Exploring the critical role of SDHA in breast cancer proliferation: implications for novel therapeutic strategies.

Genes encoding growth-inhibitory proteins are postulated to be candidate tumor suppressors. The identification of such proteins may benefit the early diagnosis and therapy of tumors. Here we report the cloning and functional characterization of a novel human bone marrow stromal cell (BMSC)-derived growth inhibitor (BDGI) by large scale random sequencing of a human BMSC cDNA library. Human BDGI cDNA encodes a 477-amino acid residue protein that shares high homology with rat and mouse pregnancy-induced growth inhibitors. The C-terminal of BDGI is identical to a novel human pregnancy-induced growth inhibitor, OKL38. BDGI is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved family of BDGI-like proteins. BDGI overexpression inhibits the proliferation, decreases anchorage-dependent growth, and reduces migration of MCF-7 human breast cancer cells, whereas down-regulation of BDGI expression promotes the proliferation of MCF-7 and HeLa cervix epitheloid carcinoma cells. Interestingly, the inhibitory effect of BDGI on MCF-7 cells is more potent than that of OKL38. We demonstrate that BDGI induces cell cycle arrest in S phase and subsequent apoptosis of MCF-7 cells, which is likely to account for the antiproliferative effects of BDGI. This process may involve up-regulation of p27Kip1 and down-regulation of cyclin A, Bcl-2, and Bcl-xL. The inhibitory effect of BDGI on cell proliferation and the induction of apoptosis were also observed in A549 lung cancer cells but not HeLa cells. These results indicate that BDGI might be a growth inhibitor for human tumor cells, especially breast cancer cells, possibly contributing to the development of new therapeutic strategies for breast cancer.

Inhibition of the Activity of Cyclophilin A Impedes Prolactin Receptor-Mediated Signaling, Mammary Tumorigenesis, and Metastases

Trefoil factor 1 (TFF1) is a potential prognostic biomarker with functional significance in breast cancers

To detect differential levels of FAM129A and CXCL14 in breast cancer samples, and to explore their influences on breast cancer proliferation. Differential levels of FAM129A and CXCL14 in breast cancer samples were examined by qRT-PCR. The correlation between FAM129A level and clinic pathological factors in breast cancer patients was analyzed. The regulatory effects of FAM129A and CXCL14 on proliferative potential in highly invasive breast cancer cell lines MCF-7 and SKBR-3 were assessed by CCK-8 and EdU assay. The interaction between FAM129A and CXCL14 was explored by bioinformatics analysis and Dual-Luciferase reporter assay. FAM129A was upregulated in breast cancer samples, and it was positively correlated to TNM staging in breast cancer patients. Knockdown of FAM129A markedly attenuated in vitro proliferative ability in breast cancer. CXCL14 was lowly expressed in breast cancer tissues and cell lines, which was able to inhibit breast cancer proliferation. FAM129A could bind CXCL14 and negatively regulate its level in breast cancer samples. Rescue experiments demonstrated that knockdown of CXCL14 could abolish the inhibited proliferative ability in breast cancer cells with FAM129A knockdown. FAM129A is upregulated in breast cancer samples with highly invasive potential, and it is linked to TNM staging. It aggravates the malignant proliferation of breast cancer cells by targeting and downregulating CXCL14.

Because activated estrogen (ER) and androgen (AR) receptors stimulate cell proliferation in breast and prostate cancer, inhibiting their actions represents a major therapeutic goal. Most efforts to modulate ER and AR activity have focused on inhibiting the synthesis of estrogens or androgens or on the identification of small molecules that act by competing with agonist hormones for binding in the ligand-binding pocket of the receptor. An alternative approach is to implement screens for small molecule inhibitors that target other sites in the pathway of steroid receptor action. Many of these second-site inhibitors directly target ER or AR; others have still unknown sites of action. Small molecule inhibitors that target second sites represent new leads with clinical potential; they serve as novel modulators of receptor action; and they can reveal new and as yet unidentified interactions and pathways that modulate ER and AR action.

Background: Breast cancer is one of the most common malignancies in women with high mortality rate worldwide. Clinical evidence suggests that antiestrogens have potential to inhibit the progression of hormone-dependent breast cancer. Here we showed that a novel selective estrogen receptor degrader (SERD), EC372, reduced estrogen receptor-α (ER-α), progesterone receptor (PR) and stabilized the expression of ER-β, which resulted in apoptosis in ER-positive breast cancer cells. Methods: We studied the effect of EC372 on DNA damage, cell cycle arrest, apoptosis, epithelial-to-mesenchymal transition (EMT), and mitochondrial membrane potential (MMP) in breast cancer cells. We also performed in vitro assays to evaluate the effect of EC372 on cell proliferation, colony formation, invasion, migration, cell adhesion in 2-dimensional (2-D) and sphere-forming abilities of tumor cells in 3-dimensional (3-D) cultures. Results: Our data show that EC372 inhibited the growth of T47D and MCF-7 human breast cancer cells. EC372 induced apoptosis in both cell lines as marked by cell shrinkage and apoptotic bodies. We found that treatment of EC372 downregulated the levels of ER-α and PR and stabilized the levels of ER-β, which subsequently resulted in cell cycle arrest, apoptosis, and suppressed the growth of breast cancer cells. We have also shown that EC372 reduced cell proliferation, colony formation, 3-D tumor spheroid size, migration, and invasive ability of breast cancer cells. Our cell cycle analysis data indicated that EC372 induced G1 arrest, as evidenced by a decrease in the protein levels of Cyclin D, phospho-Rb (S-807/811), and CDK-6 and increased Rb and p21 levels. We have further shown that EC372 induced apoptosis as evidenced by an increase in the levels of Annexin-V and by upregulation of proapoptotic molecules such as BAK, BAX, BID, cytochrome C, PARP1/cleaved PARP, and caspase 3, with a decrease in the levels of antiapoptotic molecules including BCl2, BCLxL, and survivin. In complement to our results, EC372 reduced the mitochondrial membrane potential (MMP) that triggered the activation of caspase 3 cascade, cleavage of PARP proteins, DNA damage, and subsequent cell death of breast cancer cells. Based on our in vitro results, EC372 is a unique and novel SERD, which inhibits tumor growth, migration, and invasion of breast cancer cells. Studies evaluating the potential of EC372 on in vivo tumor growth and metastasis are under way. Conclusions: This is the first comprehensive study on the mechanism by which EC372 inhibits tumor growth and promotes apoptosis of human breast cancer cells. Our collective data suggest that EC372 inhibits cell growth of ER-positive breast cancer cell lines through downregulating the levels of ER-α and PR, and stabilized the levels of ER-β. Based on our results, we suggest that EC372 is a potent SERD with anticancer effects that could be developed as a novel therapeutic agent to treat ER-positive breast cancer. Citation Format: Deepak Parashar, Anjali Geethadevi, Jyotsna Mishra, Bindu Nair, Bindu Santhamma, Klaus Nickisch, Pradeep Chaluvally-Raghavan. A novel selective estrogen receptor degrader, EC-372, inhibits tumor growth and metastasis of breast cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A195.

Targeting Prosurvival Bcl-2 Family Members Increases Radiation Sensitivity in Breast Cancer Cells

Aerobic glycolysis is a hallmark of tumor cells. SGLT1 plays a vital role in glucose metabolism. However, whether SGLT1 could promote cell growth and proliferation in breast cancer remains unclear. Here, we investigated the expression of SGLT1 in breast cancer and examined its role in malignant behavior and prognosis. Further, we examined the SGLT1 expression in breast cancer tissues and its relationship with clinicopathologic characteristics. We clarified that SGLT1 was overexpressed in HER2+ breast cancer cell lines and was affected by HER2 status. We further found that SGLT1 affected breast cancer cell proliferation and patient survival by mediating cell survival pathway activation. SGLT1 was overexpressed in HER2+ breast cancers and associated with lymph node metastasis and HER2+ status. Inhibition of HER2 decreased SGLT1 expression, and the extracellular acidification rate was also reduced in the UACC812 and SKBR3 cell lines. These changes could be reversed by proteasome inhibitor treatment. Knockdown of SGLT1 blocked PI3K/Akt/mTOR signaling, thereby inhibiting cell proliferation. Further, we demonstrated that high SGLT1 was significantly correlated with shorter survival in all breast cancer patients and specifically in HER2+ breast cancer patients. Therefore, we conclude that SGLT1 is overexpressed in HER2+ breast cancer, thereby promoting cell proliferation and shortening survival by activating PI3K/Akt/mTOR signaling. This study submits that SGLT1 is promising not only as a novel biomarker of HER2+ breast cancer subtype but also as a potential drug target.

Breast cancer is the leading cause of cancer-related death in women worldwide, and its developmental mechanisms involve complex factors. Recent studies have shown that ferroptosis is closely related to the occurrence and progression of breast cancer. However, the role of circular RNAs (circRNAs) in regulating ferroptosis in breast cancer remains unclear. In this study, we investigated the regulatory role of circSCD1 (hsa_circ_0019512) in breast cancer. We examined the expression of circSCD1 in breast cancer cell lines and explored its impact on cell viability and colony formation. We also evaluated the involvement of circSCD1 in ferroptosis by measuring the levels of malondialdehyde (MDA), glutathione (GSH), reactive oxygen species (ROS), and intracellular iron. In vivo xenograft experiments were performed to confirm the role of circSCD1 in promoting tumor growth and inhibiting ferroptosis.Furthermore, we investigated the mechanism by which circSCD1 regulates SCD1 protein stability through ubiquitination and identified the interaction between circSCD1 and the deubiquitinase OTUB1. Our results showed that circSCD1 was upregulated in breast cancer cell lines and promoted cell viability and colony formation. Knockdown of circSCD1 increased MDA and ROS levels, decreased GSH levels, and enhanced ferroptosis in breast cancer cells. In vivo, circSCD1 knockdown significantly reduced tumor size and weight, while its overexpression enhanced tumor growth. Mechanistically, circSCD1 interacted with OTUB1 to inhibit the ubiquitination and degradation of SCD1 protein, thereby stabilizing its expression. Rescue experiments demonstrated that SCD1 overexpression partially reversed the effects of circSCD1 knockdown on cell proliferation and ferroptosis. Our findings suggest that circSCD1 plays a crucial role in promoting breast cancer cell growth and inhibiting ferroptosis by regulating SCD1 protein stability. Targeting the circSCD1/OTUB1/SCD1 axis may provide a potential therapeutic strategy for breast cancer treatment.

BackgroundBreast cancer is the most common tumor in women worldwide. Diabetes mellitus is a global chronic metabolic disease with increasing incidence. Diabetes mellitus has been reported to positively regulate the development of many tumors. However, the specific mechanism of hyperglycemic environment regulating breast cancer remains unclear. PFKFB3 (6-phosphofructose-2-kinase/fructose-2, 6-bisphosphatase 3) is a key regulatory factor of the glycolysis process in diabetes mellitus, as well as a promoter of breast cancer. So, we want to explore the potential link between PFKFB3 and the poor prognosis of breast cancer patients with hyperglycemia in this study.MethodsCell culture was utilized to construct different-glucose breast cancer cell lines. Immunohistochemistry was adopted to analyze the protein level of PFKFB3 in benign breast tissues, invasive ductal carcinoma with diabetes and invasive ductal carcinoma without diabetes. The Kaplan–Meier plotter database and GEO database (GSE61304) was adopted to analyze the survival of breast cancer patients with different PFKFB3 expression. Western blot was adopted to analyze the protein level of PFKFB3, epithelial–mesenchymal transition (EMT)-related protein and extracellular regulated protein kinases (ERK) in breast cancer cells. Gene Set Cancer Analysis (GSCA) was utilized to investigate the potential downstream signaling pathways of PFKFB3. TargetScan and OncomiR were utilized to explore the potential mechanism of PFKFB3 overexpression by hyperglycemia. Transfections (including siRNAs and miRNA transfection premiers) was utilized to restrain or mimic the expression of the corresponding RNA. Cell functional assays (including cell counting, MTT, colony formation, wound-healing, and cell migration assays) were utilized to explore the proliferation and migration of breast cancer cells.ResultsIn this study, we demonstrated that the expression of PFKFB3 in breast cancer complicated with hyperglycemia was higher than that in breast cancer with euglycemia through cell experiment in vitro and histological experiment. PFKFB3 overexpression decreased the survival period of breast cancer patients and was correlated with a number of clinicopathological parameters of breast cancer complicated with diabetes. PFKFB3 promoted the proliferation and migration of breast cancer in a hyperglycemic environment and might be regulated by miR-26. In addition, PFKFB3 stimulated epithelial-mesenchymal transition of breast cancer in a hyperglycemic environment. In terms of downstream mechanism exploration, we predicted and verified the cancer-promoting effect of PFKFB3 in breast cancer complicated with hyperglycemia through RAS/MAPK pathway.ConclusionsIn conclusion, PFKFB3 could be overexpressed by hyperglycemia and might be a potential therapeutic target for breast cancer complicated with diabetes.

Targeting Mitochondrial Glutaminase Activity Inhibits Oncogenic Transformation

Estrogen, the female dominant hormone, has been found to play critical roles in cancers for over 100 years. In 1896, estrogen was found to promote the tumor growth of breast cancer; and in 1937, estrogen was found to prevent the tumor growth of liver cancer. After the estrogen receptor alpha (ERα) was identified in 1958, ERα-mediated estrogen signaling was found to promote or prevent the growth of breast cancer cells or liver cancer cells, respectively. Recent studies showed that ERα-mediated estrogen signaling in promoting the growth of breast cancer cells was dependent of forkhead box protein A1 (FOXA1). Our recent study showed that ERα-mediated estrogen signaling in preventing the growth of liver cancer cells also depended on FOXA factors (FOXA1 and FOXA2). Thus, FOXA-dependent ERα-mediated estrogen signaling plays opposite roles in the growth of breast cancer and liver cancer cells. Here, we applied genomic approaches to identify 184 FOXA/ER dual target genes that showed opposite expression in response to estrogen-mediated stimulation or suppression of cell growth in breast or liver cancer cells. Gene ontology analysis showed that the majority of these FOXA/ER dual target genes were involved in the processes of cell proliferation and growth, cell death, tissue development, and cancer. Manipulations of the expression of these target genes were able to reverse the growth of breast and liver cancer cells. Thus, these 184 FOXA/ER dual target genes provide us a novel set of potential biomarkers and therapeutic targets for both breast cancer and liver cancer. Note: This abstract was not presented at the conference. Citation Format: Zhaoyu Li. Comparative genomics study of FOXA/ER dual regulation in breast cancer and liver cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-08.

Breast cancer is the most common and the second leading cause of death in women worldwide while ovarian cancer is the leading cause of gynecological cancers. Breast and ovarian cancer cells have increased proliferation, invasion/metastasis and resistance to programmed cell death due to mutations, altered signaling pathways and deregulated of control mechanism. One of the differences between normal and cancer cells is the changes in the regulation and the function of ion channels. Ion channels are important signaling molecules expressed in a wide range of tissues where they have significant involvement in determining a variety of cellular functions: solute transport, volume control, enzyme activity, secretion, invasion, gene expression, excitation-contraction coupling and intercellular communication. During the malignant transformation, a series of genetic alterations occur, which may also affect the expression and activity of ion channels. This abnormal ion channel activity has been hypothesized to support cell proliferation and tumor growth. Voltage-gated sodium channels (VGNaC) are classically described as critical elements of action potential initiation and propagation in excitable cells. Recent studies showed that VGNaC expression was significantly up-regulated in metastatic human breast cancer cells and tissues, and VGNaC activity potentiated cellular directional motility, endocytosis and invasion. However, the mechanisms of the VGNaC and its functional relevance to breast cancer proliferation are currently not well-known. In this study, we investigated that the effects of downregulation NaV1.5 channel expression by specific RNA interference (ie siRNA) in breast and ovarian cancer cells in vitro. In our work, we first determined the effects of Adult NaV1.5 siRNA and analyzed the effects of Adult and Neonatal (neonatal splice variant of NaV1.5) NaV1.5 silencing by siRNA in BCa cells by Real-Time PCR. Our Real-Time PCR results showed that Adult NaV1.5 channel siRNA reduced the level of NaV1.5 mRNA for MDA-MB-231 BCa cells. We found that knockdown of NaV1.5 channel by siRNA significantly inhibited MDA-MB-231 breast and SKOV3-IP1, SKOV3-TR and HEYA8 ovarian cancer cell proliferation and colony formation. We are currently investigating downstream cellular pathways involved in NaV1.5 mediated proliferation and other cellular functions. In conclusion, our results demonstrate that targeting of NaV1.5 in breast and ovarian cancer cells decreases cell proliferation and colony formation and it may be used as a new therapeutic target in these cancers. Citation Format: Mumin A. Erdogan, Bulent Ozpolat. Targeting of Voltage-gated sodium channel NaV1.5 inhibits cell proliferation and colony formation in breast and ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 514. doi:10.1158/1538-7445.AM2013-514

Aberrant activity of the phosphatidylinositol 3-kinase (PI3K) pathway supports growth of many tumors including those of breast, lung, and prostate. Resistance of breast cancer cells to targeted chemotherapies including tyrosine kinase inhibitors (TKI) has been linked to persistent PI3K activity, which may in part be due to increased membrane expression of epidermal growth factor (EGF) receptors (HER2 and HER3). Recently we found that proteins of the RGS (regulator of G protein signaling) family suppress PI3K activity downstream of the receptor by sequestering its p85alpha subunit from signaling complexes. Because a substantial percentage of breast tumors have RGS16 mutations and reduced RGS16 protein expression, we investigated the link between regulation of PI3K activity by RGS16 and breast cancer cell growth. RGS16 overexpression in MCF7 breast cancer cells inhibited EGF-induced proliferation and Akt phosphorylation, whereas shRNA-mediated extinction of RGS16 augmented cell growth and resistance to TKI treatment. Exposure to TKI also reduced RGS16 expression in MCF7 and BT474 cell lines. RGS16 bound the amino-terminal SH2 and inter-SH2 domains of p85alpha and inhibited its interaction with the EGF receptor-associated adapter protein Gab1. These results suggest that the loss of RGS16 in some breast tumors enhances PI3K signaling elicited by growth factors and thereby promotes proliferation and TKI evasion downstream of HER activation.

Our previous study reported several alternative splicing variants of arginine N-methyltransferase 2(PRMT2), which lose different exons in the C-terminals of the wild-type PRMT2 gene. Particularly, due to frame-shifting, PRMT2β encodes a novel amino acid sequence at the C-terminus of the protein, the function of which is not understood. In the present study, we determined the role of PRMT2β in breast cancer cell proliferation, apoptosis and its effect on the Akt signaling pathway. Stable breast cancer MCF7 cell line with lentivirus-mediated PRMT2β overexpression was obtained after selection by puromycin for 2weeks. The effect of lentivirus-mediated PRMT2β overexpression on breast cancer cellular oncogenic properties was evaluated by MTT, colony formation, cell cycle analysis and apoptosis assays in MCF7 cells. Luciferase activity assay and western blot analysis were performed to characterize the effects of PRMT2β on cyclinD1 promoter activities and the Akt signaling pathway. Tissue microarray was performed to investigate the association of PRMT2β with breast cancer progression. Lentivirus-mediated PRMT2β overexpression suppressed the cell proliferation and colony formation of breast cancer MCF7 cells. PRMT2β overexpression induced cell cycle arrest and apoptosis of MCF7 cells. Furthermore, PRMT2β was revealed to suppress the transcription activity of the cyclinD1 promoter, and PRMT2β was also found to inhibit cyclinD1 expression via the suppression of Akt/GSK-3β signaling in breast cancer cells. Clinically, it was revealed that PRMT2β expression was negatively correlated with human epidermal growth factor receptor2 (HER2) (p=0.033) in breast tumors. Our results revealed that PRMT2β, a novel splice variant of PRMT2, plays potential antitumor effect by suppressing cyclinD1 expression and inhibiting Akt signaling activity. This also opens a new avenue for treating breast cancer.