Velusamy Rangasamy

Mixed Lineage Kinase-3/JNK1 Axis Promotes Migration of Human Gastric Cancer Cells following Gastrin Stimulation

Gastrin is a gastrointestinal peptide hormone, secreted by the gastric G cells and can exist as a fully processed amidated form (G17) or as unprocessed forms. All forms of gastrin possess trophic properties towards the gastrointestinal mucosa. An understanding of the signaling pathways involved is important to design therapeutic approaches to target gastrin-mediated cellular events. The studies described here were designed to identify the signaling pathways by which amidated gastrin (G17) mediates cancer cell migration. These studies indicated a time- and dose-dependent increase in gastric cancer cell migration after G17 stimulation, involving cholecystokinin 2 receptor. G17-induced migration was preceded by activation of MAPK pathways and was antagonized after pretreatment with SP600125, a pharmacological inhibitor of c-Jun-NH(2)-terminal kinase (JNK) pathway. Knockdown of endogenous JNK1 expression via small interference RNA (JNK1-siRNA) inhibited G17-induced phosphorylation of c-Jun and migration, and overexpression of wild-type JNK1 or constitutive active JNK1 promoted G17-induced migration. Studies designed to identify the MAPK kinase kinase member mediating JNK activation indicated the involvement of mixed lineage kinase-3 (MLK3), which was transiently activated upon G17 treatment. Inhibition of MLK3 pathway via a pan-MLK inhibitor or knockdown of MLK3 expression by MLK3-siRNA antagonized G17-induced migration. Incubation with G17 also resulted in an induction of matrix metalloproteinase 7 promoter activity, which is known to mediate migration and invasion pathways in cancer cells. Modulation of MLK3, JNK1, and c-Jun pathways modulated G17-induced matrix metalloproteinase 7 promoter activation. These studies indicate that the MLK3/JNK1 axis mediates G17-induced gastric cancer cell migration, which can be targeted for designing novel therapeutic strategies for treating gastric malignancies.

Mixed-lineage kinase 3 phosphorylates prolyl-isomerase Pin1 to regulate its nuclear translocation and cellular function

Nuclear protein peptidyl-prolyl isomerase Pin1-mediated prolyl isomerization is an essential and novel regulatory mechanism for protein phosphorylation. Therefore, tight regulation of Pin1 localization and catalytic activity is crucial for its normal nuclear functions. Pin1 is commonly dysregulated during oncogenesis and likely contributes to these pathologies; however, the mechanism(s) by which Pin1 catalytic activity and nuclear localization are increased is unknown. Here we demonstrate that mixed-lineage kinase 3 (MLK3), a MAP3K family member, phosphorylates Pin1 on a Ser138 site to increase its catalytic activity and nuclear translocation. This phosphorylation event drives the cell cycle and promotes cyclin D1 stability and centrosome amplification. Notably, Pin1 pSer138 is significantly up-regulated in breast tumors and is localized in the nucleus. These findings collectively suggest that the MLK3-Pin1 signaling cascade plays a critical role in regulating the cell cycle, centrosome numbers, and oncogenesis.

TRAF2-MLK3 interaction is essential for TNF-α-induced MLK3 activation

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase that is activated by tumor necrosis factor-α (TNF-α) and specifically activates c-Jun N-terminal kinase (JNK) on TNF-α stimulation. The mechanism by which TNF-α activates MLK3 is still not known. TNF receptor-associated factors (TRAFs) are adapter molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK. Here, we report that MLK3 associates with TRAF2, TRAF5 and TRAF6; however only TRAF2 can significantly induce the kinase activity of MLK3. The interaction domain of TRAF2 maps to the TRAF domain and for MLK3 to its C-terminal half (amino acids 511-847). Endogenous TRAF2 and MLK3 associate with each other in response to TNF-α treatment in a time-dependent manner. The association between MLK3 and TRAF2 mediates MLK3 activation and competition with the TRAF2 deletion mutant that binds to MLK3 attenuates MLK3 kinase activity in a dose-dependent manner, on TNF-α treatment. Furthermore the downstream target of MLK3, JNK was activated by TNF-α in a TRAF2-dependent manner. Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-α-induced activation of MLK3 and its downstream target, JNK.

Estrogen Suppresses MLK3-Mediated Apoptosis Sensitivity in ER+ Breast Cancer Cells

Little knowledge exists about the mechanisms by which estrogen can impede chemotherapy-induced cell death of breast cancer cells. 17beta-Estradiol (E(2)) hinders cytotoxic drug-induced cell death in estrogen receptor-positive (ER(+)) breast cancer cells. We noted that the activity of the proapoptotic mixed lineage kinase 3 (MLK3) kinase was relatively higher in estrogen receptor-negative (ER(-)) breast tumors, suggesting that E(2) might inhibit MLK3 activity. The kinase activities of MLK3 and its downstream target, c-Jun NH(2)-terminal kinase, were rapidly inhibited by E(2) in ER(+) but not in ER(-) cells. Specific knockdown of AKT1/2 prevented MLK3 inhibition by E(2), indicating that AKT mediated this event. Furthermore, MLK3 inhibition by E(2) involved phosphorylation of MLK3 Ser(674) by AKT, attenuating the proapoptotic function of MLK3. We found that a pan-MLK inhibitor (CEP-11004) limited Taxol-induced cell death and that E(2) accentuated this limitation. Taken together, our findings indicate that E(2) inhibits the proapoptotic function of MLK3 as a mechanism to limit cytotoxic drug-induced death of ER(+) breast cancer cells.

Mixed Lineage Kinase 3 Modulates β-Catenin Signaling in Cancer Cells

Background: β-Catenin mediates a wide variety of cellular processes, but the signaling pathways regulating β-catenin downstream events are not fully understood. The role of MLK3 in modulating β-catenin pathway has not been reported earlier. Results: MLK3 can induce β-catenin stabilization but inhibit conventional β-catenin/TCF transcriptional activation. Conclusion: These provide a new mechanism of regulating β-catenin/TCF axis. Significance: MLK3 can be targeted in regulating the growth of β-catenin overexpressing tumors. Expression of β-catenin is strictly regulated in normal cells via the glycogen synthase kinase 3β (GSK3β)- adenomatous polyposis coli-axin-mediated degradation pathway. Mechanisms leading to inactivation of this pathway (example: activation of Wnt/β-catenin signaling or mutations of members of the degradation complex) can result in β-catenin stabilization and activation of β-catenin/T-cell factor (TCF) signaling. β-Catenin-mediated cellular events are diverse and complex. A better understanding of the cellular signaling networks that control β-catenin pathway is important for designing effective therapeutic strategies targeting this axis. To gain more insight, we focused on determining any possible cross-talk between β-catenin and mixed lineage kinase 3 (MLK3), a MAPK kinase kinase member. Our studies indicated that MLK3 can induce β-catenin expression via post-translational stabilization in various cancer cells, including prostate cancer. This function of MLK3 was dependent on its kinase activity. MLK3 can interact with β-catenin and phosphorylate it in vitro. Overexpression of GSK3β-WT or the S9A mutant was unable to antagonize MLK3-induced stabilization, suggesting this to be independent of GSK3β pathway. Surprisingly, despite stabilizing β-catenin, MLK3 inhibited TCF transcriptional activity in the presence of both WT and S37A β-catenin. These resulted in reduced expression of β-catenin/TCF downstream targets Survivin and myc. Immunoprecipitation studies indicated that MLK3 did not decrease β-catenin/TCF interaction but promoted interaction between β-catenin and KLF4, a known repressor of β-catenin/TCF transcriptional activity. In addition, co-expression of MLK3 and β-catenin resulted in significant G2/M arrest. These studies provide a novel insight toward the regulation of β-catenin pathway, which can be targeted to control cancer cell proliferation, particularly those with aberrant activation of β-catenin signaling.

Mixed Lineage Kinase–c-Jun N-Terminal Kinase Axis A Potential Therapeutic Target in Cancer

Mixed lineage kinases (MLKs) are members of the mitogen-activated protein kinase kinase kinase (MAP3K) family and are reported to activate MAP kinase pathways. There have been at least 9 members of the MLK family identified to date, although the physiological functions of all the family members are yet unknown. However, MLKs in general have been implicated in neurodegenerative diseases, including Parkinson and Alzheimer diseases. Recent reports suggest that some of the MLK members could play a role in cancer via modulating cell migration, invasion, cell cycle, and apoptosis. This review article will first describe the biology of MLK members and then discuss the current progress that relates to their functions in cancer.

Abstract 4957: Mixed Lineage Kinase 3 mediates vanadium-induced cell death in cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Vanadium is a transition metal and occurs naturally in a variety of minerals. In mammals, vanadium has been shown to promote cancer cell death and can also mimic insulin action, most probably through the inhibition of cellular tyrosine phosphatase and subsequent activation of signaling pathways. The mechanism by which vanadium causes cancer cell death is still not known. Interestingly, it has been shown that various mitogen activated protein kinase (MAPK) members are activated upon vanadium treatment. However, the contribution of any specific MAPK member that mediates vanadium-induced cell death has not been clearly delineated. It has been reported that JNK is activated by vanadium and therefore we investigated the role of Mixed Lineage Kinase 3 (MLK3) on vanadium-induced cell death. MLK3 is a MAP3K member that is known to specifically activate JNK pathway. We have earlier reported that MLK3 activation promotes cell death in cancer and neuronal cells. Our results demonstrated that endogenous MLK3 was activated by vanadium and MLK3 was highly phosphorylated on tyrosine residues. We also identified Src as a tyrosine kinase that phosphorylated MLK3 on specific tyrosine residues and phosphorylation of these tyrosine residues by Src was essential for MLK3 activation. Vanadium-induced cancer cell death was mediated by MLK3 or MLKs, because CEP-1347, a pan MLK inhibitor, prevented this cell death. Furthermore, the tyrosine phospho-deficient mutants of MLK3 also prevented vanadium-induced cell death. Interestingly, the activity of Src was seen to be in parallel with MLK3 kinase activity in human primary breast tumors. Taken together, our data demonstrate that tyrosine phosphorylation of MLK3 by oncogenic Src leads to MLK3 activation, which subsequently promotes vanadium-induced cell death in cancer cells. Our results also implicate a paradoxical pro-apoptotic role of Src in cancer cells, and thus it is conceivable that in tumors where both Src and MLK3/MLKs activities are higher, targeting of this axis might promote cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4957. doi:1538-7445.AM2012-4957

Abstract 251: Role of ß-catenin in prostate cancer cell apoptosis

Introduction: Evasion of apoptosis is a hallmark of cancer cells, and apoptotic signaling pathways are attractive targets for cancer therapy. TNF-related apoptosis inducing ligand (TRAIL) has gained much importance recently due to its ability to preferentially induce apoptosis in transformed cells and not in normal cells. Since many tumor cells develop TRAIL resistance, developing combinatorial treatment strategies to enhance TRAIL sensitivity is a major challenge of current cancer research. The reasons behind TRAIL resistance are multiple, and might be linked with aberrant activation of various oncogenic pathways. s-catenin is a well established oncogene being overexpressed in various cancers and might be involved in TRAIL resistance. Normally, it functions as a component of both the adherens junction complex and the Wnt/wingless signaling pathway. Our earlier studies have shown that treatment of cancer cells with Troglitazone (TZD), a synthetic ligand for Peroxisome Proliferator-activator receptor gamma can reduce cell proliferation and s-catenin expression. When combined with TZD, TRAIL was capable of inducing potent apoptosis in various TRAIL-resistant cancer cells. Aims: The present study was designed to determine the involvement of s-catenin during TRAIL-TZD-induced apoptosis of prostate cancer cells. Experimental procedures: Different prostate cancer cell lines (LNCaP, 22RV1, PC-3, DU145) were used to determine the effect of TRAIL-TZD on apoptosis and s-catenin pathway, utilizing Western Blots, Immunoprecipitation, and luciferase assays. Results: Treatment with a combination of TRAIL and TZD resulted in significant apoptosis (PARP, caspase-3 cleavage) and s-catenin cleavage in LNCaP but not in PC-3 and DU145 cells, and 22RV1 showed partial PARP cleavage. TRAIL-TZD-induced s-catenin cleavage was antagonized by caspase inhibitors, suggesting this to be downstream of caspase activation. Luciferase assays with s-catenin/TCF-responsive reporter indicated a significant reduction of luciferase activity with this treatment. Interestingly the cleaved s-catenin fragment retained strong interaction with E-Cadherin, as indicated by immunoprecipitation studies. Detergent fractionation of the cell extracts treated with the drug combination revealed that s-catenin cleavage was enriched in the cytosolic fraction. To understand any participation of HDACs, TRAIL treatment was performed in combination with various HDAC inhibitors. These results showed a strong increase in apoptosis and s-catenin cleavage with TRAIL and valproic acid treatment, suggesting an involvement of HDACs in this apoptotic resistance. Conclusion: These results show that targeting s-catenin signaling pathway might be effective means of increasing TRAIL sensitivity in prostate cancer, and combining TRAIL with TZD or valproic acid might be a promising treatment regimen for drug-resistant cells, especially those with aberrant s-catenin expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 251. doi:1538-7445.AM2012-251

Abstract 5200: Role of MLK3 in breast cancer cell migration and metastasis

Effectively treating breast cancer metastasis is one of the biggest challenges in breast cancer therapy. The overall poor prognosis associated with cancer cell migration to distal organs underscores the critical need to understand the biology of the disease in order to develop more effective therapies. Members of protein kinase family have often been targeted for therapeutic purposes in different types of cancer, including breast cancer, and several protein kinase inhibitors are currently in clinical trials for specific types of cancer. The members of Mixed Lineage Kinases (MLKs) are novel family of MAP3Ks and their physiological function is not known, however, we and others have shown that several of MLK family members activate Jun- N-terminal Kinases (JNKs) and MLK activation regulates survival in neuronal cells. Recently, we have seen that MLK3 is highly expressed in invasive breast cancer cell lines and in primary breast tumors, compared to the normal mammary tissue from same patient. Since MLK3 was initially identified from platelet cell line, CMK11-5, and association of venous thrombosis and occult cancer was recognized almost 140 years ago, we hypothesized that MLK3 or other MLKs are important in proteinase receptor-mediated signaling. Furthermore, the downstream effectors of proteinase receptors (PARs) that mediate the breast cancer cell growth, migration and metastasis to distal organs are currently unknown. We examined the role of MLK3 in thrombin mediated signaling in a highly invasive breast cancer cell line, MDA-MB-231. Thrombin treatment of these cells activated MLK3 kinase in a time dependent manner. MLK3 was activated within 2 minutes of thrombin treatment and the activity peaked at 5 minutes, and was associated with consequent activation of its downstream target JNK. Furthermore, pretreatment of MDA-MB-231 with an MLK inhibitor, CEP-11004 completely blocked thrombin-induced JNK activation. Importantly, we also discovered that thrombin induces MLK-dependent migration in MDA-MB-231 cell line was blocked by MLKs inhibitor, CEP-11004. These results collectively suggest that MLK3 is an important target of proteinase receptors-mediated migration/invasion/metastatic pathways, and help us to identify MLK family members’ inhibitors as viable drugs to treat metastatic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5200.