RAD51AP1 Deficiency Reduces Tumor Growth by Targeting Stem Cell Self-Renewal.

RAD51-associated protein 1 (RAD51AP1) plays an integral role in homologous recombination by activating RAD51 recombinase. Homologous recombination is essential for preserving genome integrity and RAD51AP1 is critical for D-loop formation, a key step in homologous recombination. Although RAD51AP1 is involved in maintaining genomic stability, recent studies have shown that RAD51AP1 expression is significantly upregulated in human cancers. However, the functional role of RAD51AP1 in tumor growth and the underlying molecular mechanism(s) by which RAD51AP1 regulates tumorigenesis have not been fully understood. Here, we use Rad51ap1-knockout mice in genetically engineered mouse models of breast cancer to unravel the role of RAD51AP1 in tumor growth and metastasis. RAD51AP1 gene transcript was increased in both luminal estrogen receptor-positive breast cancer and basal triple-negative breast cancer, which is associated with poor prognosis. Conversely, knockdown of RAD51AP1 (RADP51AP1 KD) in breast cancer cell lines reduced tumor growth. Rad51ap1-deficient mice were protected from oncogene-driven spontaneous mouse mammary tumor growth and associated lung metastasis. In vivo, limiting dilution studies provided evidence that Rad51ap1 plays a critical role in breast cancer stem cell (BCSC) self-renewal. RAD51AP1 KD improved chemotherapy and radiotherapy response by inhibiting BCSC self-renewal and associated pluripotency. Overall, our study provides genetic and biochemical evidences that RAD51AP1 is critical for tumor growth and metastasis by increasing BCSC self-renewal and may serve as a novel target for chemotherapy- and radiotherapy-resistant breast cancer. SIGNIFICANCE: This study provides in vivo evidence that RAD51AP1 plays a critical role in breast cancer growth and metastasis by regulating breast cancer stem cell self-renewal.

Multi-omic Profiling Reveals Dynamics of the Phased Progression of Pluripotency.

Pluripotency is highly dynamic and progresses through a continuum of pluripotent stem cell states. The two states that bookend the pluripotency continuum, naive and primed, are well characterized, but our understanding of the intermediate states and transitions between them remains incomplete. Here, we dissect the dynamics of pluripotent state transitions underlying pre- to post-implantation epiblast differentiation. Through comprehensive mapping of the proteome, phosphoproteome, transcriptome, and epigenome of embryonic stem cells transitioning from naive to primed pluripotency, we find that rapid, acute, and widespread changes to the phosphoproteome precede ordered changes to the epigenome, transcriptome, and proteome. Reconstruction of the kinase-substrate networks reveals signaling cascades, dynamics, and crosstalk. Distinct waves of global proteomic changes mark discrete phases of pluripotency, with cell-state-specific surface markers tracking pluripotent state transitions. Our data provide new insights into multi-layered control of the phased progression of pluripotency and a foundation for modeling mechanisms regulating pluripotent state transitions (www.stemcellatlas.org).

Combined Inhibition of DNMT and HDAC Blocks the Tumorigenicity of Cancer Stem-like Cells and Attenuates Mammary Tumor Growth.

Recently, impressive technical advancements have been made in the isolation and validation of mammary stem cells and cancer stem cells (CSC), but the signaling pathways that regulate stem cell self-renewal are largely unknown. Furthermore, CSCs are believed to contribute to chemo- and radioresistance. In this study, we used the MMTV-Neu-Tg mouse mammary tumor model to identify potential new strategies for eliminating CSCs. We found that both luminal progenitor and basal stem cells are susceptible to genetic and epigenetic modifications, which facilitate oncogenic transformation and tumorigenic potential. A combination of the DNMT inhibitor 5-azacytidine and the HDAC inhibitor butyrate markedly reduced CSC abundance and increased the overall survival in this mouse model. RNA-seq analysis of CSCs treated with 5-azacytidine plus butyrate provided evidence that inhibition of chromatin modifiers blocks growth-promoting signaling molecules such as RAD51AP1 and SPC25, which play key roles in DNA damage repair and kinetochore assembly. Moreover, RAD51AP1 and SPC25 were significantly overexpressed in human breast tumor tissues and were associated with reduced overall patient survival. In conclusion, our studies suggest that breast CSCs are intrinsically sensitive to genetic and epigenetic modifications and can therefore be significantly affected by epigenetic-based therapies, warranting further investigation of combined DNMT and HDAC inhibition in refractory or drug-resistant breast cancer. Cancer Res; 76(11); 3224-35. ©2016 AACR.

The crucial role of vitamin C and its transporter (SVCT2) in bone marrow stromal cell autophagy and apoptosis.

Vitamin C is an antioxidant that plays a vital role in various biological processes including bone formation. Previously, we reported that vitamin C is transported into bone marrow stromal cells (BMSCs) through the sodium dependent Vitamin C Transporter 2 (SVCT2) and this transporter plays an important role in osteogenic differentiation. Furthermore, this transporter is regulated by oxidative stress. To date, however, the exact role of vitamin C and its transporter (SVCT2) in ROS regulated autophagy and apoptosis in BMSCs is poorly understood. In the present study, we observed that oxidative stress decreased survival of BMSCs in a dose-dependent manner and induced growth arrest in the G1 phase of the cell cycle. These effects were accompanied by the induction of autophagy, confirmed by P62 and LC3B protein level and punctate GFP-LC3B distribution. The supplementation of vitamin C significantly rescued the BMSCs from oxidative stress by regulating autophagy. Knockdown of the SVCT2 transporter in BMSCs synergistically decreased cell survival even under low oxidative stress conditions. Also, supplementing vitamin C failed to rescue cells from stress. Our results reveal that the SVCT2 transporter plays a vital role in the mechanism of BMSC survival under stress conditions. Altogether, this study has given new insight into the role of the SVCT2 transporter in oxidative stress related autophagy and apoptosis in BMSCs.

DNMT1 is essential for mammary and cancer stem cell maintenance and tumorigenesis.

Mammary stem/progenitor cells (MaSCs) maintain self-renewal of the mammary epithelium during puberty and pregnancy. DNA methylation provides a potential epigenetic mechanism for maintaining cellular memory during self-renewal. Although DNA methyltransferases (DNMTs) are dispensable for embryonic stem cell maintenance, their role in maintaining MaSCs and cancer stem cells (CSCs) in constantly replenishing mammary epithelium is unclear. Here we show that DNMT1 is indispensable for MaSC maintenance. Furthermore, we find that DNMT1 expression is elevated in mammary tumours, and mammary gland-specific DNMT1 deletion protects mice from mammary tumorigenesis by limiting the CSC pool. Through genome-scale methylation studies, we identify ISL1 as a direct DNMT1 target, hypermethylated and downregulated in mammary tumours and CSCs. DNMT inhibition or ISL1 expression in breast cancer cells limits CSC population. Altogether, our studies uncover an essential role for DNMT1 in MaSC and CSC maintenance and identify DNMT1-ISL1 axis as a potential therapeutic target for breast cancer treatment.

The niacin/butyrate receptor GPR109A suppresses mammary tumorigenesis by inhibiting cell survival.

GPR109A, a G-protein-coupled receptor, is activated by niacin and butyrate. Upon activation in colonocytes, GPR109A potentiates anti-inflammatory pathways, induces apoptosis, and protects against inflammation-induced colon cancer. In contrast, GPR109A activation in keratinocytes induces flushing by activation of Cox-2-dependent inflammatory signaling, and the receptor expression is upregulated in human epidermoid carcinoma. Thus, depending on the cellular context and tissue, GPR109A functions either as a tumor suppressor or a tumor promoter. However, the expression status and the functional implications of this receptor in the mammary epithelium are not known. Here, we show that GPR109A is expressed in normal mammary tissue and, irrespective of the hormone receptor status, its expression is silenced in human primary breast tumor tissues, breast cancer cell lines, and in tumor tissues of three different murine mammary tumor models. Functional expression of this receptor in human breast cancer cell lines decreases cyclic AMP production, induces apoptosis, and blocks colony formation and mammary tumor growth. Transcriptome analysis revealed that GPR109A activation inhibits genes, which are involved in cell survival and antiapoptotic signaling, in human breast cancer cells. In addition, deletion of Gpr109a in mice increased tumor incidence and triggered early onset of mammary tumorigenesis with increased lung metastasis in MMTV-Neu mouse model of spontaneous breast cancer. These findings suggest that GPR109A is a tumor suppressor in mammary gland and that pharmacologic induction of this gene in tumor tissues followed by its activation with agonists could be an effective therapeutic strategy to treat breast cancer.

Molecular mechanism of SLC5A8 inactivation in breast cancer.

SLC5A8 is a putative tumor suppressor that is inactivated in more than 10 different types of cancer, but neither the oncogenic signaling responsible for SLC5A8 inactivation nor the functional relevance of SLC5A8 loss to tumor growth has been elucidated. Here, we identify oncogenic HRAS (HRAS(G12V)) as a potent mediator of SLC5A8 silencing in human nontransformed normal mammary epithelial cell lines and in mouse mammary tumors through DNMT1. Further, we demonstrate that loss of Slc5a8 increases cancer-initiating stem cell formation and promotes mammary tumorigenesis and lung metastasis in an HRAS-driven murine model of mammary tumors. Mammary-gland-specific overexpression of Slc5a8 (mouse mammary tumor virus-Slc5a8 transgenic mice), as well as induction of endogenous Slc5a8 in mice with inhibitors of DNA methylation, protects against HRAS-driven mammary tumors. Collectively, our results provide the tumor-suppressive role of SLC5A8 and identify the oncogenic HRAS as a mediator of tumor-associated silencing of this tumor suppressor in mammary glands. These findings suggest that pharmacological approaches to reactivate SLC5A8 expression in tumor cells have potential as a novel therapeutic strategy for breast cancer treatment.