Chiuan-Ren Yeh

New therapy targeting differential androgen receptor signaling in prostate cancer stem/progenitor vs. non-stem/progenitor cells

The androgen deprivation therapy (ADT) to systematically suppress/reduce androgens binding to the androgen receptor (AR) has been the standard therapy for prostate cancer (PCa); yet, most of ADT eventually fails leading to the recurrence of castration resistant PCa. Here, we found that the PCa patients who received ADT had increased PCa stem/progenitor cell population. The addition of the anti-androgen, Casodex, or AR-siRNA in various PCa cells led to increased stem/progenitor cells, whereas, in contrast, the addition of functional AR led to decreased stem/progenitor cell population but increased non-stem/progenitor cell population, suggesting that AR functions differentially in PCa stem/progenitor vs. non-stem/progenitor cells. Therefore, the current ADT might result in an undesired expansion of PCa stem/progenitor cell population, which explains why this therapy fails. Using various human PCa cell lines and three different mouse models, we concluded that targeting PCa non-stem/progenitor cells with AR degradation enhancer ASC-J9 and targeting PCa stem/progenitor cells with 5-azathioprine and γ-tocotrienol resulted in a significant suppression of the tumors at the castration resistant stage. This suggests that a combinational therapy that simultaneously targets both stem/progenitor and non-stem/progenitor cells will lead to better therapeutic efficacy and may become a new therapy to battle the PCa before and after castration resistant stages.

Altered prostate epithelial development in mice lacking the androgen receptor in stromal fibroblasts.

Androgens and the androgen receptor (AR) play important roles in the development of male urogenital organs. We previously found that mice with total AR knockout (ARKO) and epithelial ARKO failed to develop normal prostate with loss of differentiation. We have recently knocked out AR gene in smooth muscle cells and found the reduced luminal infolding and IGF‐1 production in the mouse prostate. However, AR roles of stromal fibroblasts in prostate development remain unclear.

Estrogen receptor α in cancer-associated fibroblasts suppresses prostate cancer invasion via modulation of thrombospondin 2 and matrix metalloproteinase 3

The prostate cancer (PCa) microenvironment contains active stromal cells known as cancer-associated fibroblasts (CAF) that may play important roles in influencing tumor progression. Here we studied the role of CAF estrogen receptor alpha (ERα) and found that it could protect against PCa invasion. Immunohistochemistry on prostatectomy specimens showed that PCa patients with ERα-positive stroma had a significantly lower risk for biochemical recurrence. In vitro invasion assays further confirmed that the stromal ERα was able to reduce PCa cell invasion. Dissection of the molecular mechanism revealed that the CAF ERα could function through a CAF-epithelial interaction via selectively upregulating thrombospondin 2 (Thbs2) and downregulating matrix metalloproteinase 3 (MMP3) at the protein and messenger RNA levels. Chromatin immunoprecipitation assays further showed that ERα could bind to an estrogen response element on the promoter of Thbs2. Importantly, knockdown of Thbs2 led to increased MMP3 expression and interruption of the ERα mediated invasion suppression, providing further evidence of an ERα-Thbs2-MMP3 axis in CAF. In vivo studies using athymic nude mice injected with CWR22Rv1 (22Rv1) PCa epithelial cells and CAF cells ± ERα also confirmed that mice coimplanted with PCa cells and CAF ERα+ cells had less tumor foci in the pelvic lymph nodes, less metastases, and tumors showed less angiogenesis, MMP3, and MMP9 (an MMP3 downstream target) positive staining. Together, these data suggest that CAF ERα could play protective roles in suppressing PCa metastasis. Our results may lead to developing new and alternative therapeutic approaches to battle PCa via controlling ERα signaling in CAF.

Recruited mast cells in the tumor microenvironment enhance bladder cancer metastasis via modulation of ERβ/CCL2/CCR2 EMT/MMP9 signals

Early clinical studies suggested that infiltrating mast cells could be associated with a poor outcome in bladder cancer (BCa) patients. The mechanisms of how mast cells influence the BCa progression, however, are unclear. Using the human clinical BCa sample survey and in vitro co-culture systems, we found BCa cells could recruit more mast cells than the surrounding non-malignant urothelial cells. The consequences of this better recruitment of mast cells toward BCa cells could then enhance BCa cell invasion. Mechanism dissection revealed that the enhanced BCa cell invasion could function via up-regulation of the estrogen receptor beta (ERβ) in both mast cells and BCa cells, which resulted in the increased CCL2/CCR2/EMT/MMP9 signals. Using the pre-clinical mouse BCa model, we further validated the mast cell-promoted BCa invasion. Interruption of the newly identified ERβ/CCL2/CCR2/EMT/MMP9 pathway via either ERβ-siRNA, ERβ antagonist PHTPP, or CCR2 antagonist can effectively reverse the mast cell-enhanced BCa cells invasion. Together, our finding could lead to the development of an alternative new therapeutic approach to better treat BCa metastasis.

Estrogen receptor α in cancer associated fibroblasts suppresses prostate cancer invasion via reducing CCL5, IL6 and macrophage infiltration in the tumor microenvironment

BackgroundCancer associated fibroblasts (CAF) play important roles in tumor growth that involves inflammation and epithelial cell differentiation. Early studies suggested that estrogen receptor alpha (ERα) was expressed in stromal cells in normal prostates and prostate cancer (PCa), but the detailed functions of stromal ERα in the PCa remain to be further elucidated.MethodsMigration and invasion assays demonstrated the presence of high levels of ERα in CAF cells (CAF.ERα(+)) suppressed PCa invasion via influencing the infiltration of tumor associated macrophages. ERα decreased CAF CCL5 secretion via suppressing the CCL5 promoter activity was examined by luciferase assay. ERα decreased CCL5 and IL-6 expression in conditioned media that was collected from CAF cell only or CAF cell co-cultured with macrophages as measured by ELISA assay.ResultsBoth in vitro and in vivo studies demonstrated CAF.ERα(+) led to a reduced macrophage migration toward PCa via inhibiting CAF cells secreted chemokine CCL5. This CAF.ERα(+) suppressed macrophage infiltration affected the neighboring PCa cells invasion and the reduced invasiveness of PCa cells are at least partly due to reduced IL6 expression in the macrophages and CAF.ConclusionOur data suggest that CAF ERα could be applied as a prognostic marker to predict cancer progression, and targeting CCL5 and IL6 may be applied as an alternative therapeutic approach to reduce M2 type macrophages and PCa invasion in PCa patients with low or little ERα expression in CAF cells.

Reduced prostate branching morphogenesis in stromal fibroblast, but not in epithelial, estrogen receptor α knockout mice.

Early studies suggested that estrogen receptor alpha (ERα) is involved in estrogen-mediated imprinting effects in prostate development. We recently reported a more complete ERα knockout (KO) mouse model via mating β-actin Cre transgenic mice with floxed ERα mice. These ACTB-ERαKO male mice showed defects in prostatic branching morphogenesis, which demonstrates that ERα is necessary to maintain proliferative events in the prostate. However, within which prostate cell type ERα exerts those important functions remains to be elucidated. To address this, we have bred floxed ERα mice with either fibroblast-specific protein (FSP)-Cre or probasin-Cre transgenic mice to generate a mouse model that has deleted ERα gene in either stromal fibroblast (FSP-ERαKO) or epithelial (pes-ERαKO) prostate cells. We found that circulating testosterone and fertility were not altered in FSP-ERαKO and pes-ERαKO male mice. Prostates of FSP-ERαKO mice have less branching morphogenesis compared to that of wild-type littermates. Further analyses indicated that loss of stromal ERα leads to increased stromal apoptosis, reduced expression of insulin-like growth factor-1 (IGF-1) and FGF10, and increased expression of BMP4. Collectively, we have established the first in vivo prostate stromal and epithelial selective ERαKO mouse models and the results from these mice indicated that stromal fibroblast ERα plays important roles in prostatic branching morphogenesis via a paracrine fashion. Selective deletion of the ERα gene in mouse prostate epithelial cells by probasin-Cre does not affect the regular prostate development and homeostasis.

The Wedelolactone Derivative Inhibits Estrogen Receptor-Mediated Breast, Endometrial, and Ovarian Cancer Cells Growth

Estrogen and estrogen receptor (ER)-mediated signaling pathways play important roles in the etiology and progression of human breast, endometrial, and ovarian cancers. Attenuating ER activities by natural products and their derivatives is a relatively practical strategy to control and reduce breast, endometrial, and ovarian cancer risk. Here, we found 3-butoxy-1,8,9-trihydroxy-6H-benzofuro[3,2-c]benzopyran-6-one (BTB), a new derivative of wedelolactone, could effectively inhibit the 17-estradiol (E2)-induced ER transactivation and suppress the growth of breast cancer as well as endometrial and ovarian cancer cells. Our results indicate that 2.5 μM BTB effectively suppresses ER-positive, but not ER-negative, breast, endometrial, and ovarian cancer cells. Furthermore, our data indicate that BTB can modulate ER transactivation and suppress the expression of E2-mediated ER target genes (Cyclin D1, E2F1, and TERT) in the ER-positive MCF-7, Ishikawa, and SKOV-3 cells. Importantly, this BTB mediated inhibition of ER activity is selective since BTB does not suppress the activities of other nuclear receptors, including glucocorticoid receptor and progesterone receptor, suggesting that BTB functions as a selective ER signaling inhibitor with the potential to treat breast, endometrial, and ovarian cancers.

Infiltrating T cells promote renal cell carcinoma (RCC) progression via altering the estrogen receptor β-DAB2IP signals

Previous studies indicated the T cells, one of the most common types of immune cells existing in the microenvironment of renal cell carcinoma (RCC), may influence the progression of RCC. The potential linkage of T cells and the estrogen receptor beta (ERβ), a key player to impact RCC progression, however, remains unclear. Our results demonstrate that RCC cells can recruit more T cells than non-malignant kidney cells. Using an in vitro matrigel invasion system, we found infiltrating T cells could promote RCC cells invasion via increasing ERβ expression and transcriptional activity. Mechanism dissection suggested that co-culturing T cells with RCC cells released more T cell attraction factors, including IFN-γ, CCL3 and CCL5, suggesting a positive regulatory feed-back mechanism. Meanwhile, infiltrating T cells may also promote RCC cell invasion via increased ERβ and decreased DAB2IP expressions, and knocking down DAB2IP can then reverse the T cells-promoted RCC cell invasion. Together, our results suggest that infiltrating T cells may promote RCC cell invasion via increasing the RCC cell ERβ expression to inhibit the tumor suppressor DAB2IP signals. Further mechanism dissection showed that co-culturing T cells with RCC cells could produce more IGF-1 and FGF-7, which may enhance the ERβ transcriptional activity. The newly identified relationship between infiltrating T cells/ERβ/DAB2IP signals may provide a novel therapeutic target in the development of agents against RCC.

Infiltrating neutrophils promote renal cell carcinoma (RCC) proliferation via modulating androgen receptor (AR) → c-Myc signals

Early studies found critical roles for neutrophils in renal cell carcinoma (RCC) progression. However, detailed mechanisms of how infiltrating neutrophils in the kidney tumor microenvironment impact RCC progression remain unclear. Here we found more neutrophils were infiltrated in human RCC lesions than those found in surrounding normal kidney tissues. Similarly, in vitro studies also revealed that RCC cells recruited more neutrophil HL-60N cells than normal kidney epithelial cells. Furthermore, in vitro and in vivo experiments also showed that the infiltrated neutrophils could promote RCC cell growth. Mechanism studies showed that co-culture of RCC cells with neutrophil HL-60N cells could selectively upregulate the androgen receptor (AR) signals, which might then alter the c-Myc signals. Interruption approaches using AR-siRNA to knock down AR in RCC cells blocked neutrophil-enhanced RCC cell proliferation. In vivo data using an orthotopically xenografted RCC mouse model also confirmed that infiltrated neutrophils could promote RCC proliferation via modulating the expressions of related cytokines. Together, these results conclude that infiltrated neutrophils may function through modulating the AR → c-Myc signals to promote RCC cell proliferation. Targeting this newly identified infiltrating neutrophil → AR → c-Myc signal pathway in the kidney tumor microenvironment may provide a new potential therapy to better suppress RCC progression.

Estrogen receptor β promotes renal cell carcinoma progression via regulating LncRNA HOTAIR-miR-138/200c/204/217 associated CeRNA network

Recent studies indicated that the estrogen receptor beta (ERβ) could affect the progression of prostate and bladder tumors, however, its roles in the renal cell carcinoma (RCC), remain to be elucidated. Here, we provide clinical evidence that ERβ expression is correlated in a negative manner with the overall survival/disease-free survival in RCC patients. Mechanism dissection revealed that targeting ERβ with ERβ-shRNA and stimulating the transactivation of ERβ with 17β-estradiol or environmental endocrine disrupting chemicals, all resulted in altering the lncRNA HOTAIR expression. The ERβ-modulated HOTAIR is able to function via antagonizing several microRNAs, including miR-138, miR-200c, miR-204, or miR-217 to impact various oncogenes, including ADAM9, CCND2, EZH2, VEGFA, VIM, ZEB1, and ZEB2, to promote RCC proliferation and invasion. Together, the identification of the ERβ-HOTAIR axis may provide us new biomarkers and/or therapeutic targets to better suppress RCC progression in the future.