MLK3 promotes prooncogenic signaling in hepatocellular carcinoma via TGFß pathway.

Advanced hepatocellular carcinoma (HCC) is a lethal disease, with limited therapeutic options. Mixed Lineage Kinase 3 (MLK3) is a key regulator of liver diseases, although its role in HCC remains unclear. Analysis of TCGA databases suggested elevated MAP3K11 (MLK3 gene) expression, and TMA studies showed higher MLK3 activation in human HCCs. To understand MLK3's role in HCC, we utlized carcinogen-induced HCC model and compared between wild-type and MLK3 knockout (MLK3-/-) mice. Our studies showed that MLK3 kinase activity is upregulated in HCC, and MLK3 deficiency alleviates HCC progression. MLK3 deficiency reduced proliferation in vivo and MLK3 inhibition reduced proliferation and colony formation in vitro. To obtain further insight into the mechanism and identify newer targets mediating MLK3-induced HCCs, RNA-sequencing analysis was performed. These showed that MLK3 deficiency modulates various gene signatures, including EMT, and reduces TGFB1&2 expressions. HCC cells overexpressing MLK3 promoted EMT via autocrine TGFß signaling. Moreover, MLK3 deficiency attenuated activated hepatic stellate cell (HSC) signature, which is increased in wild-type. Interestingly, MLK3 promotes HSC activation via paracrine TGFß signaling. These findings reveal TGFß playing a key role at different steps of HCC, downstream of MLK3, implying MLK3-TGFß axis to be an ideal drug target for advanced HCC management.

Crosstalk between PD-L1 and Jak2-Stat3/ MAPK-AP1 signaling promotes oral cancer progression, invasion and therapy resistance.

BACKGROUND: Programmed cell death ligand-1 (PD-L1)is an antitumor immunity molecule and a great target to cure oral cancer; nonetheless, the limited success can be attributed to many complex pathways and tumor-related interferences. METHODS: In the present study, 150 human oral squamous cell carcinoma (OSCC) tissue samples, including 17 adjacent normals, 56 primary tumors, 47 invasive tumors, and 30 therapy-resistant (RT) samples, were included. The parental/cisplatin-resistant (CisR-SCC4/9) cells were utilized for overexpression (Jak1-3 wild type and catalytically inactive), knockdown (PD-L1 siRNA), targeting MAPK/PI3K/Jak-Stat pathways (SMIs) and checking microsomes. The expression of PD-L1, transcription factors (TFs), signaling pathways, survival/apoptosis, therapy resistance, and invasiveness-related molecules/their activity were determined by RT-PCR, Immunohistochemistry, Western blot, Gelatin Zymography, and MTT assay. RESULTS: Advanced OSCC tumors (invasive and drug-resistance), CisR-SCC4/9 cells, and secretory exosomes (CisR-SCC4/9) were found with increased PD-L1 expression. PD-L1 mRNA/protein showed a positive correlation with different TFs (AP1 > Stat3 > c-myc > NFκB) in tumor samples. The PD-L1 expression was more influenced by Jak-Stat/ MAPK-AP1 pathways over PI3K. The ectopic expression of Jak1-3 suggests Jak2 inducted PD-L1 level over Jak1/Jak3. Finally, PD-L1 directly supports survival (Bcl-xL, Bax, cleaved caspase-3), invasion (MMP2/9), and drug-resistance (ALDH-1A1/-3A1) program in OSCC through its link with several molecules. CONCLUSIONS: PD-L1 was regulated mainly by the Jak2-Stat3/ MAPK-AP1 pathway, and besides the routine immunological functions, it supports OSCC survival, invasion, and therapy resistance. PD-L1 can be used as an indicator of severity and can be targeted along with Jak2-Stat3/ MAPK-AP1 for a better outcome OSCC.

Linchpin-directed precise labeling of lysine in native proteins, purification, and analysis.

The proteins are critical building blocks of living systems and serve as a tool for their investigation and intervention. Their precision engineering enables its tuning and expands the functional landscape. Among various proteinogenic amino acids, high-frequency lysine offers a promising bioconjugation target. However, it is also among the most challenging candidates for homogeneous single-site modification. The linchpin-directed modification (LDM) addresses this concern by offering chemoselective, site-selective, and modular protein bioconjugation. The protocol outlines a general method for single-site modification of a native protein. At first, the selected LDM reagent constructs a stable bioconjugate through acylation of the Lys side chain. Subsequently, its chemically orthogonal handle creates an opportunity to install desired probes directly. Alternatively, the same group enables bioconjugate enrichment through ordered immobilization. The subsequent release, coupled with probe installation, renders analytically pure single-site tagged protein bioconjugate. The analysis of these constructs involves intact MS of protein bioconjugate, peptide mapping, and MS-MS for the site of modification and homogeneity.

Pluripotency transcription factor Nanog and its association with overall oral squamous cell carcinoma progression, cisplatin-resistance, invasion and stemness acquisition.

BACKGROUND: Cisplatin-resistant oral squamous cell carcinoma (OSCC) cells acquire stem-like characteristics and are difficult to treat. Nanog is a transcription factor and needed for maintenance of pluripotency, but its transcription-promoting role in OSCC progression and cisplatin resistance is poorly understood. METHODS: Here, 110 fresh human tissue specimens of various stages, including invasive (N1-3 )/chemoradiation-resistant OSCC samples, cisplatin-resistant (CisR-SCC-4/-9) OSCC cells/parental cells, photochemical ECGC, and siRNA (Nanog) were used. RESULTS: Nanog overexpression was associated with overall progression, chemoresistance, and invasion of OSCC. Nanog recruitment to c-Myc, Slug, E-cadherin, and Oct-4 gene promoter was observed. Positive correlation of Nanog protein expression with c-Myc, Slug, cyclin D1, MMP-2/-9, and Oct-4 and negative correlation with E-cadherin gene expression were found. Knockdown of Nanog and treatment of epicatechin-3-gallate reversed cisplatin resistance and diminished invasion/migration potential. CONCLUSION: Nanog directly participated in the regulation of Slug, E-cadherin, Oct-4, and c-Myc genes, causing cisplatin resistance/recurrence of OSCC.

Role and regulation of proapoptotic Bax in oral squamous cell carcinoma and drug resistance.

BACKGROUND: Bax, a proapoptotic protein but its regulation during oral cancer progression and resistance remains elusive. METHODS: A total of 127 samples including adjacent normal, primary tumor, and resistance to chemoradiation therapy (RCRT) samples from oral squamous cell carcinoma (OSCC) patients were used. The status of Bax was analyzed at DNA/mRNA/protein levels and the results were correlated with p53 and Akt expression in tissue samples/cisplatin-resistant oral tongue SCC (SCC9/SCC4-CisR) cell line. RESULTS: Frequent progressive decrease of Bax expression with infrequent promoter methylation, polymorphisms G(-248)A, and mutations was observed in OSCC progression/resistance. Furthermore, by targeting Akt pathway, induction of Bax-dependent cell death was observed and this was further enhanced with nimbolide treatment in SCC9/SCC4-CisR cells. CONCLUSION: Hence, the Bax gene alteration and its deregulation through p53/Akt pathway are important for OSCC progression and drug resistance. Akt Inhibitor VIII and nimbolide synergistically induce Bax, and it is therefore beneficial for chemosensitizing cisplatin-resistant human OSCC.

Crosstalk between Raf-MEK-ERK and PI3K-Akt-GSK3ß signaling networks promotes chemoresistance, invasion/migration and stemness via expression of CD44 variants (v4 and v6) in oral cancer.

BACKGROUND: The cell-surface glycoprotein CD44 is an important oral cancer stem cell (OCSC) marker and plays significant role in oral squamous cell carcinoma (OSCC) aggressiveness, however, the regulation of CD44 is incompletely understood. METHODS: In the present study, 145 fresh human OSCC tissue specimens, including 18 adjacent normal, 42 noninvasive (N0), 53 invasive tumor samples (N1-3) and 32 chemo-radiation resistant samples (RCRT), were included. The expression of CD44 standard (CD44s) and variants (CD44v4, CD44v6); the activation of pERK1/2, GSK3ß, NICD (Notch) pathways; the cell viability; and the MMP-9/-2 activity were assessed using RT-PCR, immunohistochemistry, Western blotting, MTT assay and gelatin zymography. OSCC cell lines, including parental (SCC9/SCC4) and Cisplatin-resistant (CisR-SCC9/-SCC4) cells, were used. Knock down of CD44v4/CD44v6 (by siRNA) or inactivation of MAPK/PI3K pathways using specific PD98059/LY294002 was achieved for in vitro analysis of chemoresistance and invasion/migration. RESULTS: Elevated CD44 variants were associated with overall OSCC progression, chemoresistance and invasion. Positive correlations were observed, mainly between the expression of CD44v4 and the activation of ERK1/2 causing chemoresistance, whereas CD44v6 expression and inactivation of GSK3ß caused invasiveness of OSCC. Cisplatin resistant, CisR-SCC9/SCC4 cell lines showed OCSC properties. Inhibition of MEK/ERK1/2 by SMI or knock down (KD) of CD44v4 by siRNA reversed cisplatin-resistance, whereas blocking the PI3K/Akt/GSK3ß pathway by SMI or KD of CD44v6 isoforms by respective siRNA diminished invasion/metastasis potential. CONCLUSION: Collectively, our results demonstrated that CD44v4 expression is more linked with ERK1/2 activation and promote cisplatin resistance, whereas CD44v6 expression is associated primarily with PI3K/Akt/GSK3ß activation and driving tumor invasion/migration.

Glycogen synthase kinase-3ß mediated regulation of matrix metalloproteinase-9 and its involvement in oral squamous cell carcinoma progression and invasion.

PURPOSE: Oral squamous cell carcinoma (OSCC)-related deaths mainly result from invasion of the tumor cells into local cervical lymph nodes. It has been reported that progressive basement membrane loss promotes the metastatic and invasive capacities of OSCCs. Matrix metalloproteinase-9 (MMP-9) is known to play a central role in tumor progression and invasion. However, the role of MMP-9 in OSCC invasion has so far remained paradoxical and little is known about its regulation. Here, we aimed to assess MMP-9 expression regulation and its activation by glycogen synthase kinase-3ß during human OSCC progression and invasion. METHODS: In the present study, 178 human OSCC samples, including 118 fresh samples (18 adjacent normal, 42 noninvasive and 58 invasive tumor samples) and 60 archival human tissue microarray (TMA) tongue cancer samples, were included. mRNA expression, protein expression, MMP-9/-2 activity, protein-protein interaction and Snail, c-Myc, ß-catenin and TIMP1 expression were assessed using RT-PCR, immunohistochemistry, Western blotting, co-immunoprecipitation and gelatin zymography analyses, respectively. Wnt5a and LPA mediated MMP-9 regulation was assessed in OCSCC-derived SCC-9 cells exogenously expressing GSK3ß (WT) or non phosphoryable GSK3ß (S9A). RESULTS: We observed a progressive up-regulation/activation of MMP-9 at various stages of oral tumor progression/invasion. Positive correlations were observed between MMP-9 and c-Myc expression, MMP-9 and MMP-2 activity, MMP-9 and TIMP1 expression and MMP-9 activity and TIMP1-MMP-9 interaction. In contrast, a negative correlation between phosphorylated ß-catenin and MMP-9 expression was observed. Conversely, we found that in oral tongue SCC MMP-9 expression was positively correlated with inactivation of GSK3 signaling. Finally, we found that Wnt5a and LPA mediated increased MMP-9 and decreased GSK3ß activities in tongue SCC-derived SCC-9 cells. MMP-9 regulation by GSK3ß was confirmed by using phosphoryable/regulatory GSK3ß (WT construct) and not by non-phosphoryable GSK3ß (S9A construct). CONCLUSIONS: Collectively, our results show that MMP-9 overexpression and activation are important events occurring during OSCC progression/invasion and that this overexpression/activation is regulated by c-Myc, active MMP-2 and inactive GSK3ß mediated pathways.

The elevated activation of NFκB and AP-1 is correlated with differential regulation of Bcl-2 and associated with oral squamous cell carcinoma progression and resistance.

OBJECTIVES: Oral cancer is the sixth most common cancer in the world. Failure of chemoradiation therapy is a major concern for treating oral cancer patients. The objective of this study is to determine the B cell lymphoma-2 (bcl-2) expression and its regulation by nuclear factor κB (NFκB) and activator protein 1 (AP-1) in oral cancer progression and chemoradiation resistance. MATERIALS AND METHODS: In the present study, a total of 123 (n = 123) human samples were included. Briefly, 64 fresh samples were from adjacent normal (AN), primary oral tumors without treatment (PT), and tumors with resistance to chemoradiation therapy with local recurrence (RCRT). Fifty-nine samples were human tongue cancers and normal samples (TMA). Messenger RNA (mRNA) expression levels of bcl-2 and protein levels of bcl-2, NFκB, AP-1, and inactive GSK3α/ß were measured by semiquantitative RT-PCR, immunohistochemistry, Western blot, and ChIP analysis. RESULTS: Increased bcl-2 expression was observed in PT compared to AN. The RCRT tumors showed maximum expression of bcl-2 mRNA and protein over the PT and AN groups. Bcl-2 protein and mRNA expression were positively correlated with NFκB and AP-1 expression. AP-1 expression was strongly correlated with bcl-2 in the RCRT group of tumors. Further, inactive GSK3α/ß showed a positive trend with bcl-2 expression in oral tongue cancer specimens. CONCLUSION: Collectively, our results demonstrated cumulative effect of AP-1 and NFĸB for bcl-2 gene regulation in overall PT progression and chemoradiation resistance. The study provides evidence of increased bcl-2 mRNA/protein fueled by NFĸB in PT and AP-1 in RCRT. These regulations of bcl-2 by NFκB and AP-1 are important in OSCC progression and chemoradiation resistance.

Reversion-inducing cysteine-rich protein with Kazal motifs and its regulation by glycogen synthase kinase 3 signaling in oral cancer.

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) and glycogen synthase kinase (GSK3) are novel tumor suppressors, and emerging evidence has suggested their active role in oral cancer pathogenesis. In the present study, 112 human samples, including 55 fresh samples of 14 adjacent normal tissues, 25 noninvasive oral tumors, and 18 invasive tumors, were included. The messenger RNA (mRNA) expression, protein expression, and promoter methylation of the RECK gene, as well as the expression of GSK3ß, phospho/total ß-catenin, and c-myc, were measured by RT-PCR, bisulphate modification-PCR, immunohistochemistry, and Western blot analysis. Additionally, ectopic expression of in/active GSK3ß was performed in cell culture experiments. This study provided information on the progressive silencing of RECK gene expression at the protein and mRNA levels paralleled with promoter hypermethylation at various stages of oral tumor invasion. RECK expression and the hypermethylation of the RECK gene promoter were negatively and positively correlated with pS9GSK3ß/c-myc expression, respectively. Further, a negative trend of RECK protein expression with nuclear ß-catenin expression was observed. Induced expression of active GSK3ß reversed the RECK silencing in SCC9 cells. Collectively, our results demonstrated that the silencing of the RECK gene, possibly regulated by the GSK3ß pathway, is an important event in oral cancer invasion and this pathway could be exploited for therapeutic interventions.