Xiaohe Yang

p53 inactivation upregulates p73 expression through E2F-1 mediated transcription.

While p73 overexpression has been associated with increased apoptosis in cancer tissues, p73 overexpressing tumors appear to be of high grade malignancy. Why this putative tumor suppressor is overexpressed in cancer cells and what the function of overexpressed p73 is in breast cancers are critical questions to be addressed. By investigating the effect of p53 inactivation on p73 expression, we found that both protein and mRNA levels of TAp73 were increased in MCF-7/p53siRNA cells, MCF-7/p53mt135 cells and HCT-116/p53−/− cells, as compared to wild type control, suggesting that p53 inactivation by various forms upregulates p73. We showed that p53 knockdown induced p73 was mainly regulated at the transcriptional level. However, although p53 has a putative binding site in the TAp73 promoter, deletion of this binding site did not affect p53 knockdown mediated activation of TAp73 promoter. Chromatin immuno-precipitation (ChIP) data demonstrated that loss of p53 results in enhanced occupancy of E2F-1 in the TAp73 promoter. The responsive sequence of p53 inactivation mediated p73 upregulation was mapped to the proximal promoter region of the TAp73 gene. To test the role of E2F-1 in p53 inactivation mediated regulation of p73 transcription, we found that p53 knockdown enhanced E2F-1 dependent p73 transcription, and mutations in E2F-1 binding sites in the TAp73 promoter abrogated p53 knockdown mediated activation of TAp73 promoter. Moreover, we demonstrated that p21 is a mediator of p53-E2F crosstalk in the regulation of p73 transcription. We concluded that p53 knockdown/inactivation may upregulate TAp73 expression through E2F-1 mediated transcriptional regulation. p53 inactivation mediated upregulation of p73 suggests an intrinsic rescuing mechanism in response to p53 mutation/inactivation. These findings support further analysis of the correlation between p53 status and p73 expression and its prognostic/predictive significance in human cancers.

Alcohol promotes migration and invasion of triple-negative breast cancer cells through activation of p38 MAPK and JNK.

Although alcohol is an established breast cancer risk factor, the underlying mechanisms remain unclear. Previous studies examined the general association between alcohol consumption and breast cancer risk; however, the risk for different breast cancer subtypes has been rarely reported. Triple‐negative breast cancer (TNBC) is a subtype of breast cancer lacking hormone receptors and HER2 expression, and having poor prognosis. Understanding the molecular mechanisms of TNBC etiology remains a significant challenge. In this study, we investigated cellular responses to alcohol in two TNBC cell lines, MDA‐MB‐231 and MDA‐MB‐468. Our results showed that alcohol at low concentrations (0.025–0.1% v/v) induced cell proliferation, migration, and invasion in 1% FBS‐containing medium. Molecular analysis indicated that these phenotypic changes were associated with alcohol‐induced reactive oxygen species production and increased p38 and JNK phosphorylation. Likewise, p38 or JNK inhibition attenuated alcohol‐induced cell migration and invasion. We revealed that alcohol treatment activated/phosphorylated NF‐κB regulators and increased transcription of NF‐κB‐targeted genes. While examining the role of acetaldehyde, the major alcohol metabolite, in alcohol‐associated responses in TNBC cells, we saw that acetaldehyde induced cell migration, invasion, and increased phospho‐p38, phospho‐JNK, and phospho‐IκBα in a pattern similar to alcohol treatment. Taken together, we established that alcohol promotes TNBC cell proliferation, migration, and invasion in vitro. The underlying mechanisms involve the induction of oxidative stress and the activation of NF‐κB signaling. In particular, the activation of p38 and JNK plays a pivotal role in alcohol‐induced cellular responses. These results will advance our understanding of alcohol‐mediated development and promotion of TNBC.

Metformin as an anti-cancer agent: actions and mechanisms targeting cancer stem cells

Metformin, a first line medication for type II diabetes, initially entered the spotlight as a promising anti-cancer agent due to epidemiologic reports that found reduced cancer risk and improved clinical outcomes in diabetic patients taking metformin. To uncover the anti-cancer mechanisms of metformin, preclinical studies determined that metformin impairs cellular metabolism and suppresses oncogenic signaling pathways, including receptor tyrosine kinase, PI3K/Akt, and mTOR pathways. Recently, the anti-cancer potential of metformin has gained increasing interest due to its inhibitory effects on cancer stem cells (CSCs), which are associated with tumor metastasis, drug resistance, and relapse. Studies using various cancer models, including breast, pancreatic, prostate, and colon, have demonstrated the potency of metformin in attenuating CSCs through the targeting of specific pathways involved in cell differentiation, renewal, metastasis, and metabolism. In this review, we provide a comprehensive overview of the anti-cancer actions and mechanisms of metformin, including the regulation of CSCs and related pathways. We also discuss the potential anti-cancer applications of metformin as mono- or combination therapies.

Short-term early exposure to lapatinib confers lifelong protection from mammary tumor development in MMTV-erbB-2 transgenic mice

BackgroundAlthough chemopreventative agents targeting the estrogen/estrogen receptor (ER) pathway have been effective for ER+ breast cancers, prevention of hormone receptor-negative breast cancers, such as Her2/erbB-2+ breast cancers, remains a significant issue. Previous studies have demonstrated that administration of EGFR/erbB-2-targeting lapatinib to MMTV-erbB-2 transgenic mice inhibited mammary tumor development. The prevention, however, was achieved by prolonged high dose exposure. The tolerance to high dose/long-term drug administration may hinder its potential in clinical settings. Therefore, we aimed to test a novel, short-term chemopreventative strategy using lapatinib during the premalignant risk window in MMTV-erbB-2 mice.MethodsWe initially treated cultured cells with lapatinib to explore the anti-proliferative effects of lapatinib in vitro. We used a syngeneic tumor graft model to begin exploring the in vivo anti-tumorigenic effects of lapatinib in MMTV-erbB-2 mice. Then, we tested the efficacy of brief exposure to lapatinib (100xa0mg/kg/day for 8xa0weeks), beginning at 16xa0weeks of age, in the prevention of mammary tumor development in MMTV-erbB-2 mice.ResultsIn the syngeneic tumor transplant model, we determined that lapatinib significantly inhibited tumor cell proliferation. Furthermore, we demonstrated that short-term lapatinib exposure resulted in life-long protective effects, as supported by increased tumor latency in lapatinib-treated mice compared to the control mice. We further established that delayed tumor development in the treated mice was preceded by decreased BrdU nuclear incorporation and inhibited mammary morphogenesis. Molecular analysis indicated that lapatinib inhibited phosphorylation and expression of EGFR, erbB-3, erbB-2, Akt1, and Erk1/2 in premalignant mammary tissues. Also, lapatinib drastically inhibited the phosphorylation and expression of ERα and the transcription of ER target genes in premalignant mammary tissues. We also determined that lapatinib suppressed the stemness of breast cancer cell lines, as evidenced by decreased tumorsphere formation and ALDH+ cell populations.ConclusionsTaken together, these data demonstrate that brief treatment with EGFR/erbB-2-targeting agents before the onset of tumors may provide lifelong protection from mammary tumors, through the concurrent inhibition of erbB-2 and ER signaling pathways and consequential reprogramming. Our findings support further clinical testing to explore the benefit of shorter lapatinib exposure in the prevention of erbB-2-mediated carcinogenesis.

FGFR inhibitor, AZD4547, impedes the stemness of mammary epithelial cells in the premalignant tissues of MMTV-ErbB2 transgenic mice

The fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases (RTKs) regulates signaling pathways involved in cell proliferation and differentiation. Currently, the anti-tumor properties of FGFR inhibitors are being tested in preclinical and clinical studies. Nevertheless, reports on FGFR inhibitor-mediated breast cancer prevention are sparse. In this study, we investigated the anti-cancer benefits of AZD4547, an FGFR1-3 inhibitor, in ErbB2-overexpressing breast cancer models. AZD4547 (1–5u2009µM) demonstrated potent anti-proliferative effects, inhibition of stemness, and suppression of FGFR/RTK signaling in ErbB2-overexpressing human breast cancer cells. To study the in vivo effects of AZD4547 on mammary development, mammary epithelial cell (MEC) populations, and oncogenic signaling, MMTV-ErbB2 transgenic mice were administered AZD4547 (2–6u2009mg/kg/day) for 10 weeks during the ‘risk window’ for mammary tumor development. AZD4547 significantly inhibited ductal branching and MEC proliferation in vivo, which corroborated the in vitro anti-proliferative properties. AZD4547 also depleted CD24/CD49f-sorted MEC populations, as well as the CD61highCD49fhigh tumor-initiating cell-enriched population. Importantly, AZD4547 impaired stem cell-like characteristics in primary MECs and spontaneous tumor cells. Moreover, AZD4547 downregulated RTK, mTOR, and Wnt/β-catenin signaling pathways in premalignant mammary tissues. Collectively, our data provide critical preclinical evidence for AZD4547 as a potential breast cancer preventative and therapeutic agent.

Buformin inhibits the stemness of erbB-2-overexpressing breast cancer cells and premalignant mammary tissues of MMTV-erbB-2 transgenic mice

BackgroundMetformin, an FDA-approved drug for the treatment of Type II diabetes, has emerged as a promising anti-cancer agent. Other biguanide analogs, including buformin and phenformin, are suggested to have similar properties. Although buformin was shown to reduce mammary tumor burden in carcinogen models, the anti-cancer effects of buformin on different breast cancer subtypes and the underlying mechanisms remain unclear. Therefore, we aimed to investigate the effects of buformin on erbB-2-overexpressing breast cancer with in vitro and in vivo models.MethodsMTT, cell cycle, clonogenic/CFC, ALDEFLUOR, tumorsphere, and Western blot analyses were used to determine the effects of buformin on cell growth, stem cell populations, stem cell-like properties, and signaling pathways in SKBR3 and BT474 erbB-2-overexpressing breast cancer cell lines. A syngeneic tumor cell transplantation model inoculating MMTV-erbB-2 mice with 78617 mouse mammary tumor cells was used to study the effects of buformin (1.2xa0g buformin/kg chow) on tumor growth in vivo. MMTV-erbB-2 mice were also fed buformin for 10xa0weeks, followed by analysis of premalignant mammary tissues for changes in morphological development, mammary epithelial cell (MEC) populations, and signaling pathways.ResultsBuformin significantly inhibited SKBR3 and BT474 cell growth, and in vivo activity was demonstrated by considerable growth inhibition of syngeneic tumors derived from MMTV-erbB-2 mice. In particular, buformin suppressed stem cell populations and self-renewal in vitro, which was associated with inhibited receptor tyrosine kinase (RTK) and mTOR signaling. Consistent with in vitro data, buformin suppressed mammary morphogenesis and reduced cell proliferation in MMTV-erbB-2 mice. Importantly, buformin decreased MEC populations enriched with mammary reconstitution units (MRUs) and tumor-initiating cells (TICs) from MMTV-erbB-2 mice, as supported by impaired clonogenic and mammosphere formation in primary MECs. We further demonstrated that buformin-mediated in vivo inhibition of MEC stemness is associated with suppressed activation of mTOR, RTK, ER, and β-catenin signaling pathways.ConclusionsOverall, our results provide evidence for buformin as an effective anti-cancer drug that selectively targets TICs, and present a novel prevention and/or treatment strategy for patients who are genetically predisposed to erbB-2-overexpressing breast cancer.

Ganetespib induces G2/M cell cycle arrest and apoptosis in gastric cancer cells through targeting of receptor tyrosine kinase signaling

Heat shock protein 90 (HSP90) regulates several important cellular processes via its repertoire of client proteins. These client proteins have been found to play fundamental roles in signal transduction, cell proliferation, cell cycle progression and survival, as well as other features of malignant cells, such as invasion, tumor angiogenesis and metastasis. Thus, HSP90 is an emerging target for cancer therapy. To this end, we evaluated ganetespib (STA-9090), a novel and potent HSP90 inhibitor, for its activity in gastric cancer cell lines. Ganetespib significantly inhibited the proliferation of AGS and N87 human gastric cancer cell lines and potently induced G2/M cell cycle arrest and apoptosis. Upregulation of cleaved poly(ADP-ribose) polymerase (c-PARP), c-caspase-3, c-caspase-8 and c-caspase-9 and suppression of gastric cancer-associated HSP90 client proteins, including ErbB2, Erk, Akt, mTOR, GSK3 and Src, were observed in ganetespib-treated cells. These findings demonstrate that the ganetespib-induced mechanism of cell growth inhibition involves the activation of death receptor and mitochondrial pathways and the inhibition of receptor tyrosine kinase signaling pathways. Our study implicates ganetespib as a potential strategy for gastric cancer treatment, which warrants further preclinical and clinical investigation.

Ganetespib targets multiple levels of the receptor tyrosine kinase signaling cascade and preferentially inhibits ErbB2-overexpressing breast cancer cells

Although ErbB2-targeted therapeutics have significantly improved ErbB2+ breast cancer patient outcomes, therapeutic resistance remains a significant challenge. Therefore, the development of novel ErbB2-targeting strategies is necessary. Importantly, ErbB2 is a sensitive client protein of heat shock protein 90 (HSP90), which regulates client protein folding, maturation, and stabilization. HSP90 inhibition provides an alternative therapeutic strategy for ErbB2-targeted degradation. In particular, ganetespib, a novel HSP90 inhibitor, is a promising agent for ErbB2+ cancers. Nevertheless, the anti-cancer efficacy and clinical application of ganetespib for ErbB2+ breast cancer is largely unknown. In our study, we examined the anti-cancer effects of ganetespib on ErbB2+ BT474 and SKBR3 breast cancer cells, and isogenic paired cancer cell lines with lentivirus-mediated ErbB2 overexpression. Ganetespib potently inhibited cell proliferation, cell cycle progression, survival, and activation/phosphorylation of ErbB2 and key downstream effectors in ErbB2+ breast cancer cells. Moreover, ganetespib decreased the total protein levels of HSP90 client proteins and reduced ErbB2 protein half-life. ErbB2-overexpressing cancer cells were also more sensitive to ganetespib-mediated growth inhibition than parental cells. Ganetespib also strikingly potentiated the inhibitory effects of lapatinib in BT474 and SKBR3 cells. Ultimately, our results support the application of ganetespib-mediated HSP90 inhibition as a promising therapeutic strategy for ErbB2+ breast cancer.

microRNA Regulation in Estrogen Receptor-Positive Breast Cancer and Endocrine Therapy

As de novo and acquired resistance to standard first line endocrine therapies is a growing clinical challenge for estrogen receptor-positive (ER+) breast cancer patients, understanding the mechanisms of resistance is critical to develop novel therapeutic strategies to prevent therapeutic resistance and improve patient outcomes. The widespread post-transcriptional regulatory role that microRNAs (miRNAs) can have on various oncogenic pathways has been well-documented. In particular, several miRNAs are reported to suppress ERα expression via direct binding with the 3’ UTR of ESR1 mRNA, which can confer resistance to estrogen/ERα-targeted therapies. In turn, estrogen/ERα activation can modulate miRNA expression, which may contribute to ER+ breast carcinogenesis. Given the reported oncogenic and tumor suppressor functions of miRNAs in ER+ breast cancer, the targeted regulation of specific miRNAs is emerging as a promising strategy to treat ER+ breast cancer and significantly improve patient responsiveness to endocrine therapies. In this review, we highlight the major miRNA-ER regulatory mechanisms in context with ER+ breast carcinogenesis, as well as the critical miRNAs that contribute to endocrine therapy resistance or sensitivity. Collectively, this comprehensive review of the current literature sheds light on the clinical applications and challenges associated with miRNA regulatory mechanisms and novel miRNA targets that may have translational value as potential therapeutics for the treatment of ER+ breast cancer.

Abstract 3557: Cancerous inhibitor of PP2A is a novel molecular target and resistance factor of Lapatinib

Deregulation of erbB-2 and EGFR is frequently detected in breast cancer, which has been associated with poor prognosis. Lapatinib, an orally active small molecule acting as a dual inhibitor of erbB-2 and EGFR, has been used for the treatment of patients with advanced or metastatic erbB-2+ breast cancer. Lapatinib resistance, however, is emerging as a clinical challenge. Understanding the molecular mechanisms of Lapatinib-mediated anti-cancer activities and identifying relevant resistance factors are of pivotal significance in breast cancer treatment. Cancerous inhibitor of PP2A (CIP2A) is a recently identified oncoprotein that is overexpressed in breast and other types of cancers. Through interacting with protein phosphatase 2A (PP2A), CIP2A regulates c-Myc and factors involved in cell proliferation and survival. This study aimed to investigate the role of CIP2A in Lapatinib-mediated inhibition of erbB-2 overexpressing breast cancer cells. Our data showed that Lapatinib downregulated CIP2A in erbB-2 overexpressing SK-BR-3 and 78617 cells, which was correlated with concurrent inactivation of erbB-2, EGFR, Akt, Erk1/2 and mTOR. Overexpression of CIP2A rendered the cells resistant to Lapatinib induced apoptosis and growth inhibition. Conversely, CIP2A knockdown via siRNA sensitized the cells to Lapatinib-mediated effects. We also demonstrated that Lapatinib-induced downregulation of CIP2A can be abolished by LY294002, suggesting that CIP2A downregulation was regulated by Akt. Analysis with cycloheximide (CHX) and MG132 indicated that Lapatinib modulates CIP2A protein stability and promotes its degradation through prosteasome pathway. More importantly, we found that Lapatinib induced differential downregulation of CIP2A between the parental and Lapatinib-resistant (LR) BT-474 cells. Decrease in CIP2A downregulation in BT-474-LR cells suggests its association with Lapatinib resistance. Knockdown of CIP2A via lentiviral siRNA system significantly sensitized Lapatinib induced growth inhibition and apoptosis. Taken together, our results demonstrate that CIP2A is a molecular target of Lapatinib, which plays a critical role in Lapatinib-induced cellular responses. Lapatinib-induced downregulation of CIP2A involves the inhibition of the Akt-CIP2A feedback loop. Correlation between Lapatinib resistance and decreased CIP2A downregulation, as well as CIP2A knockdown-mediated sensitization of BT-474-LR cells indicate that CIP2A is a resistance factor of Lapatinib. Further investigation on Lapatinib-mediated regulation of CIP2A will advance our understanding of Lapatinib-associated antitumor activities and drug resistance. Citation Format: Ming Zhao, Amanda Blackwelder, Harry Lee, Xiaohe Yang. Cancerous inhibitor of PP2A is a novel molecular target and resistance factor of Lapatinib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3557. doi:10.1158/1538-7445.AM2015-3557