In current study, we provide evidence for the first time that MKP1 inhibitor TPI-3 sensitized cancer cells to IFNα2b and 5FU in vitro and in mice. TPI-3 sensitized WM9 human melanoma cells in vitro and WM9 xenografts to the biotherapeutic IFNα2b (). It also sensitized MC-26 colon cancer cells to colon cancer chemotherapeutic 5FU in vitro and in mice (). These actions of the compound were likely mediated via targeting MKP1 given that TPI-3 was an inhibitor of recombinant MKP1 () and capable of increasing MKP1 phospho-substrates in both WM9 and MC-26 cells ( and ). Our data thus indicate that targeting MKP1 could be an effective strategy for sensitizing differential cancers to both bio- and chemo-therapeutics in vitro and in vivo. Moreover, the tolerance of TPI-3 and its combinations with cancer therapeutics suggests safety for the strategy and supports further translational evaluation. Additional studies are clearly needed to define the role of MKP1 as the target of TPI-3. In particular, assessment of the responses of cancer cells with differential MKP1 expression levels to TPI-3 could be of significance and may provide mechanistic insights.
The effectiveness and tolerance of TPI-3 in sensitizing melanoma and colon cancer to therapeutics in mice also suggested the potential of the compound as a lead for developing MKP1-targeted agents to overcome drug resistance. The oral availability ( and ) of TPI-3 further increases its attractiveness for translational development. In addition, its chemical characteristics (data not shown) show no violation of the Lipinski's Rule of Five and the Extensions (48
), indicating good druggability. Moreover, the higher potency of TPI-3 in comparison to its structurally related TPI-2, L6a1 or L6a2 suggests potential and insights for improving potency via structural modifications. In addition, TPI-3 analogs may be exploited for developing more selective inhibitors. TPI-3 displayed selective actions for MKP1 since it was inactive for SHP-1 under the experimental conditions ( and ). It is also encouraging that L6a1, although inactive for MKP1 ( and ), selectively increased pERK1/2 (), suggesting that it targets a pERK1/2- specific phosphatase (e.g., MKP3) (18
). The limited structural differences between TPI-3 and L6a1 () implicate feasibility of modulating target specificity via minor chemical variations. Whereas the full spectrum of TPI-3-targeted phosphatases remains to be established, it is worth noting that several inhibitors targeting multi-kinases have been approved as effective and tolerated cancer therapeutics (e.g., imatinib and sunitinib), suggesting translational potential for inhibitors with multi-phosphatases targets. Our data together provide a basis for developing refined inhibitors against MKPs (or other phosphatases) and for biological evaluations of new candidate inhibitors.
Our results also provide the first evidence implicating MKP1 over-expression as a drug-resistant mechanism in melanoma. MKP1 over-expression was detected in human melanoma cell lines and advanced human melanoma tissues (). Its functional significance in therapeutic resistance was suggested by the sensitization of WM9 melanoma to IFNα2b in vitro
and in mice (). Advanced melanoma responds poorly (~10%) to each of the three approved melanoma treatments (IFNα2b, IL-2 and dacarbazine) 4 (49
). As indicated by the TPI-3 activity in sensitizing WM9 melanoma to IFNα2b (), MKP1-targeted agents might be combined with melanoma therapeutics as more efficacious treatment options. Indeed, TPI-3 also near significantly increased WM9 tumor growth inhibition induced by the dacarbazine analog temozolomide or its combination with methoxyamine (50
) (data not shown). Moreover, the capacity of TPI-3 to sensitize WM9 melanoma () that harbors the B-RafV600E mutant (our unpublished data) with heightened downstream pERK1/2 levels () also suggests that TPI-3 and other MKP1 inhibitors might be complementary with B-Raf inhibitors in anti-tumor actions and could be exploited for combination treatments. Mutations of B-Raf, N-Ras and c-kit have been reported in melanoma, leading to active oncogenic molecules. Since MKP1 substrates are signaling molecules downstream of the onco-proteins, targeting MKP1 with TPI-3 is expected to be effective in melanoma with each of the mutant genotypes. Comparative evaluation of TPI-3 responses by melanoma cells with differential genotypes will be informative in this regard and could have implications for MKP1-targeted agents. Interestingly, low MKP1 expression levels were detected in ~ 10% samples of the advanced melanoma tissues () and may correlate with better clinical responses that might be exploited for pre-selecting potential responding cases.
Among the three MKP1 phospho-substrates, pJNK was the only one that was consistently induced by TPI-3 in Jurkat, WM9 and MC-26 cells (, and ). Thus pJNK was implicated as a key mediator for TPI-3 in pro-apoptotic action and anti-tumor action and could be further evaluated to define the mechanism of action of the compound and its significance as a biomarker. Since MKP1 negatively regulates innate and adaptive immune responses (18
), targeting the phosphatase might lead to immune cell activation. Indeed, TPI-2 and TPI-3 were capable of increasing mouse splenocyte IFNγ+ cells in vitro
(our unpublished data) although immune cell activation was apparently not required for TPI-3 sensitization of WM9 melanoma to IFNα2b since the sensitization occurred in vitro
in the absence of immune cells and in T-cell deficient nude mice (). Despite its effectiveness in inducing cancer cell death in vitro
(), TPI-3 did not exhibit significant activity as a single agent against WM9 xenografts and had only a modest effect on MC-26 tumors although it did sensitize both to cancer therapeutics ( and ). This might be related to the lower doses (≤ 10 ng/ml) of TPI-3 required for sensitization in comparison to its death-induction doses (~100 ng/ml) ( and ). Taken together, our results provide a strong basis for elucidating the mechanism of action of MKP1 inhibition and for potential translation in future studies.