Panjamurthy Kuppusamy

Mechanistic Elucidation of the Antitumor Properties of Withaferin A in Breast Cancer

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.

Benzyl Isothiocyanate potentiates p53 signaling and antitumor effects against breast cancer through activation of p53-LKB1 and p73-LKB1 axes.

Functional reactivation of p53 pathway, although arduous, can potentially provide a broad-based strategy for cancer therapy owing to frequent p53 inactivation in human cancer. Using a phosphoprotein-screening array, we found that Benzyl Isothiocynate, (BITC) increases p53 phosphorylation in breast cancer cells and reveal an important role of ERK and PRAS40/MDM2 in BITC-mediated p53 activation. We show that BITC rescues and activates p53-signaling network and inhibits growth of p53-mutant cells. Mechanistically, BITC induces p73 expression in p53-mutant cells, disrupts the interaction of p73 and mutant-p53, thereby releasing p73 from sequestration and allowing it to be transcriptionally active. Furthermore, BITC-induced p53 and p73 axes converge on tumor-suppressor LKB1 which is transcriptionally upregulated by p53 and p73 in p53-wild-type and p53-mutant cells respectively; and in a feed-forward mechanism, LKB1 tethers with p53 and p73 to get recruited to p53-responsive promoters. Analyses of BITC-treated xenografts using LKB1-null cells corroborate in vitro mechanistic findings and establish LKB1 as the key node whereby BITC potentiates as well as rescues p53-pathway in p53-wild-type as well as p53-mutant cells. These data provide first in vitro and in vivo evidence of the integral role of previously unrecognized crosstalk between BITC, p53/LKB1 and p73/LKB1 axes in breast tumor growth-inhibition.

Interacting post-muscarinic receptor signaling pathways potentiate matrix metalloproteinase-1 expression and invasion of human colon cancer cells.

M3 muscarinic receptor (M3R) expression is increased in colon cancer; M3R activation stimulates colon cancer cell invasion via cross-talk with epidermal growth factor receptors (EGFR), post-EGFR activation of mitogen-activated protein kinase (MAPK) extracellular signal-related kinase 1/2 (ERK1/2), and induction of matrix metalloproteinase-1 (MMP1) expression. MMP1 expression is strongly associated with tumor metastasis and adverse outcomes. Here, we asked whether other MAPKs regulate M3R agonist-induced MMP1 expression. In addition to activating ERK1/2, we found that treating colon cancer cells with acetylcholine (ACh) stimulated robust time- and dose-dependent phosphorylation of p38 MAPK. Unlike ERK1/2 activation, ACh-induced p38 phosphorylation was EGFR-independent and blocked by inhibiting protein kinase C-α (PKC-α). Inhibiting activation of PKC-α, EGFR, ERK1/2, or p38-α/β alone attenuated, but did not abolish ACh-induced MMP1 expression, a finding that predicted potentiating interactions between these pathways. Indeed, ACh-induced MMP1 expression was abolished by incubating cells with either an EGFR or MEK/ERK1/2 inhibitor combined with a p38-α/β inhibitor. Activating PKC-α and EGFR directly with the combination of phorbol 12-myristate 13-acetate (PMA) and EGF potentiated MMP1 gene and protein expression, and cell invasion. PMA- and ACh-induced MMP1 expression were strongly diminished by inhibiting Src and abolished by concurrently inhibiting both p38-α/β and Src, indicating that Src mediates the cross-talk between PKC-α and EGFR signaling. Using siRNA knockdown, we identified p38-α as the relevant p38 isoform. Collectively, these studies uncover novel functional interactions between post-muscarinic receptor signaling pathways that augment MMP1 expression and drive colon cancer cell invasion; targeting these potentiating interactions has therapeutic potential.

ADIPOQ/adiponectin induces cytotoxic autophagy in breast cancer cells through STK11/LKB1-mediated activation of the AMPK-ULK1 axis

ABSTRACT ADIPOQ/adiponectin, an adipocytokine secreted by adipocytes in the breast tumor microenvironment, negatively regulates cancer cell growth hence increased levels of ADIPOQ/adiponectin are associated with decreased breast cancer growth. However, its mechanisms of action remain largely elusive. We report that ADIPOQ/adiponectin induces a robust accumulation of autophagosomes, increases MAP1LC3B-II/LC3B-II and decreases SQSTM1/p62 in breast cancer cells. ADIPOQ/adiponectin-treated cells and xenografts exhibit increased expression of autophagy-related proteins. LysoTracker Red-staining and tandem-mCherry-GFP-LC3B assay show that fusion of autophagosomes and lysosomes is augmented upon ADIPOQ/adiponectin treatment. ADIPOQ/adiponectin significantly inhibits breast cancer growth and induces apoptosis both in vitro and in vivo, and these events are preceded by macroautophagy/autophagy, which is integral for ADIPOQ/adiponectin-mediated cell death. Accordingly, blunting autophagosome formation, blocking autophagosome-lysosome fusion or genetic-knockout of BECN1/Beclin1 and ATG7 effectively impedes ADIPOQ/adiponectin induced growth-inhibition and apoptosis-induction. Mechanistic studies show that ADIPOQ/adiponectin reduces intracellular ATP levels and increases PRKAA1 phosphorylation leading to ULK1 activation. AMPK-inhibition abrogates ADIPOQ/adiponectin-induced ULK1-activation, LC3B-turnover and SQSTM1/p62-degradation while AMPK-activation potentiates ADIPOQ/adiponectins effects. Further, ADIPOQ/adiponectin-mediated AMPK-activation and autophagy-induction are regulated by upstream master-kinase STK11/LKB1, which is a key node in antitumor function of ADIPOQ/adiponectin as STK11/LKB1-knockout abrogates ADIPOQ/adiponectin-mediated inhibition of breast tumorigenesis and molecular analyses of tumors corroborate in vitro mechanistic findings. ADIPOQ/adiponectin increases the efficacy of chemotherapeutic agents. Notably, high expression of ADIPOQ receptor ADIPOR2, ADIPOQ/adiponectin and BECN1 significantly correlates with increased overall survival in chemotherapy-treated breast cancer patients. Collectively, these data uncover that ADIPOQ/adiponectin induces autophagic cell death in breast cancer and provide in vitro and in vivo evidence for the integral role of STK11/LKB1-AMPK-ULK1 axis in ADIPOQ/adiponectin-mediated cytotoxic autophagy.

Concomitant activation of ETS-like transcription factor-1 and Death Receptor-5 via extracellular signal-regulated kinase in withaferin A-mediated inhibition of hepatocarcinogenesis in mice

Hepatocellular carcinoma (HCC) has the second lowest 5-year survival rate (~16%) of all tumor types partly owing to the lack of effective therapeutic agents. Withaferin A (WA) is a bioactive molecule derived from Withania somnifera and the present study is designed to systemically investigate the anti-HCC efficacy of WA. WA inhibited growth, migration and invasion of HCC cells. Using a phospho-kinase screening array, we discovered that WA increased phosphorylation of ERK and p38 in HCC. Further analyses revealed a key role of ERK leading to increased phosphorylation of p90-ribosomal S6 kinase (RSK) and a concomitant activation of ETS-like transcription factor-1(ELK1) and Death Receptor protein-5 (DR5) in HCC. Importantly, oral administration of WA effectively inhibited HepG2-xenografts and DEN-induced-HCC in C57BL/6 mice. Analyses of WA-treated HepG2-xenografts and DEN-induced-HCC tumors showed elevated levels of ERK, RSK, ELK1 and DR5 along with decreased expression of Ki67. In silico analyses of HCC, utilizing published profiling studies showed an inverse correlation between DR5 and Ki67. These data showed the efficacy of WA as an effective agent for HCC inhibition and provided first in vitro and in vivo evidence supporting the key role of a novel crosstalk between WA, ERK/RSK, ELK1, and DR5 in HCC inhibition.

Abstract 3301: A three-pronged attack on cancer cells: Induction of non-protective autophagy, inhibition of lysosomal acidification and promotion of energetic impairment

Background and Aim: Cancer cells undergo cytoprotective autophagy and evade chemotherapy therefore many clinical trials are investigating the efficacy of autophagy inhibition in combination with chemotherapy. At the functional level, autophagic process can be cytoprotective, cytotoxic, cytostatic or nonprotective. We investigated strategies to convert cellular autophagic response to non-protective autophagy which does not interfere with therapeutic regimens exploiting bioactive molecules. Methods: Utilizing in vivo xenograft models, we established that Withaferin A (WA), a bioactive molecule from Withania Somnifera inhibits breast tumorigenesis. Autophagy studies were conducted utilizing immunoblot, RT-PCR, and immunofluorescence analyses for autophagy markers, transmission electron microscopy and confocal imaging. The fusion of autophagosome and lysosome was examined by using GFP-LC3/LysoTracker-red and GFP-LC3/mCherryRAB7A. Protein degradation activity of lysosomes and ATP levels were examined by DQ-BSA assay, Cathepsin activity and quantitative ATP assay. Results: WA inhibited growth and induced apoptosis in breast cancer cells resulting in inhibition of breast carcinogenesis in vivo. Although WA increased tumor suppressor LKB1 which is known to be involved in autophagy, WA-mediated increased cleavage of Light Chain 3 type II (LC3-II) and punctated LC3-II staining was LKB1-independent. The redistribution of EGFP-LC3 from cytosol to autophagosome indicated increased formation of autophagosomes in WA-treated cells. However, WA-induced increased autophagosome-formation was not mediated by increased activation of autophagy by upstream processes but was due to blockade of lysosomal-degradation as evident by higher level of sequestosome 1 (SQSTM1/p62) and decreased turnover of LC3. WA was found to be a potent lysosomal deacidification agent capable of blocking autophagic flux. Accordingly, inhibiting autophagy by blocking formation of autophagosomes or elevating lysosomal pH did not interfere with WA-mediated growth-inhibition. WA blocked autophagic flux decreasing recycling of cellular fuels leading to reduced energy supply. Investigating this alternative mechanism, we discovered that indeed, WA reduced ATP levels and increased phosphorylation of AMP-activated protein kinase (AMPK). Modulating substrates for tricarboxylic acid (TCA) cycle with methyl pyruvate protected WA-treated cells while 2DG potentiated WA-induced cell death. Conclusion: Our results indicate that WA induces a non-protective autophagy and blocks energy fuels in cancer cells by reducing ATP levels and inhibiting lysosomal acidification hence offering a three-pronged approach to facilitate cancer cell death. WA might be a useful strategic addition to chemotherapy regimens to evade cytoprotective effects of autophagy. Citation Format: Nethaji Muniraj, Arumugam Nagalingam, Panjamurthy Kuppusamy, Neeraj K. Saxena, Dipali Sharma. A three-pronged attack on cancer cells: Induction of non-protective autophagy, inhibition of lysosomal acidification and promotion of energetic impairment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3301. doi:10.1158/1538-7445.AM2017-3301

Abstract 3319: Elevating adipokine adiponectin level can induce cytotoxic autophagy in breast cancer cells and potentiate the efficacy of chemotherapeutic regimens: preclinical studies

Adiponectin, an adipocytokine secreted by adipocytes in the breast tumor microenvironment, negatively regulates cancer cell growth hence increased levels of adiponectin are associated with decreased breast cancer growth. However, its mechanisms of action remain largely elusive. We report that adiponectin induces a robust accumulation of autophagosomes, increases LC3II and decreases p62/SQSTM1 in breast cancer cells. Adiponectin-treated cells and xenografts exhibit increased expression of autophagy-related proteins. Lysotracker-Red-staining and tandem-mCherry-GFP-LC3 assay show that autophagosomes/lysosomes fusion is augmented upon adiponectin treatment. Adiponectin significantly inhibits breast cancer growth and induces apoptosis both in vitro and in vivo, and these events are preceded by autophagy, which is integral for adiponectin-mediated cell death. Accordingly, blunting autophagosomes-formation, blocking autophagosomes-autolysosome fusion or genetic-knockout of BECN1/Beclin1/ATG7 effectively impedes adiponectin induced growth-inhibition and apoptosis-induction. Mechanistic studies show that adiponectin reduces intracellular ATP levels and increases AMPK phosphorylation leading to ATG1 activation. AMPK-inhibition abrogates adiponectin-induced ATG1-activation, LC3II-turnover and p62-degradation while AMPK-activation potentiates adiponectin’s effects. Further, adiponectin-mediated AMPK-activation and autophagy-induction are regulated by upstream master-kinase LKB1, which is a key node in anti-tumor function of adiponectin as LKB1-knockout abrogates adiponectin-mediated inhibition of breast tumorigenesis and molecular analyses of tumors corroborate in vitro mechanistic findings. Adiponectin increases the efficacy of chemotherapeutic agents. Notably, high expression of adiponectin receptor, adiponectin and BECN1 significantly correlates with increased overall survival in chemotherapy-treated breast cancer patients. Collectively, these data uncover that adiponectin induces autophagic cell death in breast cancer and provide in vitroa and in vivo evidence for the integral role of LKB1-AMPK-ATG1 axis in adiponectin-mediated cytotoxic-autophagy. Citation Format: Seung J Chung, Ganji Purnachandra Nagaraju, Arumugam Nagalingam, Nethaji Muniraj, Panjamurthy Kuppusamy, Alyssa Walker, Juhyung woo, Balazs Győrffy, Edward Gabrielson, Neeraj K. Saxena, Dipali Sharma. Elevating adipokine adiponectin level can induce cytotoxic autophagy in breast cancer cells and potentiate the efficacy of chemotherapeutic regimens: preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3319. doi:10.1158/1538-7445.AM2017-3319

Abstract LB-113: A novel role of Withaferin A as an effective pro-apoptotic receptor agonist (PARA) and insights into the underlying molecular mechanisms

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. Results: Because many cellular signaling events involve induced-phosphorylation of key targets, in the present study we utilized phosphokinase arrays to gain insight into the intricacies of WFA-mediated signaling networks and discovered that WFA induces RSK phosphorylation in breast cancer cells. Further analysis revealed an integral role of ERK in WFA-mediated RSK activation and WFA-induced ERK/RSK signaling resulted in concomitant upregulation and activation of Elk1(ETS-like transcription factor-1) and CHOP (C-EBP homology protein) leading to transcriptional upregulation of Death Receptor protein-5 (DR5). It is interesting to note that in recent years, many phase 1 and 2 single agent and combination clinical trials have been conducted to examine the efficacy and safety of recombinant human TRAIL (dulanermin, (Amgen/Genentech)), and the agonistic monoclonal antibodies to DR5 (lexatumumab (Human Genome Sciences), conatumumab (Amgen), drozitumab (Genentech), tigatuzumab (Daiichi-Sankyo) and LBY135 (Novartis)). Studies with these pro-apoptotic receptor agonists (PARAs) have been encouraging but no full phase 3 studies have been performed suggesting the need for novel PARAs with improved properties. Here, we show that WFA is more effective than three other known DR5 activators, TRAIL, etoposide and celecoxib, in activation of DR5 in breast cancer cells hence, appears to be a good candidate for further development as an effective DR5 inducer. WFA treatment effectively inhibited breast tumorigenesis in vivo and analysis of WFA-treated xenografts and MMTV-neu mice tumors showed increased activation of ERK/RSK axis and DR5 along with elevated nuclear accumulation of Elk1 and CHOP. 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/Elk1 axes in breast tumor growth inhibition. Also, our findings may potentially open new avenues of research on the role of withaferin A as a novel pro-apoptotic receptor agonist (PARA). Citation Format: Arumugam Nagalingam, Panjamurthy Kuppusamy, Neeraj Saxena, Dipali Sharma. A novel role of Withaferin A as an effective pro-apoptotic receptor agonist (PARA) and insights into the underlying molecular mechanisms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-113. doi:10.1158/1538-7445.AM2014-LB-113

Abstract P1-07-04: A novel bioactive approach to inhibit leptin-induced epithelial-mesenchymal transition in breast cancer

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.

Abstract P2-09-12: Withaferin A-mediated inhibition of breast carcinoma involves concomitant activation of CHOP and Elk1 via ERK/ribosomal S6 kinase (RSK) signaling axis regulating death receptor 5 expression

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.