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Perspective on This Article from Metformin Prevents Liver Tumorigenesis by Inhibiting Pathways Driving Hepatic Lipogenesis

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Tumors and Metastasis in KRas+/LA1;Dicer+/2SD mice

Dicer1 functions as a tumor suppressor in mouse models. In humans, somatic mutations are associated with many cancers in adults, and patients with DICER1 syndrome with DICER1 germline mutations are susceptible to childhood cancers. Dicer is phosphorylated by the ERK-MAP kinase pathway and because this pathway is activated in human cancers, we asked whether phosphorylated Dicer1 contributed to tumor development. In human endometrioid cancers, we discovered that phosphorylated DICER1 is significantly associated with invasive disease. To test a direct involvement of Dicer1 phosphorylation in tumor development, we studied mice with phosphomimetic alterations at the two conserved serines phosphorylated by ERK and discovered that a phosphomimetic Dicer1 drives tumor development and dissemination in two independent murine cancer models (KRas+/LA1 and p53+/−). Our findings demonstrate that phosphomimetic Dicer1 promotes tumor development and invasion.Significance:This work highlights the relevance of Dicer1 phosphorylation in mammalian tumor development and dissemination.

PDF file - 131KB, Supplemental Figure 1 Withaferin A inhibits cell viability of breast cancer cells.

Supplementary Figure Legends 1-2 from Bidirectional Crosstalk between Leptin and Insulin-like Growth Factor-I Signaling Promotes Invasion and Migration of Breast Cancer Cells via Transactivation of Epidermal Growth Factor Receptor

Supplementary Figure 1 from Bidirectional Crosstalk between Leptin and Insulin-like Growth Factor-I Signaling Promotes Invasion and Migration of Breast Cancer Cells via Transactivation of Epidermal Growth Factor Receptor

PDF file - 141KB, Supplemental Figure 6 ERK and RSK activation is important for WFA-mediated inhibition of clonogenicity of breast cancer cells.

Withaferin A (WFA) is a steroidal lactone with antitumor effects manifested at multiple levels that are mechanistically obscure. Using a phospho-kinase screening array, we discovered that WFA activated phosphorylation of the S6 kinase RSK (ribosomal S6 kinase) in breast cancer cells. Pursuing this observation, we defined activation of extracellular signal–regulated kinase (ERK)–RSK and ETS-like transcription factor 1 (Elk1)–CHOP (C-EBP homologous protein) kinase pathways in upregulating transcription of the death receptor 5 (DR5). Through this route, WFA acted as an effective DR5 activator capable of potentiating the biologic effects of celecoxib, etoposide, and TRAIL. Accordingly, WFA treatment inhibited breast tumor formation in xenograft and mouse mammary tumor virus (MMTV)-neu mouse models in a manner associated with activation of the ERK/RSK axis, DR5 upregulation, and elevated nuclear accumulation of Elk1 and CHOP. Together, our results offer mechanistic insight into how WFA inhibits breast tumor growth. Cancer Res; 74(9); 2617–29. ©2014 AACR.

Supplementary Figure 2 from Bidirectional Crosstalk between Leptin and Insulin-like Growth Factor-I Signaling Promotes Invasion and Migration of Breast Cancer Cells via Transactivation of Epidermal Growth Factor Receptor

PDF file - 99KB, Supplementary Figure 8 CHOP is important for Withaferin A mediated inhibition of breast cancer cell growth.

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PDF file - 115KB, Supplemental Figure 3 WFA increases phosphorylation of ERK and RSK.

PDF file - 147KB, Supplemental Figure 4 WFA activates RSK in an ERK-dependent manner.

PDF file - 126KB, Supplemental Figure 7 Withaferin A induces phosphorylation of Elk1 in ERK-dependent manner.

PDF file - 156KB, Supplementary Figure 9 WFA increases DR5 expression in multiple breast cancer cell lines and CHOP and Elk1 contribute to WFA-induced upregulation of Death receptor 5 in breast cancer cells.

PDF file - 112KB, Supplemental Figure 5 WFA activates RSK in an ERK-dependent manner. ERK and RSK activation is important for WFA-mediated cleavage of apoptotic marker, PARP.

PDF file - 89KB, Supplemental Figure 2 Withaferin A treatment inhibits expression of anti-apoptotic proteins, Survivin and XIAP and increases PARP cleavage.

Background and Aim: Obese state is associated with increased breast cancer growth, metastasis, and poor overall survival. In this study, we seek to decipher the underlying molecular mechanisms by which obesity/hyperleptinemia reduces the efficacy of tamoxifen. Methods: The impact of obesity on tamoxifen was evaluated utilizing clonogenicity, high-fat-diet- induced obese mice and leptin-treated xenograft-models. Mechanistic studies involved immunoblotting, real-time PCR, immunocytochemistry, chromatin immunoprecipitation assay, phosphokinase array and in silico analysis. Results: Obese mice with hyperleptinemia exhibit increased tumor progression and respond poorly to tamoxifen compared to non-obese mice. Exogenous leptin abrogates tamoxifen-mediated growth inhibition and potentiates breast tumor growth even in the presence of tamoxifen. Mechanistically, leptin induces nuclear translocation of phosphorylated-ER and increases the expression of ER-responsive genes while reducing tamoxifen-mediated gene repression by abrogating tamoxifen-induced recruitment of corepressors NCoR, SMRT and Mi2. Further, we found that coactivator Med1 potentially associates with 48 (out of 75) obesity-signature genes. Interestingly, leptin upregulates Med1 expression by decreasing miR-205 and increases its functional activation via phosphorylation that is mediated by activation of Her2 and EGFR. It is important to note that Med1 silencing abrogates the negative effects of leptin on tamoxifen efficacy. Additionally, honokiol or adiponectin treatment effectively inhibits leptin-induced Med1 expression and improve tamoxifen efficacy in hyperleptinemic state.Conclusion: In conclusion, these studies show the molecular mechanisms by which obese/hyperleptinemic state may contribute to poor response to tamoxifen implicating leptin-miR205-Med1 and leptin-Her2-EGFR-Med1 axes and present bioactive compound honokiol and adipocytokine adiponectin as agents that can block leptin’s negative effect on tamoxifen. Citation Format: Arumugam Nagalingam, Nethaji Muniraj, Sumit Siddharth, Dimiter Avtanski, Sheetal Parida, Pajamurthy Kuppusamy, Neeraj K Saxena, Nethaji Muniraj. Hyperleptinemia in obese state renders luminal breast cancers refractory to tamoxifen coordinating a crosstalk between Med1, miR205 and Erb B kinases [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-32.

Background and Aim: Obese state is associated with increased breast cancer growth, metastatic progression, poor overall survival and poor response to therapy. Analyses of clinical trial population showed that obesity associates with poor outcomes in patients with estrogen receptor (ER)-positive breast cancer treated with standard chemohormonal therapy. A characteristic feature of obesity is hyperleptinemia (high leptin levels) which mediates the biological effects of obesity. In this study, we seek to decipher the underlying molecular mechanisms by which obesity/hyperleptinemia reduces the efficacy of tamoxifen. Results: Obese mice models exhibiting hyperleptinemia supported larger tumors and responded poorly to tamoxifen therapy. Kaplan meier analyses showed that high leptin and high leptin receptor levels associates with poor prognosis in ER positive breast cancer. Indeed, leptin treatment abrogated tamoxifen-mediated inhibition of clonogenicity, and soft-agar colony formation. Leptin treatment increased breast tumor growth and significantly reduced the efficacy of tamoxifen. At the molecular level, leptin induced nuclear translocation of pER (ser 118 and ser 167) and increased the expression of ER-responsive genes while reducing tamoxifen-mediated gene repression. Tamoxifen is known to recruit corepressors to ER-responsive genes. ChIP analysis revealed that tamoxifen-induced recruitment of NCoR, SMRT and Mi2 corepressors was abrogated in the presence of leptin. We found that Med1, anchor subunit of human mediator complex, interacts with various key effector molecules of leptin-signaling network and potentially associates with 48 (out of 75) obesity-signature genes indicating its functional importance. Further, we found that leptin upregulated Med1 via decreasing miR-205 and increased its functional activation via phosphorylation, nuclear translocation and promoter-recruitment. Med1 phosphorylation was mediated by activation of Her2 and EGFR in response to leptin. It is important to note that silencing of Med1 abrogated the negative effects of leptin on tamoxifen efficacy. Bioactive strategies using honokiol or adiponectin treatment effectively inhibited leptin-induced Med1 expression and increased the efficacy of tamoxifen even in the presence of leptin. Conclusion: In conclusion, these studies show the molecular mechanisms by which obese state may contribute to poor response to endocrine therapy. We show that activation of Med1 via leptin-induced EGFR and Her2 kinases act as the key node mediating the negative effects of leptin on tamoxifen efficacy. A combination regimen of honokiol and adiponectin with tamoxifen may increase its efficacy in obese state. Citation Format: Arumugam Nagalingam, Nethaji Muniraj, Sumit Siddharth, Dimiter. B Avtanski, Sheetal Parida, Panjamurthy Kuppusamy, Balázs Győrffy, Neeraj Saxena, Dipali Sharma. Hyperleptinemia in obese state renders luminal breast cancers refractory to tamoxifen coordinating a crosstalk between Med1, miR205 and Erb B kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 310.

Background and Aim: Honokiol, a natural phenolic compound isolated from an extract of seed cones from Magnolia grandiflora, is widely known for its therapeutic potential as an antioxidant, anti-inflammatory, antithrombosis, and anti-depressant agent. Our recent studies show that honokiol impedes breast carcinogenesis. Cancer cells undergo cytoprotective autophagy and evade chemotherapy therefore many clinical trials are investigating the efficacy of autophagy inhibition in combination with chemotherapy. In the present study, we investigated the involvement of autophagic process in honokiol-mediated functional networks in breast cancer and explored the efficacy of combination regimens involving honokiol and chloroquine. Methods: Autophagy studies were conducted utilizing immunoblot, RT-PCR, immunofluorescence analyses, confocal imaging and transmission electron microscopy for autophagy markers. Autophagic flux was analyzed using a plasmid tfLC3B and acridine orange staining. The fusion of autophagosome and lysosome was examined by using GFP-LC3/LysoTracker-red. Functional impact of autophagic process was evaluated using genetic knockout (KO) of AGT7 and BECN1 in MCF7 cells as well as combined treatment with autophagy inhibitors (3-MA, Baf-1 and CQ) and honokiol. Alterations in ATP levels were measured by ATPliteTM luminescence assay. In vivo studies using mammary gland implantation of cancer cells in NOD-SCID mice were conducted to evaluate the efficacy of combination regimen of Honokiol and Chloroquine. Results: We found that Honokiol induces autophagy flux, increases accumulation of autophagosomes and elevates LC3B-II-conversion. Utilizing tandem-mCherry-GFP-LC3B assay and LysoTracker Red-staining, we observed that honokiol increases the autophagosome/lysosome fusion in breast cancer cells. We found that Honokiol induces autophagic response in a STK11-dependent manner as STK11-null breast cancer cells do not exhibit LC3B-II-puncta in response to Honokiol. Next, we explored the functional impact of autophagy in Honokiol mediated breast cancer inhibition and found that inhibiting autophagosome formation, abrogating autophagosome-lysosome fusion or genetic-knockout of BECN1/Beclin1 and ATG7 effectively increases the efficacy of Honokiol. These results clearly showed the cytoprotective nature of Honokiol-mediated autophagy and put forth the notion that a combined strategy of autophagy-inhibition with Honokiol would be more effective. Indeed, our in vivostudies showed that a combined treatment with Honokiol and Chloroquine can effectively inhibit primary tumor growth as well as metastatic progression. Conclusion: Together, these results implicate that honokiol is a potent inducer of cytoprotective autophagy and a combined treatment of honokiol and chloroquine is a promising therapeutic strategy for breast cancer. Citation Format: Nethaji Muniraj, Marey Shriver, Arumugam Nagalingam, Sumit Siddharth, Sheetal Parida, Neeraj K Saxena, Dipali Sharma. Induction of STK11-dependent cytoprotective autophagy in breast cancer cells upon Honokiol treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4271.

Introduction: Despite significant progress towards screening efforts and targeted therapies, breast cancer is still a leading cause of cancer-related mortality among women. Novel therapeutic strategies are clearly needed for effective targeting of breast cancer. Natural products continue to generate interest for identification of potential chemopreventive and therapeutic agents. Withaferin A (WFA) is a bioactive small molecule that has been proposed as a new-generation molecule capable of eliciting growth inhibitory effect on cancer cells. The molecular mechanisms by which WFA mediates inhibition of breast cancer remain elusive. The present study was designed to systematically elucidate the underlying mechanisms by which WFA inhibits growth and metastatic potential of breast cancer cells. Methods: Efficacy of WFA treatment on cell growth was evaluated by using clonogenicity, and anchorage–independent growth. Western blot and immunofluorescence analysis were used to examine activation of ERK/RSK (extracellular signal-regulated kinase/ ribosomal S6 kinase) and downstream signaling axes. Functional importance of ERK/RSK and CHOP (C/EBP homologous protein) in the biologic effects of WFA was examined by using overexpression, phospho-deficient constructs and specific inhibitors. Finally, mouse xenografts, immunohistochemical, RT-PCR and western blot analysis of tumors were used. Results: Analysis of the underlying molecular mechanisms revealed that WFA treatment induced phosphorylation of RSK2 in an ERK-dependent manner. Further, WFA-induced ERK/RSK signaling resulted in concomitant upregulation of CHOP and Elk-1(ETS-like transcription factor 1). By using ERK1/2 siRNA and FMK-MEA inhibitor, we found that ERK/RSK signaling axis is required for WFA-mediated modulation of CHOP and Elk-1. Intriguingly, we discovered that WFA triggers the nuclear translocation of CHOP and Elk1 resulting in transcriptional upregulation of Death Receptor protein (DR5). CHOP and Elk1 overexpression potentiated while phospho-deficient Elk-1 inhibited WFA-induced DR5 expression exhibiting that CHOP/Elk-1 cooperatively regulate DR5 expression. Furthermore, using nontoxic doses of WFA, we showed that WFA treatment effectively inhibited breast tumorigenesis in vivo. Analysis of WFA-treated breast tumors showed increased activation of ERK/RSK axis and DR5 along with higher nuclear accumulation of CHOP and Elk-1. Conclusion: These data provided first in vitro and in vivo evidence of the integral role of a previously unrecognized crosstalk between WFA and ERK/RSK and CHOP/Elk-1 axes in breast tumor growth inhibition. WFA treatment could potentially be a rational therapeutic strategy for breast carcinoma. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-12.

Introduction: Molecular effects of obesity, a well-established risk factor for breast cancer progression, are mediated by adipocytokine leptin. Recent studies from our lab reveal that leptin induces epithelial-mesenchymal transition (EMT) and tumorsphere formation via concomitant activation of Akt/GSK3b and MTA1/Wnt1 signaling axes leading to β catenin activation. Given the important role of leptin in breast cancer growth and metastasis, novel strategies to antagonize biological effects of leptin are much desired. We showed previously that honokiol, a bioactive polyphenol from Magnolia grandiflora, inhibits breast carcinogenesis. The present study provides first evidence for the efficacy of honokiol against oncogenic effects of leptin including EMT. Methods: Efficacy of honokiol to inhibit oncogenic effects of leptin was evaluated by using clonogenicity, anchorage–independent growth, matrigel-invasion and spheroid-migration assays. RT-PCR, western blot and immunofluorescence analyses were used to examine the molecular changes associated with EMT as well as underlying molecular pathways. Functional importance of MTA1/Wnt1/ β catenin axis was examined by using overexpression, phospho-deficient constructs and specific inhibitors. Finally, mouse xenografts, immuniohistochemical, RT-PCR and western blot analysis of tumors was used. Results: Honokiol treatment circumvents leptin-induced EMT-associated phenotypic changes. A biochemical hallmark of EMT is loss of expression of epithelial markers with a concurrent increase in mesenchymal marker expression. Honokiol elicits increased expression of E-cadherin, occludin and keratin-18 (epithelial markers) and decreased expression of vimentin, fibronectin and N-cadherin (mesenchymal markers) in leptin-treated cells providing molecular evidence for reversal of EMT. Honokiol also inhibits expression and nuclear translocation of transcriptional effectors of EMT; Snail, Slug, Zeb1 and Zeb2. Analysis of underlying molecular mechanisms reveals that Honokiol effectively inhibits leptin-induced MTA1/Wnt1/ β catenin axis. Furthermore, using nontoxic doses of honokiol, we show that honokiol treatment effectively inhibits leptin-induced breast tumorigenesis in vivo. Analysis of breast tumors show that honokiol treatment reverses leptin-associated signaling (MTA1/Wnt1/ β catenin axis) along with decreased expression of mesenchymal markers and increased expression of epithelial markers. Conclusions: In this study, we demonstrate for the first time that HNK is able to abolish leptin-induced EMT and provide in vitro and in vivo evidence for the integral role of a previously unrecognized crosstalk between honokiol and MTA1/Wnt1/ β catenin axis. Thus, HNK may be used as a non-toxic and non-endocrine rational therapeutic strategy for breast carcinoma in obese patients with high leptin levels. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-04.