Abstract
PTPN6 (SHP1) is a tyrosine phosphatase that negatively controls the activity of multiple signaling pathways including STAT signaling, however role of mutated PTPN6 is not much known. Here we investigated whether PTPN6 might also be a potential target for diffuse large B cell lymphoma (DLBCL) and performed Sanger sequencing of the PTPN6 gene. We have identified missense mutations within PTPN6 (N225K and A550V) in 5% (2/38) of DLBCL tumors. Site directed mutagenesis was performed to mutate wild type (WT) PTPN6 and stable cell lines were generated by lentiviral transduction of PTPN6(WT), PTPN6(N225K) and PTPN6(A550V) constructs, and effects of WT or mutated PTPN6 on STAT3 signaling were analyzed. WT PTPN6 dephosphorylated STAT3, but had no effect on STAT1, STAT5 or STAT6 phosphorylation. Both PTPN6 mutants were unable to inhibit constitutive, as well as cytokines induced STAT3 activation. Both PTPN6 mutants also demonstrated reduced tyrosine phosphatase activity and exhibited enhanced STAT3 transactivation activity. Intriguingly, a lack of direct binding between STAT3 and WT or mutated PTPN6 was observed. However, compared to WT PTPN6, cells expressing PTPN6 mutants exhibited increased binding between JAK3 and PTPN6 suggesting a more dynamic interaction of PTPN6 with upstream regulators of STAT3. Consistent with this notion, both the mutants demonstrated increased resistance to JAK3 inhibitor, WHIP-154 relative to WT PTPN6. Overall, this is the first study, which demonstrates that N225K and A550V PTPN6 mutations cause loss-of-function leading to JAK3 mediated deregulation of STAT3 pathway and uncovers a mechanism that tumor cells can use to control PTPN6 substrate specificity.
Highlights
PTPN6, known as SHP1, is a ubiquitously expressed SH2 domain-containing Protein tyrosine phosphatases (PTP).[1,2,3] In humans, the PTPN6 gene is encoded by 17 exons and has 2 promoter regions.[4]
To further improve the clinical outcomes in diffuse large B cell lymphoma (DLBCL), current research is aimed on increasing the understanding of the pathophysiology of this disease by focusing on the study of the molecular mechanisms involved in the pathogenesis of lymphoma
The approval of the JAK1/JAK2 inhibitor Ruxolitinib in patients with myeloproliferative neoplasms[37] has increased investigations as to whether inhibiting Janus kinase (JAK)/ STAT signaling will be effective in DLBCL
Summary
PTPN6, known as SHP1, is a ubiquitously expressed SH2 domain-containing PTP.[1,2,3] In humans, the PTPN6 gene is encoded by 17 exons and has 2 promoter regions.[4]. Www.impactjournals.com/oncotarget binding to cell surface receptors triggering the activity of receptor-associated Janus kinase (JAK) family members.[33] Stimulation of cells expressing WT PTPN6 with IFN-α, IL-2, IL-6 or IL-10 resulted in decreased phosphorylation of STAT3 but not STAT5 as compared to the cells transduced with empty vector (Figure 2E). A dose dependent effect of WHI-P154 on STAT3 phosphorylation was observed in cells expressing PTPN6 mutants, and complete dephosphorylation was observed at 3-5 fold higher concentrations of WHI-P154, suggesting that loss of PTPN6 activity due to mutations may cause resistance to the JAK3 inhibition (Figure 4C). Overexpression of WT PTPN6 slightly protected against cell proliferation as compared to the vector alone, overexpression of PTPN6 mutants promoted cell proliferation (Figure 5D) Taken together these results indicate that via disrupting phosphatase activity of PTPN6, genetic mutations in PTPN6 potentiate oncogenic effects of STAT3
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