Cooperativity of c-MYC with Krüppel-Like Factor 6 Splice Variant 1 induces phenotypic plasticity and promotes prostate cancer progression and metastasis.

Metastasis remains a major cause of morbidity and mortality in men with prostate cancer, and the functional impact of the genetic alterations, alone or in combination, driving metastatic disease remains incompletely understood. The proto-oncogene c-MYC, commonly deregulated in prostate cancer. Transgenic expression of c-MYC is sufficient to drive the progression to prostatic intraepithelial neoplasia and ultimately to moderately differentiated localized primary tumors, however, c-MYC-driven tumors are unable to progress through the metastatic cascade, suggesting that a "second-hit" is necessary in the milieu of aberrant c-MYC-driven signaling. Here, we identified cooperativity between c-MYC and KLF6-SV1, an oncogenic splice variant of the KLF6 gene. Transgenic mice that co-expressed KLF6-SV1 and c-MYC developed progressive and metastatic prostate cancer with a histological and molecular phenotype like human prostate cancer. Silencing c-MYC expression significantly reduced tumor burden in these mice supporting the necessity for c-MYC in tumor maintenance. Unbiased global proteomic analysis of tumors from these mice revealed significantly enriched vimentin, a dedifferentiation and pro-metastatic marker, induced by KLF6-SV1. c-MYC-positive tumors were also significantly enriched for KLF6-SV1 in human prostate cancer specimens. Our findings provide evidence that KLF6-SV1 is an enhancer of c-MYC-driven prostate cancer progression and metastasis, and a correlated genetic event in human prostate cancer with potential translational significance.

Papillary urothelial carcinoma of the vagina: A case presentation and review of the literature.

A 92-year-old woman with history of right nephroureterectomy for low grade Ta urothelial carcinoma (UC) of the renal pelvis and proximal ureter and high grade Ta and carcinoma-in-situ of the bladder presented for surveillance cystoscopy in 2019. She was found to have no evidence of disease within bladder or upper tract, however demonstrated a large papillary lesion within the vagina. This lesion stained for p40+, p16 patchy+, and GATA3+, all markers of UC, and the same molecular makeup of her prior bladder tumor.

Mechanisms of Osteoblastic Bone Metastasis in Prostate Cancer: Role of Prostatic Acid Phosphatase.

Prostate cancer (PCa) preferentially metastasizes to bone, leading to complications including severe pain, fractures, spinal cord compression, bone marrow suppression, and a mortality of ∼70%. In spite of recent advances in chemo-, hormonal, and radiation therapies, bone-metastatic, castrate-resistant PCa is incurable. PCa is somewhat unique among the solid tumors in its tendency to produce osteoblastic lesions composed of hypermineralized bone with multiple layers of poorly organized type I collagen fibrils that have reduced mechanical strength. Many of the signaling pathways that control normal bone homeostasis are at play in pathologic PCa bone metastases, including the receptor activator of nuclear factor-κB/receptor activator of nuclear factor-κB ligand/osteoprotegerin system. A number of PCa-derived soluble factors have been shown to induce the dysfunctional osteoblastic phenotype. However, therapies directed at these osteoblastic-stimulating proteins have yielded disappointing clinical results to date. One of the soluble factors expressed by PCa cells, particularly in bone metastases, is prostatic acid phosphatase (PAP). Human PAP is a prostate epithelium-specific secretory protein that was the first tumor marker ever described. Biologically, PAP exhibits both phosphatase activity and ecto-5'-nucleotidase activity, generating extracellular phosphate and adenosine as the final products. Accumulating evidence indicates that PAP plays a causal role in the osteoblastic phenotype and aberrant bone mineralization seen in bone-metastatic, castrate-resistant PCa. Targeting PAP may represent a therapeutic approach to improve morbidity and mortality from PCa osteoblastic bone metastases.

Small-Molecule Activators of Protein Phosphatase 2A for the Treatment of Castration-Resistant Prostate Cancer.

Primary prostate cancer is generally treatable by androgen deprivation therapy, however, later recurrences of castrate-resistant prostate cancer (CRPC) that are more difficult to treat nearly always occur due to aberrant reactivation of the androgen receptor (AR). In this study, we report that CRPC cells are particularly sensitive to the growth-inhibitory effects of reengineered tricyclic sulfonamides, a class of molecules that activate the protein phosphatase PP2A, which inhibits multiple oncogenic signaling pathways. Treatment of CRPC cells with small-molecule activators of PP2A (SMAP) in vitro decreased cellular viability and clonogenicity and induced apoptosis. SMAP treatment also induced an array of significant changes in the phosphoproteome, including most notably dephosphorylation of full-length and truncated isoforms of the AR and downregulation of its regulatory kinases in a dose-dependent and time-dependent manner. In murine xenograft models of human CRPC, the potent compound SMAP-2 exhibited efficacy comparable with enzalutamide in inhibiting tumor formation. Overall, our results provide a preclinical proof of concept for the efficacy of SMAP in AR degradation and CRPC treatment.Significance: A novel class of small-molecule activators of the tumor suppressor PP2A, a serine/threonine phosphatase that inhibits many oncogenic signaling pathways, is shown to deregulate the phosphoproteome and to destabilize the androgen receptor in advanced prostate cancer. Cancer Res; 78(8); 2065-80. ©2018 AACR.

Prostatic Acid Phosphatase Alters the RANKL/OPG System and Induces Osteoblastic Prostate Cancer Bone Metastases.

Prostate cancer (PCa) is unique in its tendency to produce osteoblastic (OB) bone metastases. There are no existing therapies that specifically target the OB phase that affects 90% of men with bone metastatic disease. Prostatic acid phosphatase (PAP) is secreted by PCa cells in OB metastases and increases OB growth, differentiation, and bone mineralization. The purpose of this study was to investigate whether PAP effects on OB bone metastases are mediated by autocrine and/or paracrine alterations in the receptor activator of nuclear factor κ-B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system. To investigate whether PAP modulated these factors and altered the bone reaction, we knocked down PAP expression in VCaP cells and stably overexpressed PAP in PC3M cells, both derived from human PCa bone metastases. We show that knockdown of PAP in VCaP cells decreased OPG while increasing RANK/RANKL expression. Forced overexpression of PAP in PC3M cells had the inverse effect, increasing OPG while decreasing RANK/RANKL expression. Coculture of PCa cells with MC3T3 preosteoblasts also revealed a role for secretory PAP in OB-PCa cross talk. Reduced PAP expression in VCaP cells decreased MC3T3 proliferation and differentiation and reduced their OPG expression. PAP overexpression in PC3M cells altered the bone phenotype creating OB rather than osteolytic lesions in vivo using an intratibial model. These findings demonstrate that PAP secreted by PCa cells in OB bone metastases increases OPG and plays a critical role in the vicious cross talk between cancer and bone cells. These data suggest that inhibition of secretory PAP may be an effective strategy for PCa OB bone lesions.

Metastatic Malignant Melanoma to Urinary Bladder: A Potential Pitfall for High-Grade Urothelial Carcinoma.

We present a case of a 61-year-old female presenting with a bladder tumor that occurred 7 years after her previous diagnosis of Clark's level III mid-back melanoma. The bladder tumor was submitted to histopathology without accompanying clinical history, and an initial diagnosis of high-grade urothelial carcinoma was rendered based on epithelioid and sarcomatoid appearing pleomorphic histopathology. We present this case to highlight the diagnostic challenge presented by the rare occurrence of metastatic melanoma to the urinary bladder and the potential pitfall of this lesion being diagnosed as high-grade urothelial carcinoma in the presence of limited clinical history.

Androgens up-regulate transcription of the Notch inhibitor Numb in C2C12 myoblasts via Wnt/ß-catenin signaling to T cell factor elements in the Numb promoter.

Androgen signaling via the androgen receptor is a key pathway that contributes to development, cell fate decisions, and differentiation, including that of myogenic progenitors. Androgens and synthetic steroids have well established anabolic actions on skeletal muscle. Wnt and Notch signaling pathways are also essential to myogenic cell fate decisions during development and tissue repair. However, the interactions among these pathways are largely unknown. Androgenic regulation of Wnt signaling has been reported. Nandrolone, an anabolic steroid, has been shown to inhibit Notch signaling and up-regulate Numb, a Notch inhibitor. To elucidate the mechanisms of interaction between nandrolone and Wnt/Notch signaling, we investigated the effects of nandrolone on Numb expression and Wnt signaling and determined the roles of Wnt signaling in nandrolone-induced Numb expression in C2C12 myoblasts. Nandrolone increased Numb mRNA and protein levels and T cell factor (Tcf) transcriptional activity via inhibition of glycogen synthase kinase 3ß. Up-regulation of Numb expression by nandrolone was blocked by the Wnt inhibitors, sFRP1 and DKK1, whereas Wnt3a increased Numb mRNA and protein expression. In addition, we observed that the proximal promoter of the Numb gene had functional Tcf binding elements to which ß-catenin was recruited in a manner enhanced by both nandrolone and Wnt3a. Moreover, site-directed mutagenesis indicated that the Tcf binding sites in the Numb promoter are required for the nandrolone-induced Numb transcriptional activation in this cell line. These results reveal a novel molecular mechanism underlying up-regulation of Numb transcription with a critical role for increased canonical Wnt signaling. In addition, the data identify Numb as a novel target gene of the Wnt signaling pathway by which Wnts would be able to inhibit Notch signaling.

KLF6-SV1 drives breast cancer metastasis and is associated with poor survival.

Metastasis is the major cause of cancer mortality. A more thorough understanding of the mechanisms driving this complex multistep process will aid in the identification and characterization of therapeutically targetable genetic drivers of disease progression. We demonstrate that KLF6-SV1, an oncogenic splice variant of the KLF6 tumor suppressor gene, is associated with increased metastatic potential and poor survival in a cohort of 671 lymph node-negative breast cancer patients. KLF6-SV1 overexpression in mammary epithelial cell lines resulted in an epithelial-to-mesenchymal-like transition and drove aggressive multiorgan metastatic disease in multiple in vivo models. Additionally, KLF6-SV1 loss-of-function studies demonstrated reversion to an epithelial and less invasive phenotype. Combined, these findings implicate KLF6-SV1 as a key driver of breast cancer metastasis that distinguishes between indolent and lethal early-stage disease and provides a potential therapeutic target for invasive breast cancer.

Nandrolone, an anabolic steroid, stabilizes Numb protein through inhibition of mdm2 in C2C12 myoblasts.

Nandrolone, an anabolic steroid, slows denervation atrophy of rat muscle, prevents denervation-induced nuclear accumulation of intracellular domain of the Notch receptor, and elevates expression of Numb. Numb acts as an inhibitor of Notch signaling and promotes myogenic differentiation of satellite cells. Turnover of Numb is regulated by mdm2, an E3 ubiquitin ligase. With these considerations in mind, we investigated the effects of nandrolone on the expression of Numb and mdm2 proteins and determined the effect of mdm2 on nandrolone-induced alterations in Numb protein in C2C12 myoblasts. When C2C12 cells were cultured in a medium favoring differentiation (Dulbecco modified Eagle medium containing 2% horse serum), nandrolone up-regulated Numb protein levels in a time-dependent manner and prolonged Numb protein half-life from 10 to 18 hours. In contrast, nandrolone reduced the expression of mdm2 protein. To determine whether the decreased mdm2 expression induced by nandrolone was responsible for the increased levels and prolonged half-life of Numb protein in this cell line, mdm2-small interfering RNA (siRNA) was employed to inhibit mdm2 expression. Compared to cells transfected with scrambled siRNA (negative control), transfection with mdm2-siRNA increased basal Numb protein expression but abolished the further increase in Numb protein levels by nandrolone. In addition, transfection of mdm2-siRNA mimicked the effect of nandrolone to prolong the half-life of Numb protein. Moreover, when C2C12 cells were forced to overexpress mdm2, there was a significant decline in the expression of both basal and inducible Numb protein. Our data suggest that nandrolone, by a novel mechanism for this agent in a muscle cell type, increases Numb protein levels in C2C12 myoblasts by stabilizing Numb protein against degradation, at least in part, via suppression of mdm2 expression.

Androgen deprivation therapy as primary treatment for prostate cancer.

CONTEXT: Hormonal therapy has been the mainstay of treatment for advanced prostate cancer for over 70 yr. The timing and extent of androgen ablative therapy for earlier stage disease remains controversial. In addition, recent studies demonstrate that so-called "castration-resistant" tumors are still dependent on androgen receptor signaling. PATIENT AND METHODS: A 66-yr-old man presented with clinical stage T1C N+ M0 prostate cancer and received primary androgen deprivation therapy. Over the course of the next 17 yr, he was treated with various forms of androgen deprivation therapy, including two newer agents, abiraterone acetate and MDV 3100. A review of the literature was conducted to identify indications, controversies, and new developments regarding hormonal therapy for prostate cancer. CONCLUSIONS: Androgen deprivation therapy remains the treatment of choice for metastatic prostate cancer; however, it is not without its adverse effects, and most men with advanced disease eventually develop castration resistance. Newer compounds that more specifically and effectively target androgen and androgen receptor signaling in prostate cancer cells may provide more long-lasting remissions in advanced disease.

Prostatic acid phosphatase is expressed in human prostate cancer bone metastases and promotes osteoblast differentiation.

Prostate cancer (PCa) bone metastases are a major cause of morbidity and mortality. There are no effective therapies for PCa bone metastases that prolong survival. Prostatic acid phosphatase (PAP) is a secretory protein expressed by PCa cells. We demonstrate that PAP is strongly expressed in PCa bone metastases in 7/7 patients, while prostate-specific antigen (PSA) is only weakly expressed. The human PCa cell line VCaP secretes PAP and induces an osteoblastic reaction in bone similar to that seen in human PCa bone metastases. Coculture of MC3T3 mouse preosteoblast cells with VCaP cells induces MC3T3 cell growth and differentiation as measured by alkaline phosphatase secretion, and this effect is inhibited by addition of the PAP-inhibitor, l-tartrate. Taken together, these data indicate that PAP is expressed in PCa bone metastases and may play a causal role in the osteoblastic phase of the disease.

Overview of prostate anatomy, histology, and pathology.

The human prostate is heterogeneous with regard to its embryologic origin. The two most prevalent diseases of aging males, benign prostatic hyperplasia and prostate cancer (PCa), arise from different zones within the prostate. The biology of PCa is also heterogeneous and even within a single individual there often exist prostate cancers with varying potential to progress and metastasize. Through careful study of the histology and molecular signatures of both the human and mouse-modeled disease, treatment decisions can be tailored to individual cases so as to optimize efficacy and minimize side effects from therapy.

Nandrolone reduces activation of Notch signaling in denervated muscle associated with increased Numb expression.

Nandrolone, an anabolic steroid, slows denervation-atrophy in rat muscle. The molecular mechanisms responsible for this effect are not well understood. Androgens and anabolic steroids activate Notch signaling in animal models of aging and thereby mitigate sarcopenia. To explore the molecular mechanisms by which nandrolone prevents denervation-atrophy, we investigated the effects of nandrolone on Notch signaling in denervated rat gastrocnemius muscle. Denervation significantly increased Notch activity reflected by elevated levels of nuclear Notch intracellular domain (NICD) and expression of Hey1 (a Notch target gene). Activation was greatest at 7 and 35 days after denervation but remained present at 56 days after denervation. Activation of Notch in denervated muscle was prevented by nandrolone associated with upregulated expression of Numb mRNA and protein. These data demonstrate that denervation activates Notch signaling, and that nandrolone abrogates this response associated with increased expression of Numb, suggesting a potential mechanism by which nandrolone reduces denervation-atrophy.

Prostaglandin E2 modulates components of the Wnt signaling system in bone and prostate cancer cells.

Both Wnt signaling and prostaglandin E(2) (PGE(2)) play pivotal roles in bone development, remodeling, osteoporosis and prostate cancer (PCa) bone metastases. We investigated the effects of PGE(2) on Wnt signaling in osteoblast-lineage cells and Wnt-inhibitor expression in PCa cells. We demonstrate that low dose PGE(2) (0.1 microM) promotes Wnt signaling while higher doses of PGE(2) (1.0-10 microM) inhibit these same parameters in osteoblast-lineage cells. The differential effects of low vs high-dose PGE(2) on pre-osteoblasts may be attributed to dose-dependent modulation of prostaglandin receptor (EP) subtype expression; with lower doses increasing the expression the cAMP-stimulatory EP4 receptor subtype and higher doses increasing the expression of the cAMP-inhibitory EP3 receptor subtype. Moreover, we demonstrate that high expression levels of COX-2 and PGE(2) promote the secretion of Wnt inhibitors from prostate cancer cells. These data demonstrate that there are dose-dependent effects of PGE(2) on Wnt activation in osteoblast-lineage cells and Wnt-inhibitor expression in PCa cells which may have clinical implications in the management.

Ribosome-inactivating proteins isolated from dietary bitter melon induce apoptosis and inhibit histone deacetylase-1 selectively in premalignant and malignant prostate cancer cells.

Epidemiologic evidence suggests that a diet rich in fruits and vegetables is associated with a reduced risk of prostate cancer (PCa) development. Although several dietary compounds have been tested in preclinical PCa prevention models, no agents have been identified that either prevent the progression of premalignant lesions or treat advanced disease. Momordica charantia, known as bitter melon in English, is a plant that grows in tropical areas worldwide and is both eaten as a vegetable and used for medicinal purposes. We have isolated a protein, designated as MCP30, from bitter melon seeds. The purified fraction was verified by SDS-PAGE and mass spectrometry to contain only 2 highly related single chain Type I ribosome-inactivating proteins (RIPs), alpha-momorcharin and beta-momorcharin. MCP30 induces apoptosis in PIN and PCa cell lines in vitro and suppresses PC-3 growth in vivo with no effect on normal prostate cells. Mechanistically, MCP30 inhibits histone deacetylase-1 (HDAC-1) activity and promotes histone-3 and -4 protein acetylation. Treatment with MCP30 induces PTEN expression in a prostatic intraepithelial neoplasia (PIN) and PCa cell lines resulting in inhibition of Akt phosphorylation. In addition, MCP30 inhibits Wnt signaling activity through reduction of nuclear accumulation of beta-catenin and decreased levels of c-Myc and Cyclin-D1. Our data indicate that MCP30 selectively induces PIN and PCa apoptosis and inhibits HDAC-1 activity. These results suggest that Type I RIPs derived from plants are HDAC inhibitors that can be utilized in the prevention and treatment of prostate cancer.

Urinary bladder adenocarcinoma arising in a spina bifida patient.

Urinary bladder adenocarcinomas are rare malignancies accounting for approximately 2.5% of all urothelial neoplasms. Intestinal metaplasia of the urothelium indicates the presence of intestinal-type goblet cells and was generally observed to coexist with or to precede the diagnosis of bladder adenocarcinomas. Controversy continues of whether intestinal metaplasia is an acquired precancerous lesion, secondary to different insults to the urothelium, or a concomitant lesion in glandular carcinogenesis. Patients with neurogenic bladders are particularly at risk for developing bladder cancer, mostly squamous cell carcinoma and rarely adenocarcinoma. In these patients, chronic irritation of the urothelium as well as long-term indwelling urinary catheters were the most significant risk factors. Spina bifida is a congenital developmental abnormality that may result in neurogenic bladder. There is only one previously reported case of urothelial carcinoma with associated squamous metaplasia of the bladder occurring in a spina bifida patient. We report the first case of bladder adenocarcinoma associated with intestinal metaplasia occurring in a spina bifida occulta patient. The patient had a complicated clinical course and suffered recurrent urinary tract infections, renal calculi, and urinary incontinence and was managed with intermittent as well as indwelling catheterization.

Concomitant secretion of glucocorticoid, androgens, and mineralocorticoid by an adrenocortical carcinoma: case report and review of literature.

OBJECTIVE: To present a case of concomitant secretion of cortisol, androgens, and 11-deoxycorticosterone (DOC) by an adrenocortical carcinoma and review the literature in an attempt to identify similar cases. METHODS: The patient's medical history, physical examination, laboratory data, computed tomographic scan, and histopathologic results were analyzed and summarized in a case report, and an extensive review of the literature was performed. RESULTS: Endocrinologic data showed excess cortisol production, substantially elevated testosterone and androstenedione levels, and profoundly increased DOC in the setting of suppressed aldosterone. An abdominal computed tomographic scan showed a left adrenal tumor. A left adrenalectomy was performed, and the histopathologic diagnosis was stage II adrenocortical carcinoma. The review of the pertinent literature revealed the absence of any identical cases in the past. CONCLUSION: Our patient presented with a rare case of cosecretion of cortisol, testosterone, androstenedione, and DOC by an adrenocortical carcinoma, resulting in a clinical picture consistent with Cushing's syndrome, hyperandrogenism, and primary hypermineralocorticoidism. We recommend the routine performance of a DOC assay in the setting of mineralocorticoid excess in association with low plasma aldosterone levels.

Androgens promote preosteoblast differentiation via activation of the canonical Wnt signaling pathway.

Although androgens stimulate bone formation the precise events underlying these effects have not been elucidated. Wnt signaling plays a central role in osteoblast development and bone formation. We demonstrated that dihydrotestosterone (DHT) significantly stimulates MC3T3 preosteoblast differentiation with no effect on cell growth. This effect of DHT was accompanied by increased Wnt signaling in the same cells. Moreover, the stimulatory effects of DHT on preosteoblast differentiation were inhibited by overexpression of soluble frizzed-related protein (sFRP), a naturally occurring Wnt antagonist. These results suggest that androgens promote preosteoblastic differentiation via effects on the canonical Wnt signaling pathway.

Androgen-induced Wnt signaling in preosteoblasts promotes the growth of MDA-PCa-2b human prostate cancer cells.

The high morbidity and mortality associated with prostate cancer (PCa) result from its tendency to metastasize to bone where it produces predominantly osteoblastic lesions. The Wnt signaling pathway plays an important role in embryogenesis, tumorigenesis, osteoblast development, and bone formation. Androgen signaling via the androgen receptor (AR) is critical in both PCa and bone cell growth. We examined the effects of androgens on cell growth and Wnt signaling in the AR-positive MDA-PCa-2b cell line and MC3T3 preosteoblasts, grown alone and in coculture. We show that the potent androgen dihydrotestosterone increases AR expression and transcriptional activity only in the preosteoblasts. Although dihydrotestosterone induced an 80% increase in PCa cell growth when the cells were grown alone, dihydrotestosterone had a more significant effect on MDA-PCa-2b cell proliferation (3.2-fold increase) when the PCa cells were cocultured with preosteoblasts. Dihydrotestosterone addition to preosteoblasts promoted Wnt-dependent transcriptional reporter activity associated with GSK3beta(S-9) phosphorylation and accumulation of nuclear beta-catenin as well as elevated Runx2 expression. In addition, the increased proliferation of PCa cells in coculture with MC3T3 cells in response to dihydrotestosterone was abrogated by the addition of either exogenous DKK-1 or sFRP-1 protein, two naturally occurring Wnt antagonists. Finally, we show that the paracrine growth-promoting effect of androgens is limited to MDA-PCa-2b cells. These data imply that Wnt signaling is involved in the androgen-regulated crosstalk between preosteoblasts and PCa cells and suggest that androgens may stimulate growth of some prostate tumor cells indirectly, via up-regulation of Wnt signaling in bone cells.