Deepak Raina

Human MUC1 carcinoma-associated protein confers resistance to genotoxic anticancer agents

The MUC1 transforming protein is overexpressed by most human carcinomas. The present studies demonstrate that the MUC1 C-terminal subunit (MUC1 C-ter) localizes to mitochondria in HCT116/MUC1 colon carcinoma cells and that heregulin stimulates mitochondrial targeting of MUC1 C-ter. We also show that MUC1 attenuates cisplatin-induced (1) release of mitochondrial apoptogenic factors, (2) activation of caspase-3, and (3) induction of apoptosis. Moreover, knockdown of MUC1 expression in A549 lung and ZR-75-1 breast carcinoma cells by MUC1siRNA was associated with increased sensitivity to genotoxic drugs in vitro and in vivo. These findings indicate that MUC1 attenuates the apoptotic response to DNA damage and that this oncoprotein confers resistance to genotoxic anticancer agents.

Triterpenoid CDDO-Me Blocks the NF-κB Pathway by Direct Inhibition of IKKβ on Cys-179

The novel oleanane triterpenoid 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid (CDDO) and the C-28 methyl ester (CDDO-Me) induce apoptosis of human tumor cells by disruption of redox balance and are currently in clinical trials. The present studies show that CDDO and CDDO-Me block tumor necrosis factorα-induced targeting of NF-κB p65 to the nucleus. CDDO-Me also blocked tumor necrosis factor α-induced phosphorylation of IκBα. In concert with these results, we found that CDDO-Me inhibits IκBα kinaseβ (IKKβ) activity in cells. In support of a direct mechanism, CDDO-Me inhibited recombinant IKKβ activity in vitro. The results also demonstrate that (i) CDDO and CDDO-Me form adducts with IKKβ, but not IKKβ with mutation of Cys-179 to Ala, and (ii) CDDO-Me inhibits IKKβ by a mechanism dependent on oxidation of Cys-179. These findings indicate that CDDO and CDDO-Me directly block IKKβ activity and thereby the NF-κB pathway by interacting with Cys-179 in the IKKβ activation loop.

MUC1 oncoprotein activates the IκB kinase β complex and constitutive NF-κB signalling

Nuclear factor-κB (NF-κB) is constitutively activated in diverse human malignancies by mechanisms that are not understood. The MUC1 oncoprotein is aberrantly overexpressed by most human carcinomas and, similarly to NF-κB, blocks apoptosis and induces transformation. This study demonstrates that overexpression of MUC1 in human carcinoma cells is associated with constitutive activation of NF-κB p65. We show that MUC1 interacts with the high-molecular-weight IκB kinase (IKK) complex in vivo and that the MUC1 cytoplasmic domain binds directly to IKKβ and IKKγ. Interaction of MUC1 with both IKKβ and IKKγ is necessary for IKKβ activation, resulting in phosphorylation and degradation of IκBα. Studies in non-malignant epithelial cells show that MUC1 is recruited to the TNF-R1 complex and interacts with IKKβ–IKKγ in response to TNFα stimulation. TNFα-induced recruitment of MUC1 is dependent on TRADD and TRAF2, but not the death-domain kinase RIP1. In addition, MUC1-mediated activation of IKKβ is dependent on TAK1 and TAB2. These findings indicate that MUC1 is important for physiological activation of IKKβ and that overexpression of MUC1, as found in human cancers, confers sustained induction of the IKKβ–NF-κB p65 pathway.

Direct targeting of the mucin 1 oncoprotein blocks survival and tumorigenicity of human breast carcinoma cells.

The mucin 1 (MUC1) oncoprotein is aberrantly overexpressed by approximately 90% of human breast cancers. However, there are no effective agents that directly inhibit MUC1 and induce death of breast cancer cells. We have synthesized a MUC1 inhibitor (called GO-201) that binds to the MUC1 cytoplasmic domain and blocks the formation of MUC1 oligomers in cells. GO-201, and not an altered version, attenuates targeting of MUC1 to the nucleus of human breast cancer cells, disrupts redox balance, and activates the DNA damage response. GO-201 also arrests growth and induces necrotic death. By contrast, the MUC1 inhibitor has no effect on cells null for MUC1 expression or nonmalignant mammary epithelial cells. Administration of GO-201 to nude mice bearing human breast tumor xenografts was associated with loss of tumorigenicity and extensive necrosis, which results in prolonged regression of tumor growth. These findings show that targeting the MUC1 oncoprotein is effective in inducing death of human breast cancer cells in vitro and in tumor models.

Dependence on the MUC1-C oncoprotein in non-small cell lung cancer cells.

Non–small cell lung cancer (NSCLC) cells are often associated with constitutive activation of the phosphoinositide 3-kinase (PI3K) → Akt → mTOR pathway. The mucin 1 (MUC1) heterodimeric glycoprotein is aberrantly overexpressed in NSCLC cells and induces gene signatures that are associated with poor survival of NSCLC patients. The present results show that the MUC1 C-terminal subunit (MUC1-C) cytoplasmic domain associates with PI3K p85 in NSCLC cells. We show that inhibition of MUC1-C with cell-penetrating peptides blocks this interaction with PI3K p85 and suppresses constitutive phosphorylation of Akt and its downstream effector, mTOR. In concert with these results, treatment of NSCLC cells with the MUC1-C peptide inhibitor GO-203 was associated with downregulation of PI3K → Akt signaling and inhibition of growth. GO-203 treatment was also associated with increases in reactive oxygen species (ROS) and induction of necrosis by a ROS-dependent mechanism. Moreover, GO-203 treatment of H1975 (EGFR L858R/T790M) and A549 (K-Ras G12S) xenografts growing in nude mice resulted in tumor regressions. These findings indicate that NSCLC cells are dependent on MUC1-C both for activation of the PI3K → Akt pathway and for survival. Mol Cancer Ther; 10(5); 806–16. ©2011 AACR.

MUC1 oncoprotein is targeted to mitochondria by heregulin-induced activation of c-Src and the molecular chaperone HSP90

The MUC1 heterodimeric transmembrane glycoprotein is aberrantly overexpressed by most human carcinomas. The MUC1 C-terminal subunit localizes to mitochondria and blocks stress-induced activation of the intrinsic apoptotic pathway. How MUC1 is delivered to mitochondria is not known. The present studies demonstrate that MUC1 forms intracellular complexes with HSP70 and HSP90. We show that the MUC1 cytoplasmic domain binds directly to HSP70 in vitro. By contrast, binding of MUC1 to HSP90 in vitro is induced by c-Src-mediated phosphorylation of the MUC1 cytoplasmic domain. c-Src also increases binding of MUC1 to HSP90 in cells. In concert with these results, we show that heregulin (HRG), a ligand for ErbB receptors, activates c-Src and, in turn, stimulates binding of MUC1 to HSP90. We also show that inhibitors of c-Src or HSP90 block HRG-induced targeting of MUC1 to mitochondria and integration of MUC1 into the mitochondrial outer membrane. These findings indicate that MUC1 is delivered to mitochondria by a mechanism involving activation of the ErbB receptor → c-Src pathway and transport by the molecular chaperone HSP70/HSP90 complex.

Triterpenoid CDDO-Methyl Ester Inhibits the Janus-Activated Kinase-1 (JAK1)!Signal Transducer and Activator of Transcription-3 (STAT3) Pathway by Direct Inhibition of JAK1 and STAT3

The C-28 methyl ester of the oleane triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO-Me) induces apoptosis of human cancer cells by disrupting redox balance and is in clinical trials. CDDO-Me contains alpha,beta-unsaturated carbonyl groups that form reversible adducts with thiol nucleophiles. The present studies show that CDDO-Me blocks interleukin-6 (IL-6)-induced and constitutive activation of the Janus-activated kinase 1 (JAK1) in cells. In support of a direct mechanism, CDDO-Me forms adducts with JAK1 at Cys(1077) in the kinase domain and inhibits JAK1 activity. In concert with these results, CDDO-Me blocked IL-6-induced and constitutive activation of signal transducer and activator of transcription 3 (STAT3). Moreover, we show that CDDO-Me (a) binds directly to STAT3 by a mechanism dependent on the alkylation of Cys(259) and (b) inhibits the formation of STAT3 dimers. These findings indicate that CDDO-Me inhibits activation of the JAK1-->STAT3 pathway by forming adducts with both JAK1 and STAT3.

MUC1 oncoprotein blocks nuclear targeting of c‐Abl in the apoptotic response to DNA damage

The nonreceptor c‐Abl tyrosine kinase binds to cytosolic 14‐3‐3 proteins and is targeted to the nucleus in the apoptotic response to DNA damage. The MUC1 oncoprotein is overexpressed by most human carcinomas and blocks the induction of apoptosis by genotoxic agents. Using human carcinoma cells with gain and loss of MUC1 function, we show that nuclear targeting of c‐Abl by DNA damage is abrogated by a MUC1‐dependent mechanism. The results demonstrate that c‐Abl phosphorylates MUC1 on Tyr‐60 and forms a complex with MUC1 by binding of the c‐Abl SH2 domain to the pTyr‐60 site. Binding of MUC1 to c‐Abl attenuates phosphorylation of c‐Abl on Thr‐735 and the interaction between c‐Abl and cytosolic 14‐3‐3. We also show that expression of MUC1 with a mutation at Tyr‐60 (i) disrupts the interaction between MUC1 and c‐Abl, (ii) relieves the MUC1‐induced block of c‐Abl phosphorylation on Thr‐735 and binding to 14‐3‐3, and (iii) attenuates the MUC1 antiapoptotic function. These findings indicate that MUC1 sequesters c‐Abl in the cytoplasm and thereby inhibits apoptosis in the response to genotoxic anticancer agents.

MUC1 Oncoprotein Functions in Activation of Fibroblast Growth Factor Receptor Signaling

Activation of the fibroblast growth factor (FGF) receptor 3 (FGFR3) has been linked to the development of human cancers by mechanisms that are not well understood. The MUC1 oncoprotein is aberrantly overexpressed by certain hematologic malignancies and most human carcinomas. The present studies show that MUC1 associates with FGFR3. Stimulation of cells with FGF1 increased the interaction between MUC1 and FGFR3. FGF1 stimulation also induced c-Src-dependent tyrosine phosphorylation of the MUC1 cytoplasmic domain on a YEKV motif. FGF1-induced tyrosine phosphorylation of MUC1 was associated with increased binding of MUC1 to β-catenin and targeting of MUC1 and β-catenin to the nucleus. FGF1 also induced binding of MUC1 to the heat shock protein 90 (HSP90) chaperone by a mechanism dependent on phosphorylation of the YEKV motif. Notably, β-catenin and HSP90 compete for binding to the MUC1 cytoplasmic domain, indicating that MUC1 forms mutually exclusive complexes with these proteins. The results also show that inhibition of HSP90 with geldanamycin or 17-(allylamino)-17-demethoxygeldanamycin attenuates FGF1-induced binding of MUC1 to HSP90 and targeting of MUC1 to the mitochondrial outer membrane. These findings indicate that FGF1 induces phosphorylation of MUC1 on YEKV and thereby activates two distinct pathways: (a) nuclear localization of MUC1 and β-catenin and (b) delivery of MUC1 to mitochondria by HSP90. (Mol Cancer Res 2006;4(11):873–83)

MUC1 oncoprotein is a druggable target in human prostate cancer cells

Human prostate cancers are dependent on the androgen receptor for their progression. The MUC1 heterodimeric oncoprotein is aberrantly overexpressed in prostate cancers; however, it is not known if MUC1 is of functional importance to these tumors. To assess dependence on MUC1, we synthesized an inhibitor, designated GO-201, which interacts directly with the MUC1-C subunit at its oligomerization domain. Treatment of MUC1-positive DU145 and PC3 prostate cancer cells with GO-201, and not an altered version, resulted in inhibition of proliferation. GO-201 also induced necrotic cell death that was associated with increases in reactive oxygen species, loss of mitochondrial transmembrane potential, and depletion of ATP. By contrast, GO-201 had no effect against MUC1-negative LNCaP, CWR22Rv1, and MDA-PCa-2b prostate cancer cells. Significantly, GO-201 treatment of DU145 and PC3 xenografts growing in nude mice resulted in complete tumor regression and prolonged lack of recurrence. These findings indicate that certain prostate cancer cells are dependent on MUC1-C for growth and survival and that directly targeting MUC1-C results in their death in vitro and in tumor models. [Mol Cancer Ther 2009;8(11):3056–65]