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.

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.