members, such as, Erk, RhoA, PI3K/Akt and JNK/p38 could provide alternate therapeutic options for advanced prostate cancer.

Insensitivity to the growth inhibitory effects of activin A: an acquired capability in prostate cancer progression.

Prostate cancer (PCa), the most common non-skin cancer in men, is a worldwide health concern. Treatment options for aggressive PCa are limited to androgen deprivation therapies (ADT), which are ineffective, with robust diagnostic options also being limited. The prostate specific antigen (PSA) test, for instance, is subject to high levels of false positive results and cannot distinguish between cancer confined to the prostate and aggressive metastatic cancer. As such, additional therapeutic and diagnostic options are urgently required. In recent years, a clear association between activins and prostate cancer has become evident. Activins are members of the TGF-ß superfamily and are responsible for a plethora of physiological processes, including cell proliferation, apoptosis, immune surveillance, embryonic development, and follicle stimulating hormone (FSH) regulation. Activin A normally inhibits cancer development and progression, however, cancer cell growth in high-grade PCa is not inhibited by this protein. The mechanism for this apparent acquired capability to resist activin A-mediated growth inhibition is currently not well understood. Thus, the aim of this review is to analyse the role of activin A in PCa progression and to present mechanisms by which transformed cells may escape its effects. The overarching hypothesis is that insensitivity to the growth inhibitory effects of activin A is an acquired capability in PCa progression. Therefore, local and genetic elements that may be responsible for this change in cellular sensitivity to activin A during cancer progression will be highlighted with a view to identifying potential diagnostic or therapeutic targets.