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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Mol Cancer Ther. Author manuscript; available in PMC 2011 August 1.
Published in final edited form as:
PMCID: PMC2942023
NIHMSID: NIHMS231505

TPI-3 sensitizes melanoma and colon cancer to bio- and chemotherapeutics

Abstract

Drug resistance is a major obstacle in cancer treatments and diminishes the clinical efficacy of biological, cytotoxic or targeted therapeutics. Being an anti-apoptotic mediator of chemo-resistance in breast and lung cancer cells, MKP1 phosphatase might be targeted for overcoming chemo-resistance and improving therapeutic efficacy. In this work, TPI-3 was identified as a novel small molecule inhibitor of MKP1 and capable of sensitizing tumors to bio- and chemo-therapeutics in mice as a tolerated oral agent. Effective against recombinant MKP1, TPI-3 selectively increased MKP1 phospho-substrates in Jurkat cells and induced cell death via apoptosis at nM concentration. TPI-3 also increased MKP1 phospho-substrate in WM9 human melanoma cells and synergized with bio-therapeutic IFNα2b in growth inhibition of the melanoma cells in vitro (C.I. < 1). WM9 xenografts unresponsive to individual agents were significantly inhibited (62%, p = 0.001) in mice by a tolerated combination of oral TPI-3 (10 mg/kg, 5d/week) and IFNα2b. MKP1 expression was detected in human melanoma cell lines and tissues samples at levels up to 6 x higher than those in normal or non-malignant melanocytes. TPI-3 also interacted positively with chemotherapeutics 5FU/LV against MC-26 colon cancer cells in vitro and in mice. Our data together demonstrate pre-clinical activities of TPI-3 in overcoming cancer resistance to bio- and chemotherapeutics, implicate MKP1 as a drug-resistant molecule in melanoma and support targeting MKP1 for improving cancer therapeutic efficacy.

Keywords: Melanoma, MKP1, IFNα2b, 5FU and colon cancer

INTRODUCTION

Drug resistance is common in many types of cancers. It could be intrinsic but is often acquired following initial treatments. It is a major obstacle for clinical efficacy of biological, cytotoxic or targeted therapeutics (1-4). Identification of mediators of drug resistance and understanding their mechanism of action could provide insights for overcoming resistance and lead to novel approaches for improving cancer treatments.

MKP1 (MAPK phosphatase-1, DUSP1, and CL-100) was identified recently as a mediator of resistance to several chemotherapeutics in breast and lung cancer cells (5-7). Transient or stable over-expression of MKP1 in breast cancer cells enhanced viability in the face of treatment with alkylating agents (mechlorethamine), anthracylines (doxorubicin), and microtubule inhibitors (paclitaxel) (5). Over-expression of MKP1 rendered lung cancer cells resistant to cisplatin, whereas knocking down MKP-1 expression with siRNA sensitized lung cancer cells to cisplatin (6, 7). The clinical significance of these observations is indicated by the frequent over-expression of MKP1 in many cancer tissues including breast cancer, colon cancer, lung, prostate cancer, ovarian cancer and pancreatic cancer (6, 8-12). Potential clinical impact is underscored by the association of MKP1 over-expression with early transformation and poor prognosis (9-11).

MKP1 mediates chemo-resistance in a significant part via its anti-apoptotic activity through dephosphorylating/inactivating JNK, a pro-apoptoic signaling molecule (13). Over-expression of MKP1 in breast cancer cells reduced Caspase activation induced by chemotherapeutics (5). Knocking down MKP1 expression in cancer cells increased apoptosis (12, 14). Among the MKP1 substrates (JNK, p38 and ERK1/2) (15, 16), JNK was reported as a key molecule in MKP1 anti-apoptotic action. Inducible MKP1 expression blocked JNK activation and inhibited apoptosis (17) whereas suppression of MKP1 expression potentiates JNK activation coincident with apoptosis (14). Interestingly, MKP-1 is also a negative regulator of innate and adaptive immune responses (18).

Being an anti-apoptotic mediator of chemo-resistance in several types of cancers (6, 8-12, 19), MKP1 is a potential cancer therapeutic target. Indeed, MKP1 inhibitory compounds active at low mM range induced cancer cell death in culture and sensitized cancer cells to chemotherapeutics in vitro (20). However, the effectiveness and safety of targeting MKP1 as an anti-cancer strategy in vivo remain to be established. MKP1 inhibitors with pre-clinical anti-tumor activity in vivo have not been reported. It has not been clear whether MKP1 is significant also in cancer resistance to bio-therapeutics.

In this work, a novel MKP1 inhibitor was identified and named as TPI-3 (tyrosine phosphatase inhibitor-3). Being a small organic compound (274 Da) with defined structure (Fig 1) and of little prior interest, TPI-3 has not been reported for biological activities. The compound was included in chemical collections for high throughput screening and found inactive for the measles virus RNA polymerase or 14-3-3 molecule (pubchem). Herein, we provide pre-clinical evidence demonstrating that TPI-3 improved the efficacy of bio- and chemo-therapeutics for melanoma and colon cancer in mice as a tolerated oral agent. Our data suggest that targeting MKP1 could be an effective and tolerated strategy for overcoming resistance to multiple types of anti-cancer agents and implicate TPI-3 as a promising lead compound for further development.

Fig 1
Identification of MKP1 inhibitors TPI-2 and TPI-3. A) Chemical structure of TPI-2 (L6). B) Jurkat cells treated with TPI-2 for 30 min were analyzed by Western blotting to quantify pERK1/2 and ERK1/2 levels. C) Chemical structures of TPI-2 analogs. D) ...

MATERIALS AND METHODS

Cells, cell culture and reagents

Cancer cell lines including Jurkat (21), WM9 (22), A375 (23), MCF-7 (24), DU145 (25), SW 620 (26) and MC-26 (27) were obtained from colleagues and cultured in RPMI 1640 or DMEM medium supplemented with 10% fetal calf serum. The effects of chemical compounds on cancer cell growth in culture were quantified by MTT assays following established procedures (28). Normal human melanocytes (NHM) and human melanoma cell lines were from the institutional melanoma core.

Recombinant human IFNα2b (specific activity 2 × 108 units/mg protein, Intron A, Schering-Plough, Kenilworth, NJ), recombinant MKP1 and recombinant SHP-1 have been described previously (29). TPI-2 (L6, Chembridge, San Diego, CA), its analogs (a1-a6, Chembridge, San Diego, CA), 5FU (Sigma, St Luis, MO) and leucovorin (Sigma, St Luis, MO) were purchased from the indicated commercial sources. Antibodies to pERK1/2, ERK1/2, p-p38, pJNK, pLck-pY394, pZAP70 and actin were obtained from a commercial source (Cell Signaling, Danvers, MA).

Chemical library, databases and phosphatase assay

A library of drug-like chemical compound was from commercial source (Chembridge, San Diego, CA). Upon identification of TPI-2 as a MKP1 inhibitory lead, we sought to derive improved compounds. However, the lack of crystal or solution structure of MKP1 has prevented rational design for analogs. Moreover, the limited sequence homology (~50%) of the MKP1 protein to the other related phosphatases with resolved structures also hindered the development of a computer-assisted 3D model. Accordingly, we initially focused on identification and evaluation of analogs with chemical structural similarities to TPI-2. Analogs of TPI-2 were identified from chemical databases by computer-assisted structure analysis. Briefly, chemical structure of TPI-2 was compared to individual structures in commercial chemical databases (Chembridge, San Diego, CA and Asinex, Winston-Salem, NC) of approximately one million small organic compounds, utilizing the software at the commercial sites for calculating structural similarities with TPI-2. Six compounds with similarities ≥ 70% with TPI-2 were selected and purchased from commercial source (Chembridge, San Diego, CA). Their effects on the phosphatase activity of recombinant MKP1 in vitro were evaluated following established procedures (29).

Induction and detection of cellular phospho-proteins

Cells in culture medium were treated with agents for designated times and concentrations at 37°C and lysed on ice for 30 min in cold lysis buffer (1% NP40, 50 mM Tris, pH 7.4, 150 mM NaCl, 20 mM NaF, 0.2 mM Na3VO4) containing a cocktail of proteinase inhibitors (Sigma, 1 tablet/10 ml). The lysates were cleared by centrifuging (14,000 rpm, 10 min) in a microfuge at 4°C to remove insoluble parts, mixed with equal volume of 2 x SDS-PAGE sample buffer, boiled for 5 min and analyzed by SDS-PAGE/Western blotting with commercial antibodies as described previously (28, 30, 31). Relative intensities of phospho-protein bands were quantified through densitometry analysis.

Induction and quantification of apoptotic cells

Jurkat cells were cultured for 16 hrs in the presence of TPI-2, a1, a2 or TPI-3 (0, 0.1 and 1 μg/ml), washed 3 x in PBS and stained with the DNA dye 7-AAD (Invitrogen, Carisbad, CA) and PE-conjugated annexin V antibody (BD Biosciences, San Jose, CA) in darkness at room temperature for 30 minutes. Following staining, the samples were washed 3 times, re-suspended in 200 μl of 1% para-formaldehyde solution and analyzed (20,000 cells/sample) using a BD FACS Caliburs cytometer as described previously (32, 33).

Statistic analysis

Median effect analysis (34), which provides the most general form of studying the interactions between drugs, was utilized to analyze the interaction between TPI-3 and IFNα2β. Median effect plots were generated for IFNα2b alone, TPI-3 alone, and the combination in inhibiting the growth of WM9 melanoma cells in culture. The combination index (CI) was determined and plotted vs. fraction affected (fa). Data were analyzed in both modes, mutually exclusive and mutually nonexclusive. The interaction between two mutually nonexclusive drugs is described by the Equation CI = D1/Dx1 + D2/Dx2 + D1D2/Dx1Dx2, where Dx1 and Dx2 are the doses of drug 1 and drug 2 that are required to inhibit growth x%. D1 and D2 in combination also inhibit growth x% (i.e. drug 1 and drug 2 are iso-effective). When CI < 1, drugs are synergistic, when CI = 1, drugs are additive, and when CI > 1, drugs are antagonistic. Student's t test was used to assess the significance of the effects of different treatments against tumors in mice.

Evaluation of MKP1 expression in melanoma cell lines and melanoma tissues

The expression levels of MKP1 transcripts in normal human melanocytes (NHM) and human melanoma cell lines were quantified by gene-expression array analysis following established procedures (35). Briefly, RNA samples were harvested from cells (~80% confluence) lyzed in Trizol (Invitrogen, Carisbad, CA) and evaluated with the Illunima Sentrix Human-6_v2 Expression BeadChip technology. Data were analyzed with BeadStudio V3.2 software. Total cell lysates were also prepared (28) from cancer cell lines and endothelial cell line EA.hy926 (36). The protein levels of MKP1 in the lysates were quantified by SDS-PAGE/Western blotting (28) using a commercial antibody for the phosphatase (sc-1109, Santa Cruz Biotech, Santa Cruz, CA).

MKP1 expression in human melanoma tissue samples was investigated via immunohistochemistry (IHC) analysis of an institutional melanoma micro-tissue array. The array was consisted of formalin-fixed, paraffin-embedded tissues of human melanoma samples of radial growth phase (15) or vertical growth phase (11) and tissue samples of non-malignant human nevus (5). Sections of the array were prepared and stained for IHC (32) with a commercial MKP1 antibody (sc-1109, Santa Cruz Biotech, Santa Cruz, CA) that was reported for IHC detection of the human molecule (37). The relative MKP1 staining levels in melanoma cells and adjacent benign melanocytes in the tissue samples were estimated via microscopy.

Animal and animal studies

To evaluate the interactions of TPI-3 with IFNα2b against melanoma in vivo, athymic nude mice (nu/nu, NCR, female, 6 weeks old, Taconic Farm, Germantown, NY) were inoculated (s.c.) in the flanks with WM9 human melanoma cells (2 × 106 cells/site). On day 4 post-inoculation, the mice were subjected to treatment with vehicle control, TPI-3 (10 mg/kg, po, 5d/wk) via oral gavage, IFNα2b (500,000 U/mouse, s.c., 5d/wk) or the combination. The TPI-3 dose was chosen based on its tolerance in mice in a pilot experiment. The dose of IFNα2b used for treatment was comparable to those in previous studies (28, 38) but delivered only 5 days per week instead of the previous daily treatment. For assessing interactions between TPI-3 and 5FU-LV, Balb/c mice (female, 6 weeks old, Taconic Farm, Germantown, NY) were inoculated with MC-26 colon cancer cells (s.c., 106 cells/site). Starting on day 4 post-inoculation, the mice were treated with vehicle control, TPI-3 (10 mg/kg, po, 5d/wk), 5FU-LV (50 mg/kg-100 mg/kg, ip, weekly) or the combination. The 5FU-LV treatment was similar to those reported previously (39). Tumor volume was measured and calculated using the formula for a prolate spheroid (V = 4/3 π a2b) (40). Mouse viability and body weights were also recorded. Internal organs of the mice were inspected visually upon their termination at the end of the experiment. All studies involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) of the Cleveland Clinic.

RESULTS

Identification of MKP1 inhibitors TPI-2 and TPI-3

Given the role of MKP1 in cancer chemo-resistance, we sought to identify small molecule inhibitors for the phosphatase. Lead six (L6, Fig 1A), a compound from a chemical library, increased MKP1 phospho-substrate pERK1/2 (41) in Jurkat leukemia cells (Fig 1B), in which MKP1 and the substrate were expressed (42). This prompted us to investigate the MKP1 inhibitory activities of L6 and its analogs, six (L6a1-6, Fig 1C) of which were identified based on structural similarities to the parental compound. L6 and analog L6a6 reduced the activity of recombinant MKP1 in vitro whereas the other analogs were less or not effective (Fig 1D). These data indicated L6 and L6a6 as MKP1 inhibitors. They were named as TPI-2 and 3 (tyrosine phosphase inhibitor-2 and 3) for further investigations.

The potency and selectivity of the compounds for cellular MKP1 were evaluated via quantification of cellular MKP1 phospho-substrates levels. TPI-2 and TPI-3 increased all three of the MKP1 phospho-substrates in Jurkat cells with effective concentrations at 0.1 – 1 μg/ml (Fig 2A, left panel), suggesting a capacity to inhibit the cellular MKP1 (cellular IC50 ~ 0.1-1 μg/ml). Consistent with their lack of activities in vitro (Fig 1D), analog L6a1 only increased pERK1/2 but not the other MKP1 phospho-substrates whereas L6a2 had impact on none of the three phospho-substrates (Fig 2A, left panel). Suggesting a selective action, TPI-2 at 0.001 – 1 μg/ml had no apparent impact on SHP-1 phospho-substrates (pLck-pY394 and pZAP-70) in Jurkat cells (Fig 2B and D), in which the expression of these molecules were reported (43), or on the general cellular phospho-proteins (Fig 2C). Similarly, TPI-3 treatment also had little effects on the cellular SHP-1 substrates (Fig 2D). In further support, TPI-2 and TPI-3 at 1 μg/ml or lower concentrations had little effects on the activity of recombinant SHP-1 (data not shown). Moreover, the durability of cellular MKP1 phospho-substrates induced by TPI-3 was substantial at more than 4 hrs (Fig 2A, right panel), indicating a significant impact of TPI-3 on these substrates. These data together provided evidence that intracellular MKP1 was selectively inhibited by TPI-2 or –3 at ~1 μg/ml for duration of more than 4 hrs.

Fig 2
TPI-2 and TPI-3 selectively increase MKP1 phospho-substrates in Jurkat cells in vitro. A) Jurkat cells were treated with TPI-2 or the analogs at different doses for 10 min. Total cell lysates were analyzed by Western blotting for MKP1 phospho-substrates ...

TPI-2 and TPI-3 induce cell death via apoptosis

Being an anti-apoptotic molecule in cancer cells, MKP1 might be targeted with inhibitors to induce cell death via apoptosis. TPI-2, TPI-3 and the negative control analogs a1 and a2 were therefore evaluated for their capacities to induce Jurkat cell apoptosis and impact cancer cell viability in vitro.

Apoptotic cells, as indicated by surface annexin V expression, were induced markedly by TPI-2 (52%) and TPI-3 (58%) in Jurkat cells co-cultured with the compounds at 1 μg/ml for 16 hrs (Fig 3A). The two leads at a lower dose (0.1 μg/ml) also induced apoptotic cells with less potency (~ 23-28%) (Fig 3A). Analog a1 failed to induce cell apoptosis under comparable conditions whereas a2 was modestly effective only at 1 μg/ml (Fig 3A). Jurkat cells were completely dead when cultured in the presence of TPI-2 or TPI-3 at 0.3 μg/ml or higher levels for 6 days (Fig 3B) but were only partially inhibited (<50%) by the other analogs (Fig 3B).

Fig 3
TPI-2 and TPI-3 induce apoptosis in Jurkat cells and cell death in multiple cancer cell lines. A) Jurkat cells cultured in the presence of vehicle control, TPI-2 or its analogs for 16 hrs were stained with the DNA dye 7-AAD and PE-conjugated anti-annexin ...

TPI-2 and TPI-3 also induced cell death in a panel of cancer cell lines in vitro whereas the control a2 was not effective (Fig 3C-H). The responsive cell lines include those of melanoma (WM9 and A539), breast cancer (MCF-7), prostate cancer (DU-145) and colon cancer (SW620 and MC-26).

These results demonstrated TPI-2 and TPI-3 had pro-apoptotic activity in Jurkat cells (Fig 3) at concentrations effective for MKP1 inhibition (Fig 2) and suggested a similar action for the leads in various cancer cell lines in vitro.

TPI-3 interacts positively with IFNab in growth inhibition of WM9 melanoma in vitro and in mice

Being an anti-apoptotic mediator of chemo-resistance, MKP1 might be targeted with inhibitors for increased responses to cancer therapeutics. Accordingly, TPI-3 was evaluated of its activity to overcome drug-resistance in cancer cells. Since WM9 human melanoma cells were resistant to growth inhibition by melanoma therapeutic IFNα2b (28), the capacity of TPI-3 to sensitize the melanoma cells to IFNα2b in vitro and in vivo was determined.

TPI-3 enhanced growth inhibition of WM9 cells by IFNα2b in culture (Fig 4A) in a synergistic manner (CI < 1.0) as defined by Median Effect analysis (40) . The two agents also interacted positively against WM9 xenografts in nude mice (Fig 4B). Their combination significantly inhibited WM9 tumor growth (62%, p < 0.001), in contrast to the limited effects from single agents (Fig 4B). It remained effective beyond the point when the control mice had to be terminated due to large tumor burden and tumor ulceration (Fig 4B). TPI-3 alone and its combination were tolerated: all the mice survived to the end of study with comparable body weights (Fig 4C) and no obvious abnormalities in behaviors or gross anatomy (data not shown).

Fig 4
TPI-3 synergizes with IFNα2b in growth inhibition of WM9 melanoma cells in vitro and sensitized WM9 tumors to IFNαb2 therapy in mice. A) WM9 cells were cultured in the absence or presence of TPI-3, IFNα2b or the combinations for ...

For mechanistic insights, the impact of TPI-3 on phospho-substrates of MKP1 in WM9 cells in culture was determined. TPI-3 increased cellular pJNK levels and was effective starting at ~ 0.1 μg/ml (Fig 4D). pJNK in WM9 cells was increased also by TPI-2 but not by control analog a1 or a2 (Fig 4D). Cellular pERK1/2 and pp38 were present at high basal levels in WM9 cells and ~ 10 x higher than those in the Jurkat cells (data not shown). They were not increased further by TPI-3 treatment (Fig 4D). The samples were further evaluated for pStat1 and pStat3 molecules. Cellular levels of pStat1 and pStat3 in WM9 cells were not significantly affected by treatments with TPI-3 (0.001-1 μg/ml) under the experimental conditions (data not shown).

These results demonstrated that TPI-3 sensitized IFNα2b-refractory WM9 melanoma to the cytokine in vitro and in vivo. Consistent with targeting MKP1, TPI-3 increased pJNK levels in the melanoma cells that expressed the phosphatase. Its lack of impact on cellular pERKs and pp38 was likely due to high basal levels of the phospho-proteins, which were downstream events of the activating B-RafV600E mutation (44, 45) that was common in melanoma (46) and detected in WM9 cells (data not shown). Given the synergy between TPI-3 and IFNα2b (Fig 4), additional study is needed to assess the impact of TPI-3 on IFNα2b signaling in WM9 cells and may provide mechanistic insights.

MKP1 is expressed at significant levels in human melanoma cell lines and melanoma tissue samples

The capacity of TPI3 to sensitize WM9 melanoma to IFNα2b supported targeting MKP1 for improving melanoma treatments. However, it was unclear whether and at what levels MKP1 was expressed in human melanoma. We therefore assessed the expression of MKP1 in human melanoma cell lines and melanoma tissue samples.

MKP1 transcripts in human melanoma cell lines and normal human melanocytes (NHM) cultured under comparable conditions were quantified by gene-expression array analysis as described previously (35). The MKP1 transcripts were significantly higher (~ 1 – 6 fold) in the melanoma cell lines in comparison to NHM (Fig 5A). Expression of the MKP1 protein in human melanoma cell lines and several other cancer cell lines was verified (Fig 5B and C).

Fig 5
MKP1 is expressed at significant levels in human melanoma cell lines and tissue samples. A) MIP1 transcripts in normal human melanocytes (NHM) and human melanoma cell lines were quantified by array analysis (signals + SD). B) Expression of MKP1 protein ...

The MKP1 protein was detected in tissue samples of advanced human melanoma by immunohistochemistry (IHC) at levels ~ 3 x higher than those in benign melanocytes or melanocytic nevus cells (Fig 5D and E). MKP1 protein was present predominantly in the nuclei, as reported for other types of cancer cells (5, 7). Significant MKP1 staining (+++) was detected in ~ 90% of the tissue samples of radial or vertical growth melanoma in contrast to the weaker MKP1 staining in non-malignant human melanocytes (0/+) or nevus cells (+) (Fig 5D and E).

MKP1 was therefore expressed in human melanoma cells and melanoma tissues and often at levels several folds higher than those in normal or non-malignant melanocytes.

TPI-3 interacted positively with 5-FU/LV against MC-26 colon cancer tumors in mice

To extend the observed activity of TPI-3 to sensitize melanoma response to IFNα2b, the capacity of TPI-3 to sensitize other types of cancers to chemotherapeutics was investigated. For this, we determined the capacity TPI-3 to sensitize MC-26 colon cancer tumors in vivo to 5-FU/LV, a backbone of chemotherapy (47). Consistent with reported expression of MKP1 in colon cancer tissues (8), MKP1 protein was detected in MC-26 cells (data not shown).

MC-26 tumor growth was inhibited 78% by the combination, significantly more than by 5FU/LV (54%, p = 0.011) or TPI-3 (18%, p = 0.004) as single agents (Fig 6A). The increased inhibition of tumor growth by the combination suggested a positive interaction between TPI-3 and 5FU/LV. TPI-3 and 5FU also interacted positively in growth inhibition of MC-26 cells in culture (Fig 6B). Supporting a mechanism of action of targeting MKP1 for the sensitization, TPI-3 increased MKP1 phospho-substrates in MC-26 cells (Fig 6C). The compound also had the capacity to interact with 5FU against human colon cancer cells (HT-29) in vitro (Fig 6D).

Fig 6
TPI-3 positively interacts with 5FU-LV against MC-26 colon cancer tumors in mice and increases MKP1 phospho-substrates in MC-26 cells. A) Balb/c mice bearing 4-day established MC-26 tumors were treated with vehicle control, TPI-3 (10 mg/kg, po, M-F/wk), ...

DISCUSSION

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 (Fig 4). It also sensitized MC-26 colon cancer cells to colon cancer chemotherapeutic 5FU in vitro and in mice (Fig 6). These actions of the compound were likely mediated via targeting MKP1 given that TPI-3 was an inhibitor of recombinant MKP1 (Fig 1) and capable of increasing MKP1 phospho-substrates in both WM9 and MC-26 cells (Fig 4 and and6).6). 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 (Fig 4 and and6)6) 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 (Fig 1 and and2).2). It is also encouraging that L6a1, although inactive for MKP1 (Fig 1 and and2),2), selectively increased pERK1/2 (Fig 2A), suggesting that it targets a pERK1/2- specific phosphatase (e.g., MKP3) (18). The limited structural differences between TPI-3 and L6a1 (Fig 1) 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 (Fig 5). Its functional significance in therapeutic resistance was suggested by the sensitization of WM9 melanoma to IFNα2b in vitro and in mice (Fig 4). 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 (Fig 4), 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 (Fig 4) that harbors the B-RafV600E mutant (our unpublished data) with heightened downstream pERK1/2 levels (Fig 4) 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 (Fig 5) 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 (Fig 2, ,44 and and6).6). 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 (Fig 4). Despite its effectiveness in inducing cancer cell death in vitro (Fig 3), 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 (Fig 4 and and6).6). 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) (Fig 3 and and4).4). Taken together, our results provide a strong basis for elucidating the mechanism of action of MKP1 inhibition and for potential translation in future studies.

ACKKNOWLEDGEMENT

The authors thank Becky Haney for excellent technical assistance.

This work was supported by NIH grants CA096636 (TY), CA095020 (DJL) and CA0890344 (EB).

Abbreviations

TPI-3
Tyrosine phosphatase inhibitor-3
TPI-2
Tyrosine phosphatase inhibitor-2
MKP1
MAPK Phosphatase-I
IFNα2b
interferon-alpha-2b
5FU
5 fluorouracil
LV
leucovorin

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