Chih-Rong Shyr

Decreased Tumorigenesis and Mortality from Bladder Cancer in Mice Lacking Urothelial Androgen Receptor

Much fewer mice lacking androgen receptor (AR) in the entire body develop bladder cancer (BCa). However, the role of urothelial AR (Uro-AR) in BCa development remains unclear. In the present study, we generated mice that lacked only Uro-AR (Uro-AR(-/y)) to develop BCa by using the carcinogen BBN [N-butyl-N-(4-hydroxybutyl)-nitrosamine] and found that Uro-AR(-/y) mice had a lower incidence of BCa and a higher survival rate than did their wild-type (WT; Uro-AR(+/y)) littermates. In vitro assay also demonstrated that Uro-AR facilitates the neoplastic transformation of normal urothelial cells to carcinoma. IHC staining exhibited less DNA damage, with much higher expression of p53 and its downstream target protein PNCA in Uro-AR(-/y) than that found in WT urothelium, which suggests that Uro-AR may modulate bladder tumorigenesis through p53-PCNA DNA repair signaling. Indeed, Uro-AR(-/y) mice with the transgene, simian vacuolating virus 40 T (SV40T), in the urothelium (Uro-SV40T-AR(-/y)) had a similar incidence of BCa as did their WT littermates (Uro-SV40T-AR(+/y)), and p53 was inactivated by SV40T in both genotypes. Use of the AR degradation enhancer ASC-J9 led to suppression of bladder tumorigenesis, with few adverse effects in the BBN-induced BCa mouse model. Together, these results provide the first direct in vivo evidence that Uro-AR has an important role in promoting bladder tumorigenesis and BCa progression. Targeting AR with ASC-J9 may provide a novel approach to suppress BCa initiation.

MicroRNA-21 promotes the ovarian teratocarcinoma PA1 cell line by sustaining cancer stem/progenitor populations in vitro

IntroductionResistance of cancer stem/progenitor cells (CSPCs) to chemotherapy can lead to cancer relapse. Ovarian teratocarcinoma (OVTC) arises from germ cells and comprises pluripotent cells that can be used to study cancer cell stemness. In this study, we evaluated whether microRNA-21 (miR-21) promotes ovarian teratocarcinoma by maintaining cancer stem/progenitor populations.MethodsThe lentiviral delivery system was used to upregulate or to suppress the expression of miR-21 in the human ovarian teratocarcinoma cell line PA1 and cell growth assays were used to monitor the expression of miR-21 at different time points. Antibodies directed toward CD133, a stem cell marker, were used to identify CSPCs in the PA1 cell population, and the level of miR-21 expression was determined in enriched CSPCs. Stem cell functional assays (sphere assay and assays for CD133 expression) were used to assess the effects of miR-21 on progression of the CD133+ population.ResultsKnockdown of miR-21 in PA1 cells attenuated growth of PA1 cells whereas overexpression of miR-21 promoted cell growth. Moreover, knockdown of miR-21 resulted in a marked reduction in the CD133+ population and sphere formation of CSPCs. In contrast, overexpression of miR-21 resulted in a marked increase in the population of CD133+ cells as well as sphere formation of CSPCs.ConclusionsMicroRNA-21 plays a significant role in cancer growth by regulating stemness in cancer cells.

The expression and actions of androgen receptor in upper urinary tract urothelial carcinoma (UUTUC) tissues and the primary cultured cells

Sex hormone receptors, androgen receptor (AR) and estrogen receptors (ERs) including both ERα and ERβ, mediate the actions of sex hormones. In this study, we aimed to evaluate sex hormone receptors expression in upper urinary tract urothelial carcinomas (UUTUCs) of ureter and renal pelvis with different tumor stages and grades as well as their possible roles in tumor progression. Immunohistochemistry was used to assay the expression of AR and ERs in the primary UUTUCs. XTT viability test was applied to evaluate cell responses for anticancer drug treatment. Wound healing assay was performed to determine cell migration abilities. AR and ERβ immunoreactivities were observed in both UUTUCs, but ERα was not detected in either UUTUCs. In UUTUC of ureter specimens, higher AR expression was found in superficial or lower grade tumors. In contrast, little difference of ERβ expression was found in superficial versus muscle-invasive tumor stages or low grades versus high grades in UUTUCs of ureter specimens. Furthermore in the primary cultured cells from UUTUC specimens, the addition of functional AR reduced cell chemosensitivity, but increased cell migration. These results provide the first data showing the expression patterns of sex hormone receptors in both renal pelvis and ureter UUTUCs. From results, we concluded that there is a positive correlation for higher AR expression found in superficial or low-grade UUTUCs of ureter and identified the functional roles of AR in UUTUC progression.

Increased CK5/CK8-Positive Intermediate Cells with Stromal Smooth Muscle Cell Atrophy in the Mice Lacking Prostate Epithelial Androgen Receptor

Results from tissue recombination experiments documented well that stromal androgen receptor (AR) plays essential roles in prostate development, but epithelial AR has little roles in prostate development. Using cell specific knockout AR strategy, we generated pes-ARKO mouse with knock out of AR only in the prostate epithelial cells and demonstrated that epithelial AR might also play important roles in the development of prostate gland. We found mice lacking the prostate epithelial AR have increased apoptosis in epithelial CK8-positive luminal cells and increased proliferation in epithelial CK5-positive basal cells. The consequences of these two contrasting results could then lead to the expansion of CK5/CK8-positive intermediate cells, accompanied by stromal atrophy and impaired ductal morphogenesis. Molecular mechanism dissection found AR target gene, TGF-β1, might play important roles in this epithelial AR-to-stromal morphogenesis modulation. Collectively, these results provided novel information relevant to epithelial AR functions in epithelial-stromal interactions during the development of normal prostate, and suggested AR could also function as suppressor in selective cells within prostate.

The reduced trabecular bone mass of adult ARKO male mice results from the decreased osteogenic differentiation of bone marrow stroma cells

Male mice with androgen receptor knock-out (ARKO) show significant bone loss at a young age. However, the lasting effect of AR inactivation on bone in aging male mice remains unclear. We designed this study to evaluate the effect of AR on bone quality in aging male mice and to find the possible causes of AR inactivation contributing to the bone loss. The mice were grouped according to their ages and AR status and their trabecular bones were examined by micro-CT analysis at 6, 12, 18, and 30 weeks old. We found that bone mass consistently decreased and the bone microarchitectures continuously deteriorated in male ARKO mice at designated time points. To determine the cause of the bone loss in ARKO mice, we further examined the role of AR in bone cell fate decision and differentiation and we conducted experiments on bone marrow stromal cells (BMSC) obtained from wild type (WT) and AR knockout (KO) mice. We found that ARKO mice had higher numbers of colony formation unit-fibroblast (CFU-F), and CD44 and CD34 positive cells in bone marrow than WT mice. Our Q-RT-PCR results showed lower expression of genes linked to osteogenesis in BMSCs isolated from ARKO mice. In conclusion, AR nullification disrupted bone microarchitecture and caused trabecular bone mass loss in male ARKO mice. And the fate of BMSCs was impacted by the loss of AR. Therefore, these findings suggest that AR may accelerate the use of progenitor cells and direct them into osteogenic differentiation to affect bone metabolism.

Androgen receptor increases CD133 expression and progenitor-like population that associate with cisplatin resistance in endometrial cancer cell line.

Endometrial cancer (EMC) is a sex steroid hormone-related female malignancy. Androgen and androgen receptor (androgen/AR) signals have been implicated in EMC progression. Cancer stem/progenitor cells (CSPCs) are suspected to link to chemoresistance in patients with EMC. In this study, we examined the androgen/AR roles in cisplatin resistance and CSPC population. We found AR expression increased naive EMC side population, CSPC population, cell migration, and epithelial–mesenchymal transition. Meanwhile, it decreased cisplatin cytotoxic effect on EMC cells. Collaterally, endogenous AR expressions in EMC cells were upregulated in the cisplatin-resisting state. Moreover, AR expression could further enhance CD133 expression, CSPC-related markers, and drug-resistance gene messenger RNA expression in EMC cells. Finally, the AR-associated gene expression might go through indirect regulation. This is the first report revealing AR function on EMC cells’ CSPC and cisplatin resistance.

Androgen receptor promotes the migration and invasion of upper urinary tract urothelial carcinoma cells through the upregulation of MMP-9 and COX-2

Dysregulated androgen receptor (AR) signaling is implicated in several types of tumor, including carcinomas of the prostate, breast, liver and bladder. However, the contribution of AR to the progression of upper urinary tract urothelial carcinomas (UUTUC) has not been fully investigated. In the present study, we demonstrated that the AR is involved in the metastasis and invasiveness of UUTUC cells. We investigated the role of the AR in UUTUC by using UUTUC-derived BFTC 909 cells. The overexpression of AR promotes the migration and invasion of BFTC 909 cells. Expression of migration/invasion-related genes was increased in BFTC 909 cells overexpressing AR determined by qPCR and western blot analyses. The results showed that AR-enhanced migration and invasion of UUTUC cells are linked to the upregulation of the matrix-degrading enzyme MMP-9 and cyclooxygenase (COX)-2. Subsequently, the blocking of MMP-9 and COX-2 signaling by inhibitors suppressed AR-enhanced cell migration and invasion. The results of the present study provide evidence for the first time of the role of AR in the motility and invasion of UUT cancer cells and support the hypothesis that the AR may play a critical role in the establishment of the invasive phenotype in urothelial neoplasia of UUT. Thus, the AR may also serve as a novel biomarker and potential therapeutic target for UUT cancer.

Activins and cell migration.

Activins are the members of transforming growth factor β superfamily and act as secreted proteins; they were originally identified with a reproductive function, acting as endocrine-derived regulators of pituitary follicular stimulating hormone. In recent years, additional functions of activins have been discovered, including a regulatory role during crucial phases of growth, differentiation, and development such as wound healing, tissue repair, and regulation of branching morphogenesis. The functions of activins through activin receptors are pleiotrophic, while involving in the etiology and pathogenesis of a variety of diseases and being cell type-specific, they have been identified as important players in cancer metastasis, immune responses, inflammation, and are most likely involved in cell migration. In this chapter, we highlight the current knowledge of activin signaling and discuss the potential physiological and pathological roles of activins acting on the migration of various cell types.

The anti-tumor effect of intravesical administration of normal urothelial cells on bladder cancer

BACKGROUND AIMSnUrothelial bladder cancer (UBC) is the second most common cancer of the genitourinary tract and for advanced forms of the disease it has a high mortality rate. There are no approved new molecularly targeted agents or chemotherapeutics for advanced UBC beyond cisplatin-based chemotherapy except the recently approved anti-programmed death ligand 1 (anti-PD-1/PD-L1) antibody. With complex genetic and epigenetic alterations in tumors, despite several druggable targets identified, to cure UBC is still a challenging unmet medical need. Like other cancers, UBC to the host body is considered as a wound, aging stem cell disease and immunosuppressive disorder. Therefore, we proposed a novel cellular approach to target the host body by intravesical instilling of normal urothelial cells that could repair the injury and reduce inflammation by activating body-reparative capacity and because non-self cells are transplanted, host body immune responses could be induced in the tumor microenvironment of UBC to restrain and even eliminate tumor cells.nnnMETHODSnIn this study, we isolated and expanded normal male murine urothelial cells and intravesically administered them into the bladders of female mice of two orthotopic bladder tumor models and one urothelial injury model.nnnRESULTSnWe showed that the instillation of normal urothelial cells containing stem/progenitor cell population into bladders could have anti-tumor effect in orthotopic tumor models, possibly by activating immune responses and helping injured urothelium tissue recovery in a chemically induced urothelial injury model.nnnCONCLUSIONSnOur findings could lead to an innovative and revolutionary cell therapy modality with normal urothelial cells as an effective and safe therapeutic option for UBC.

Anabolic androgens affect the competitive interactions in cell migration and adhesion between normal mouse urothelial cells and urothelial carcinoma cells.

The urothelium is constantly rebuilt by normal urothelial cells to regenerate damaged tissues caused by stimuli in urine. However, the urothelial carcinoma cells expand the territory by aberrant growth of tumor cells, which migrate and occupy the damaged tissues to spread outside and disrupt the normal cells and organized tissues and form a tumor. Therefore, the interaction between normal urothelial cells and urothelial carcinoma cells affect the initiation and progression of urothelial tumors if normal urothelial cells fail to migrate and adhere to the damages sites to regenerate the tissues. Here, comparing normal murine urothelial cells with murine urothelial carcinoma cells (MBT-2), we found that normal cells had less migration ability than carcinoma cells. And in our co-culture system we found that carcinoma cells had propensity migrating toward normal urothelial cells and carcinoma cells had more advantages to adhere than normal cells. To reverse this condition, we used anabolic androgen, dihyrotestosterone (DHT) to treat normal cells and found that DHT treatment increased the migration ability of normal urothelial cells toward carcinoma cells and the adhesion capacity in competition with carcinoma cells. This study provides the base of a novel therapeutic approach by using anabolic hormone-enforced normal urothelial cells to regenerate the damage urothelium and defend against the occupancy of carcinoma cells to thwart cancer development and recurrence.