Arumugam Nagalingam

Adiponectin antagonizes the oncogenic actions of leptin in hepatocellular carcinogenesis.

Obesity is rapidly becoming a pandemic and is associated with increased carcinogenesis. Obese populations have higher circulating levels of leptin in contrast to low concentrations of adiponectin. Hence, it is important to evaluate the dynamic role between adiponectin and leptin in obesity‐related carcinogenesis. Recently, we reported the oncogenic role of leptin including its potential to increase tumor invasiveness and migration of hepatocellular carcinoma (HCC) cells. In the present study we investigated whether adiponectin could antagonize the oncogenic actions of leptin in HCC. We employed HCC cell lines HepG2 and Huh7, the nude mice‐xenograft model of HCC, and immunohistochemistry data from tissue‐microarray to demonstrate the antagonistic role of adiponectin on the oncogenic actions of leptin. Adiponectin treatment inhibited leptin‐induced cell proliferation of HCC cells. Using scratch‐migration and electric cell‐substrate impedance‐sensing‐based migration assays, we found that adiponectin inhibited leptin‐induced migration of HCC cells. Adiponectin treatment effectively blocked leptin‐induced invasion of HCC cells in Matrigel invasion assays. Although leptin inhibited apoptosis in HCC cells, we found that adiponectin treatment induced apoptosis even in the presence of leptin. Analysis of the underlying molecular mechanisms revealed that adiponectin treatment reduced leptin‐induced Stat3 and Akt phosphorylation. Adiponectin also increased suppressor of cytokine signaling (SOCS3), a physiologic negative regulator of leptin signal transduction. Importantly, adiponectin significantly reduced leptin‐induced tumor burden in nude mice. In HCC samples, leptin expression significantly correlated with HCC proliferation as evaluated by Ki‐67, whereas adiponectin expression correlated significantly with increased disease‐free survival and inversely with tumor size and local recurrence. Conclusion: Collectively, these data demonstrate that adiponectin has the molecular potential to inhibit the oncogenic actions of leptin by blocking downstream effector molecules. (HEPATOLOGY 2010

LKB1 is required for adiponectin-mediated modulation of AMPK-S6K axis and inhibition of migration and invasion of breast cancer cells

Adiponectin is widely known as an adipocytokine with therapeutic potential for its markedly protective function in the pathogenesis of obesity-related disorders, metabolic syndrome, systemic insulin resistance, cardiovascular disease and more recently carcinogenesis. In the present study, we show that adiponectin inhibits adhesion, invasion and migration of breast cancer cells. Further analysis of the underlying molecular mechanisms revealed that adiponectin treatment increased AMP-activated protein kinase (AMPK) phosphorylation and activity as evident by increased phosphorylation of downstream target of AMPK, acetyl-coenzyme A carboxylase and inhibition of p70S6 kinase (S6K). Intriguingly, we discovered that adiponectin treatment increases the expression of tumor suppressor gene LKB1 in breast cancer cells. Overexpression of LKB1 in breast cancer cells further increased adiponectin-mediated phosphorylation of AMPK. Using isogenic LKB1 knockdown cell line pair, we found that LKB1 is required for adiponectin-mediated modulation of AMPK–S6K axis and more importantly, inhibition of adhesion, migration and invasion of breast cancer cells. Taken together these data present a novel mechanism involving specific upregulation of tumor suppressor gene LKB1 by which adiponectin inhibits adhesion, invasion and migration of breast cancer cells. Our findings indicate the possibility of using adiponectin analogues to inhibit invasion and migration of breast cancer cells.

Benzyl isothiocyanate inhibits oncogenic actions of leptin in human breast cancer cells by suppressing activation of signal transducer and activator of transcription 3

Molecular effects of obesity, a well-established risk factor for breast cancer progression, are mediated by adipocytokine leptin. Given the important role of leptin in breast cancer growth and metastasis, novel strategies to antagonize biological effects of this adipocytokine are much desired. We showed previously that benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables (e.g. garden cress), confers significant protection against mammary carcinogenesis in a transgenic mouse model. The present study provides first evidence for the efficacy of BITC against oncogenic effects of leptin. The BITC treatment circumvented leptin-induced clonogenicity and anchorage-independent growth of MDA-MB-231 and MCF-7 human breast cancer cells. Leptin-stimulated migration and invasion of these cells was also inhibited in the presence of BITC. Analysis of the underlying molecular mechanisms revealed that BITC treatment suppressed leptin-induced Stat3 phosphorylation and cyclin D1 transactivation. The BITC-mediated inhibition of MDA-MB-231 xenograft growth correlated with a modest yet significant decrease in levels of Tyr705 phosphorylated Stat3. The BITC treatment efficiently inhibited Stat3 and SRC1 recruitment to cyclin D1 promoter in a chromatin immunoprecipitation analysis. Furthermore, overexpression of constitutively active Stat3 imparted significant protection against BITC-mediated inhibition of cyclin D1 transactivation, whereas RNA interference of Stat3 resulted in a significant increase in BITC-mediated inhibition of cyclin D1 transactivation in the presence of leptin. These results indicate that Stat3 plays an important role in BITC-mediated inhibition of leptin-induced cyclin D1 transactivation. In conclusion, BITC could potentially be a rational therapeutic strategy for breast carcinoma in obese patients with high leptin levels.

Honokiol activates AMP-activated protein kinase in breast cancer cells via an LKB1-dependent pathway and inhibits breast carcinogenesis

IntroductionHonokiol, a small-molecule polyphenol isolated from magnolia species, is widely known for its therapeutic potential as an antiinflammatory, antithrombosis, and antioxidant agent, and more recently, for its protective function in the pathogenesis of carcinogenesis. In the present study, we sought to examine the effectiveness of honokiol in inhibiting migration and invasion of breast cancer cells and to elucidate the underlying molecular mechanisms.MethodsClonogenicity and three-dimensional colony-formation assays were used to examine breast cancer cell growth with honokiol treatment. The effect of honokiol on invasion and migration of breast cancer cells was evaluated by using Matrigel invasion, scratch-migration, spheroid-migration, and electric cell-substrate impedance sensing (ECIS)-based migration assays. Western blot and immunofluorescence analysis were used to examine activation of the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) axis. Isogenic LKB1-knockdown breast cancer cell line pairs were developed. Functional importance of AMPK activation and LKB1 overexpression in the biologic effects of honokiol was examined by using AMPK-null and AMPK-wild type (WT) immortalized mouse embryonic fibroblasts (MEFs) and isogenic LKB1-knockdown cell line pairs. Finally, mouse xenografts, immunohistochemical and Western blot analysis of tumors were used.ResultsAnalysis of the underlying molecular mechanisms revealed that honokiol treatment increases AMP-activated protein kinase (AMPK) phosphorylation and activity, as evidenced by increased phosphorylation of the downstream target of AMPK, acetyl-coenzyme A carboxylase (ACC) and inhibition of phosphorylation of p70S6kinase (pS6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). By using AMPK-null and AMPK-WT (MEFs), we found that AMPK is required for honokiol-mediated modulation of pACC-pS6K. Intriguingly, we discovered that honokiol treatment increased the expression and cytoplasmic translocation of tumor-suppressor LKB1 in breast cancer cells. LKB1 knockdown inhibited honokiol-mediated activation of AMPK and, more important, inhibition of migration and invasion of breast cancer cells. Furthermore, honokiol treatment resulted in inhibition of breast tumorigenesis in vivo. Analysis of tumors showed significant increases in the levels of cytoplasmic LKB1 and phospho-AMPK in honokiol-treated tumors.ConclusionsTaken together, these data provide the first in vitro and in vivo evidence of the integral role of the LKB1-AMPK axis in honokiol-mediated inhibition of the invasion and migration of breast cancer cells. In conclusion, honokiol treatment could potentially be a rational therapeutic strategy for breast carcinoma.

Survivin upregulation, dependent on leptin-EGFR-Notch1 axis, is essential for leptin-induced migration of breast carcinoma cells

Obese breast cancer patients exhibit a higher risk for larger tumor burden and an increased likelyhood of metastasis. The molecular effects of obesity on carcinogenesis are mediated by the autocrine and paracrine effects of the adipocytokine leptin. Leptin participates in the tumor progression and metastasis of human breast. We show that leptin induces clonogenicity and increases the migration potential of breast cancer cells. We found that survivin expression is induced in response to leptin. In this study, we examine the role and leptin-mediated regulation of survivin. Leptin treatment leads to survivin upregulation, due in part to the activation of Notch1 and the release of a transcriptionally active Notch1 intracellular domain (NICD). Chromatin immunoprecipitation analysis shows that NICD gets recruited to the survivin promoter at the CSL (CBF1/RBP-Jk, Su(H), Lag-1) binding site in response to leptin treatment. Inhibition of Notch1 activity inhibits leptin-induced survivin upregulation. Leptin-induced transactivation of epidermal growth factor receptor (EGFR) is involved in leptin-mediated Notch1 and survivin upregulation, demonstrating a novel upstream role of leptin-EGFR-Notch1 axis. We further show that leptin-induced migration of breast cancer cells requires survivin, as overexpression of survivin further increases, whereas silencing survivin abrogates leptin-induced migration. Using a pharmacological approach to inhibit survivin, we show that 3-hydroxy-3-methylglutaryl-coenzyme-A-reductase inhibitors, such as lovastatin, can effectively inhibit leptin-induced survivin expression and migration. Importantly, leptin increased breast tumor growth in nude mice. These data show a novel role for survivin in leptin-induced migration and put forth pharmacological survivin inhibition as a potential novel therapeutic strategy. This conclusion is supported by in vivo data showing the overexpression of leptin and survivin in epithelial cells of high-grade ductal carcinomas in situ and in high-grade invasive carcinomas.

Honokiol inhibits epithelial—mesenchymal transition in breast cancer cells by targeting signal transducer and activator of transcription 3/Zeb1/E‐cadherin axis

Epithelial—mesenchymal transition (EMT), a critical step in the acquisition of metastatic state, is an attractive target for therapeutic interventions directed against tumor metastasis. Honokiol (HNK) is a natural phenolic compound isolated from an extract of seed cones from Magnolia grandiflora. Recent studies from our lab show that HNK impedes breast carcinogenesis. Here, we provide molecular evidence that HNK inhibits EMT in breast cancer cells resulting in significant downregulation of mesenchymal marker proteins and concurrent upregulation of epithelial markers. Experimental EMT induced by exposure to TGFβ and TNFα in spontaneously immortalized nontumorigenic human mammary epithelial cells is also completely reversed by HNK as evidenced by morphological as well as molecular changes. Investigating the downstream mediator(s) that may direct EMT inhibition by HNK, we found functional interactions between HNK, Stat3, and EMT‐signaling components. In vitro and in vivo analyses show that HNK inhibits Stat3 activation in breast cancer cells and tumors. Constitutive activation of Stat3 abrogates HNK‐mediated activation of epithelial markers whereas inhibition of Stat3 using small molecule inhibitor, Stattic, potentiates HNK‐mediated inhibition of EMT markers, invasion and migration of breast cancer cells. Mechanistically, HNK inhibits recruitment of Stat3 on mesenchymal transcription factor Zeb1 promoter resulting in decreased Zeb1 expression and nuclear translocation. We also discover that HNK increases E‐cadherin expression via Stat3‐mediated release of Zeb1 from E‐cadherin promoter. Collectively, this study reports that HNK effectively inhibits EMT in breast cancer cells and provide evidence for a previously unrecognized cross‐talk between HNK and Stat3/Zeb1/E‐cadherin axis.

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.

Med1 plays a critical role in the development of tamoxifen resistance

Understanding the molecular pathways that contribute to the development of tamoxifen resistance is a critical research priority as acquired tamoxifen resistance is the principal cause of poor prognosis and death of patients with originally good prognosis hormone-responsive breast tumors. In this report, we provide evidence that Med1, an important subunit of mediator coactivator complex, is spontaneously upregulated during acquired tamoxifen-resistance development potentiating agonist activities of tamoxifen. Phosphorylated Med1 and estrogen receptor (ER) are abundant in tamoxifen-resistant breast cancer cells due to persistent activation of extracellular signal-regulated kinases. Mechanistically, phosphorylated Med1 exhibits nuclear accumulation, increased interaction with ER and higher tamoxifen-induced recruitment to ER-responsive promoters, which is abrogated by inhibition of Med1 phosphorylation. Stable knockdown of Med1 in tamoxifen-resistant cells not only reverses tamoxifen resistance in vitro but also in vivo. Finally, higher expression levels of Med1 in the tumor significantly correlated with tamoxifen resistance in ER-positive breast cancer patients on adjuvant tamoxifen monotherapy. In silico analysis of breast cancer, utilizing published profiling studies showed that Med1 is overexpressed in aggressive subsets. These findings provide what we believe is the first evidence for a critical role for Med1 in tamoxifen resistance and identify this coactivator protein as an essential effector of the tamoxifen-induced breast cancer growth.

Honokiol activates LKB1-miR-34a axis and antagonizes the oncogenic actions of leptin in breast cancer

Leptin, a major adipocytokine produced by adipocytes, is emerging as a key molecule linking obesity with breast cancer therefore, it is important to find effective strategies to antagonize oncogenic effects of leptin to disrupt obesity-cancer axis. Here, we examine the potential of honokiol (HNK), a bioactive polyphenol from Magnolia grandiflora, as a leptin-antagonist and systematically elucidate the underlying mechanisms. HNK inhibits leptin-induced epithelial-mesenchymal-transition (EMT), and mammosphere-formation along with a reduction in the expression of stemness factors, Oct4 and Nanog. Investigating the downstream mediator(s), that direct leptin-antagonist actions of HNK; we discovered functional interactions between HNK, LKB1 and miR-34a. HNK increases the expression and cytoplasmic-localization of LKB1 while HNK-induced SIRT1/3 accentuates the cytoplasmic-localization of LKB1. We found that HNK increases miR-34a in LKB1-dependent manner as LKB1-silencing impedes HNK-induced miR-34a which can be rescued by LKB1-overexpression. Finally, an integral role of miR-34a is discovered as miR-34a mimic potentiates HNK-mediated inhibition of EMT, Zeb1 expression and nuclear-localization, mammosphere-formation, and expression of stemness factors. Leptin-antagonist actions of HNK are further enhanced by miR-34a mimic whereas miR-34a inhibitor results in inhibiting HNKs effect on leptin. These data provide evidence for the leptin-antagonist potential of HNK and reveal the involvement of LKB1 and miR-34a.

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.