Panjamurthy Kuppusamy

Hepatocellular carcinoma (HCC) incidence, as well as related mortality, has been steadily increasing in the USA and across the globe, partly due to the lack of effective therapeutic options for advanced HCC. Though sorafenib is considered standard-of-care for advanced HCC, it only improves median survival by a few months when compared to placebo. Sorafenib is also associated with several unpleasant side effects that often lead to early abatement of therapy. Here, we investigate whether a combination regimen including low-dose sorafenib and a non-toxic dose of anti-diabetic drug metformin can achieve effective inhibition of HCC. Indeed, combining metformin with low-dose sorafenib inhibited growth, proliferation, migration, and invasion potential of HCC cells. We observed a 5.3- and 1.9-fold increase in sub-G1 population in the combination treatment compared to sorafenib alone. We found that the combination of metformin enhanced the efficacy of sorafenib and inhibited the MAPK/ERK/Stat3 axis. Our in vivo studies corroborated the in vitro findings, and mice harboring HepG2-derived tumors showed effective tumor reduction upon treatment with low-dose sorafenib and metformin combination. This work sheds light on a therapeutic strategy aiming to augment sorafenib efficacy or dose-de-escalation that may prove beneficial in circumventing sorafenib resistance as well as minimizing related side effects.

Obese women with hormone receptor-positive breast cancer exhibit poor response to therapy and inferior outcomes. However, the underlying molecular mechanisms by which obesity/hyperleptinemia may reduce the efficacy of hormonal therapy remain elusive. 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 and potentiating coactivator binding. Furthermore, in silico analysis revealed that coactivator Med1 potentially associates with 48 (out of 74) obesity-signature genes. Interestingly, leptin upregulates Med1 expression by decreasing miR-205, and increases its functional activation via phosphorylation, which 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. In addition, honokiol or adiponectin treatment effectively inhibits leptin-induced Med1 expression and improves tamoxifen efficacy in hyperleptinemic state. These studies uncover the mechanistic insights how 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.

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.

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/adiponectin's 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.

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.

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.

Honokiol abrogates leptin-induced tumor progression by inhibiting Wnt1-MTA1-β-catenin signaling axis in a microRNA-34a dependent manner

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.

We have synthesized two series of bile acid tamoxifen conjugates using three bile acids lithocholic acid (LCA), deoxycholic acid (DCA), and cholic acid (CA). These bile acid–tamoxifen conjugates possess 1, 2, and 3 tamoxifen molecules attached to hydroxyl groups of bile acids having free acid and amine functionalities at the tail region of bile acids. The in vitro anticancer activities of these bile acid–tamoxifen conjugates show that the free amine headgroup based cholic acid–tamoxifen conjugate (CA-Tam₃-Am) is the most potent anticancer conjugate as compared to the parent drug tamoxifen and other acid and amine headgroup based bile acid–tamoxifen conjugates. The cholic acid–tamoxifen conjugate (CA-Tam₃-Am) bearing three tamoxifen molecules shows enhanced anticancer activities in both estrogen receptor +ve and estrogen receptor −ve breast cancer cell lines. The enhanced anticancer activity of CA-Tam₃-Am is due to more favorable irreversible electrostatic interactions followed by intercalation of these conjugates in hydrophobic core of membrane lipids causing increase in membrane fluidity. Annexin-FITC based FACS analysis showed that cells undergo apoptosis, and cell cycle analysis showed the arrest of cells in sub G₀ phase. ROS assays showed a high amount of generation of ROS independent of ER status of the cell line indicating changes in mitochondrial membrane fluidity upon the uptake of the conjugate that further leads to the release of cytochrome c, a direct and indirect regulator of ROS. The mechanistic studies for apoptosis using PCR and western analysis showed apoptotsis by intrinsic and extrinsic pathways in ER +ve MCF-7 cells and by only an intrinsic pathway in ER −ve cells. In vivo studies in the 4T1 tumor model showed that CA-Tam₃-Am is more potent than tamoxifen. These studies showed that bile acids provide a new scaffold for high drug loading and that their anticancer activities strongly depend on charge and hydrophobicity of lipid–drug conjugates.