Polyunsaturated fatty acids promote M2-like TAM deposition via dampening RhoA-YAP1 signaling in the ovarian cancer microenvironment.

BACKGROUND: Peritoneal metastases frequently occur in epithelial ovarian cancer (EOC), resulting in poor prognosis and survival rates. Tumor-associated-macrophages (TAMs) massively infiltrate into ascites spheroids and are multi-polarized as protumoral M2-like phenotype, orchestrating the immunosuppression and promoting tumor progression. However, the impact of omental conditioned medium/ascites (OCM/AS) on TAM polarization and its function in tumor progression remains elusive. METHODS: The distribution and polarization of TAMs in primary and omental metastatic EOC patients' tumors and ascites were examined by m-IHC, FACS analysis, and immunofluorescence. QPCR, immunofluorescence, FACS analysis, lipid staining assay, ROS assay, and Seahorse real-time cell metabolic assay characterized TAMs as being polarized in the ascites microenvironment. The oncogenic role of TAMs in tumor cells was demonstrated by co-cultured migration/invasion, proliferation, and spheroid formation assays. Mechanistic studies of the regulations of TAM polarization were performed by using RNA-Seq, GTPase pull-down, G-LISA activation assays, and other biochemical assays. A Yap1 macrophages (MФs) conditional knockout (cKO) mouse model demonstrated the roles of YAP1 in TAM polarization status and its pro-metastatic function. Finally, the anti-metastatic potential of targeting TAMs through restoring YAP1 by pharmacological agonist XMU MP1 was demonstrated in vitro and in vivo. RESULTS: Abundant polyunsaturated fatty acids (PUFAs) in OCM/AS suppressed RhoA-GTPase activities, which, in turn, downregulated nuclear YAP1 in MФs, leading to increased protumoral TAM polarization accompanied by elevated OXPHOS metabolism. Abolishment of YAP1 in MФs further confirmed that a higher M2/M1 ratio of TAM polarization could alleviate CD8+ T cell infiltration and cytotoxicity in vivo. Consistently, the loss of YAP1 has been observed in EOC metastatic tissues, suggesting its clinical relevance. On the contrary, restoration of YAP1 expression by pharmaceutical inhibition of MST1/2 induced conversion of M2-to-M1-like polarized MФs, elevating the infiltration of CD8+ T cells and attenuating tumor growth. CONCLUSION: This study revealed that PUFAs-enriched OCM/AS of EOC promotes M2-like TAM polarization through RhoA-YAP1 inhibition, where YAP1 downregulation is required for accelerating protumoral M2-like TAM polarization, thereby causing immunosuppression and enhancing tumor progression. Conversion of M2-to-M1-like polarized MФs through Yap1 activation inhibits tumor progression and contributes to developing potential TAMs-targeted immunotherapies in combating EOC peritoneal metastases.

Targeting estrogen receptor subtypes (ERα and ERß) with selective ER modulators in ovarian cancer.

Ovarian cancer cells express both estrogen receptor α (ERα) and ERß, and hormonal therapy is an attractive treatment option because of its relatively few side effects. However, estrogen was previously shown to have opposite effects in tumors expressing ERα compared with ERß, indicating that the two receptor subtypes may have opposing effects. This may explain the modest response to nonselective estrogen inhibition in clinical practice. In this study, we aimed to investigate the effect of selectively targeting each ER subtype on ovarian cancer growth. Ovarian cancer cell lines SKOV3 and OV2008, expressing both ER subtypes, were treated with highly selective ER modulators. Sodium 3'-(1-(phenylaminocarbonyl)-3,4-tetrazolium)-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) assay revealed that treatment with 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP) (ERα antagonist) or 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN) (ERß agonist) significantly suppressed cell growth in both cell lines. In contrast, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT) (ERα agonist) or 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]-pyrimidin-3-yl]phenol (PHTPP) (ERß antagonist) significantly enhanced cell growth. These results were confirmed on a xenograft model where SKOV3 cells were injected s.c. into ovariectomized mice. We observed that the average size of xenografts in both the DPN-treated group and the MPP-treated group was significantly smaller than that for the vehicle-treated group. In addition, we found that phospho-AKT expressions in SKOV3 cells were reduced by 80% after treatment with MPP and DPN, indicating that the AKT pathway was involved. The combined treatment with MPP and DPN had a synergistic effect in suppressing ovarian cancer cell growth. Our findings indicate that targeting ER subtypes may enhance the response to hormonal treatment in women with ovarian cancer.

The ß1-integrin-p-FAK-p130Cas-DOCK180-RhoA-vinculin is a novel regulatory protein complex at the apical ectoplasmic specialization in adult rat testes.

During spermatogenesis, step 1 spermatids (round spermatids) derive from spermatocytes following meiosis I and II at stage XIV of the epithelial cycle begin a series of morphological transformation and differentiation via 19 steps in rats to form spermatozoa. This process is known as spermiogenesis, which is marked by condensation of the genetic material in the spermatid head, formation of the acrosome and elongation of the tail. Since developing spermatids are lacking the robust protein synthesis and transcriptional activity, the cellular, molecular and morphological changes associated with spermiogenesis rely on the Sertoli cell in the seminiferous epithelium via desmosome and gap junction between Sertoli cells and step 1-7 spermatids. Interestingly, a unique anchoring junction type arises at the interface of step 8 spermatid and Sertoli cell known as apical ectoplasmic specialization (apical ES). Once it appears, apical ES is the only anchoring device restricted to the interface of step 8-19 spermatids and Sertoli cells to confer spermatid polarity, adhesion, signal communication and structural support, and to provide nutritional support during spermiogenesis, replacing desmosome and gap junction. While the adhesion protein complexes that constitute the apical ES are known, the signaling protein complexes that regulate apical ES dynamics, however, remain largely unknown. Herein we report the presence of a FAK (focal adhesion kinase)-p130Cas (p130 Crk-associated substrate)-DOCK180 (Dedicator of cytokinesis 180)-RhoA (Ras homolog gene family, member A)-vinculin signaling protein complex at the apical ES, which is also an integrated component of the ß1-integrin-based adhesion protein complex based on co-immunoprecipitation experiment. It was also shown that besides p-FAK-Tyr(397) and p-FAK-Tyr(576), ß1-integrin, p130Cas, RhoA and vinculin displayed stage-specific expression in the seminiferous epithelium during the epithelial cycle with predominant localization at the apical ES as demonstrated by immunohistochemistry. Based on these findings, functional studies can now be performed to assess the role of this ß1-integrin-p-FAK-p130Cas-DOCK180-RhoA-vinculin protein complex in apical ES dynamics during spermiogenesis.