Michaela Medová

Hsp90 transcriptionally and post-translationally regulates the expression of NDRG1 and maintains the stability of its modifying kinase GSK3β

N-myc downstream-regulated gene 1 (NRDG1) is a stress-induced protein whose putative function is suppression of tumor metastasis. A recent proteonomic study showed NDRG1 interacts with the molecular chaperone heat shock protein 90 (Hsp90). From their reported association, we investigated if NDRG1 is dependent on Hsp90 for its stability and is therefore a yet unidentified Hsp90 client protein. Here, we demonstrate that endogenous NDRG1 and Hsp90 physically associate in hepatocellular cancer cell lines. However, geldanamycin (GA)-mediated inhibition of Hsp90 did not disrupt their interaction or result in NDRG1 protein destabilization. On the contrary, inhibition of Hsp90 led to a transcriptional increase of NDRG1 protein which was associated with cell growth arrest. We also observed that GA inhibited the phosphorylation of NDRG1 by targeting its regulating kinases, serum- and glucocorticoid-induced kinase 1 (SGK1) and glycogen synthase kinase 3 beta (GSK3beta). We demonstrate that in the presence of GA, GSK3beta protein and activity were decreased thus indicating that Hsp90 is necessary for GSK3beta stability. Taken together, our data demonstrate that NDRG1 is not a classic client protein but interacts with Hsp90 and is still dually regulated by Hsp90 at a transcriptional and post-translational level. Finally, we suggest for the first time GSK3beta as a new client protein of Hsp90.

MET inhibition in tumor cells by PHA665752 impairs homologous recombination repair of DNA double strand breaks

Abnormal activation of cellular DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems has broad implications for both cancer biology and treatment. Recent studies suggest a potential link between DNA repair and aberrant activation of the hepatocyte growth factor receptor Mesenchymal‐Epithelial Transition (MET), an oncogene that is overexpressed in numerous types of human tumors and considered a prime target in clinical oncology. Using the homologous recombination (HR) direct‐repeat direct‐repeat green fluorescent protein ((DR)‐GFP) system, we show that MET inhibition in tumor cells with deregulated MET activity by the small molecule PHA665752 significantly impairs in a dose‐dependent manner HR. Using cells that express MET‐mutated variants that respond differentially to PHA665752, we confirm that the observed HR inhibition is indeed MET‐dependent. Furthermore, our data also suggest that decline in HR‐dependent DNA repair activity is not a secondary effect due to cell cycle alterations caused by PHA665752. Mechanistically, we show that MET inhibition affects the formation of the RAD51‐BRCA2 complex, which is crucial for error‐free HR repair of double strand DNA lesions, presumably via downregulation and impaired translocation of RAD51 into the nucleus. Taken together, these findings assist to further support the role of MET in the cellular DNA damage response and highlight the potential future benefit of MET inhibitors for the sensitization of tumor cells to DNA damaging agents.

MET Inhibition Results in DNA Breaks and Synergistically Sensitizes Tumor Cells to DNA-Damaging Agents Potentially by Breaching a Damage-Induced Checkpoint Arrest.

While recent studies implicate that signaling through the receptor tyrosine kinase MET protects cancer cells from DNA damage, molecular events linking MET to the DNA damage response machinery are largely unknown. Here, we studied the impact of MET inhibition by the small molecule PHA665752 on cytotoxicity induced by DNA-damaging agents. We demonstrate that PHA665752 reduces clonogenic survival of tumor cells with MET overexpression when combined with ionizing radiation and synergistically cooperates with ionizing radiation or adriamycin to induce apoptosis. In search of mechanisms underlying the observed synergism, we show that PHA665752 alone considerably increases γH2AX levels, indicating the accumulation of double-strand DNA breaks. In addition, PHA665752 treatment results in sustained high levels of γH2AX and phosphorylated ATM postirradiation, strengthening the assumption that MET inhibition attenuates postdamage DNA repair. PHA665752, alone or in combination with irradiation, leads also to a massive increase of γH2AX tyrosine phosphorylation and its subsequent interaction with the proapoptotic kinase JNK1. Finally, MET inhibition reduces activation of ATR, CHK1, and CDC25B and abrogates an associated DNA damage-induced S phase arrest. This indicates that MET inhibition compromises a critical damage-dependent checkpoint that may enable DNA-damaged cells to exit cell cycle arrest before repair is completed.

Coupling of Mutated Met Variants to DNA Repair via Abl and Rad51

Abnormal activation of DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems is a compelling likelihood with significant implications in both cancer biology and treatment. Here, we show that due to a potential substrate switch, mutated variants of the receptor for hepatocyte growth factor Met, but not the wild-type form of the receptor, directly couple to the Abl tyrosine kinase and the Rad51 recombinase, two key signaling elements of homologous recombination-based DNA repair. Treatment of cells that express the mutated receptor variants with the Met inhibitor SU11274 leads, in a mutant-dependent manner, to a reduction of tyrosine phosphorylated levels of Abl and Rad51, impairs radiation-induced nuclear translocation of Rad51, and acts as a radiosensitizer together with the p53 inhibitor pifithrin-alpha by increasing cellular double-strand DNA break levels following exposure to ionizing radiation. Finally, we propose that in order to overcome a mutation-dependent resistance to SU11274, this aberrant molecular axis may alternatively be targeted with the Abl inhibitor, nilotinib.

Differential inhibition sensitivities of MET mutants to the small molecule inhibitor SU11274

Point mutations emerge as one of the rate-limiting steps in tumor response to small molecule inhibitors of protein kinases. Here we characterized the response of the MET mutated variants, V1110I, V1238I, V1206L and H1112L to the small molecule SU11274. Our results reveal a distinct inhibition pattern of the four mutations with IC(50) values for autophosphorylation inhibition ranging between 0.15 and 1.5muM. Differences were further seen on the ability of SU11274 to inhibit phosphorylation of downstream MET transducers such as AKT, ERK, PLCgamma and STAT3 and a variety of MET-dependent biological endpoints. In all the assays, H1112L was the most sensitive to SU11274, while V1206L was less affected under the used concentration range. The differences in responses to SU11274 are discussed based on a structural model of the MET kinase domain.

Protective autophagy is involved in resistance towards MET inhibitors in human gastric adenocarcinoma cells.

MET, also known as hepatocyte growth factor receptor (HGFR), is a receptor tyrosine kinase with an important role, both in normal cellular function as well as in oncogenesis. In many cancer types, abnormal activation of MET is related to poor prognosis and various strategies to inhibit its function, including small molecule inhibitors, are currently in preclinical and clinical evaluation. Autophagy, a self-digesting recycling mechanism with cytoprotective functions, is induced by cellular stress. This process is also induced upon cytotoxic drug treatment of cancer cells and partially allows these cells to escape cell death. Thus, since autophagy protects different tumor cells from chemotherapy-induced cell death, current clinical trials aim at combining autophagy inhibitors with different cancer treatments. We found that in a gastric adenocarcinoma cell line GTL-16, where MET activity is deregulated due to receptor overexpression, two different MET inhibitors PHA665752 and EMD1214063 lead to cell death paralleled by the induction of autophagy. A combined treatment of MET inhibitors together with the autophagy inhibitor 3-MA or genetically impairing autophagy by knocking down the key autophagy gene ATG7 further decreased cell viability of gastric cancer cells. In general, we observed the induction of cytoprotective autophagy in MET expressing cells upon MET inhibition and a combination of MET and autophagy inhibition resulted in significantly decreased cell viability in gastric cancer cells.

The Novel ATP-Competitive Inhibitor of the MET Hepatocyte Growth Factor Receptor EMD1214063 Displays Inhibitory Activity against Selected MET-Mutated Variants

The receptor tyrosine kinase MET is a prime target in clinical oncology due to its aberrant activation and involvement in the pathogenesis of a broad spectrum of malignancies. Similar to other targeted kinases, primary and secondary mutations seem to represent an important resistance mechanism to MET inhibitors. Here, we report the biologic activity of a novel MET inhibitor, EMD1214063, on cells that ectopically express the mutated MET variants M1268T, Y1248H, H1112Y, L1213V, H1112L, V1110I, V1206L, and V1238I. Our results show a dose-dependent decrease in MET autophosphorylation in response to EMD1214063 in five of the eight cell lines (IC50 2–43 nmol/L). Blockade of MET by EMD1214063 was accompanied by a reduced activation of downstream effectors in cells expressing EMD1214063-sensitive mutants. In all sensitive mutant-expressing lines, EMD1214063 altered cell-cycle distribution, primarily with an increase in G1 phase. EMD1214063 strongly influenced MET-driven biologic functions, such as cellular morphology, MET-dependent cell motility, and anchorage-independent growth. To assess the in vivo efficacy of EMD1214063, we used a xenograft tumor model in immunocompromised mice bearing NIH3T3 cells expressing sensitive and resistant MET-mutated variants. Animals were randomized for the treatment with EMD1214063 (50 mg/kg/d) or vehicle only. Remarkably, five days of EMD1214063 treatment resulted in a complete regression of the sensitive H1112L-derived tumors, whereas tumor growth remained unaffected in mice with L1213V tumors and in vehicle-treated animals. Collectively, the current data identifies EMD1214063 as a potent MET small-molecule inhibitor with selective activity towards mutated MET variants. Mol Cancer Ther; 12(11); 2415–24. ©2013 AACR.

The Molecular Crosstalk between the MET Receptor Tyrosine Kinase and the DNA Damage Response-Biological and Clinical Aspects.

Radiation therapy remains an imperative treatment modality for numerous malignancies. Enduring significant technical achievements both on the levels of treatment planning and radiation delivery have led to improvements in local control of tumor growth and reduction in healthy tissue toxicity. Nevertheless, resistance mechanisms, which presumably also involve activation of DNA damage response signaling pathways that eventually may account for loco-regional relapse and consequent tumor progression, still remain a critical problem. Accumulating data suggest that signaling via growth factor receptor tyrosine kinases, which are aberrantly expressed in many tumors, may interfere with the cytotoxic impact of ionizing radiation via the direct activation of the DNA damage response, leading eventually to so-called tumor radioresistance. The aim of this review is to overview the current known data that support a molecular crosstalk between the hepatocyte growth factor receptor tyrosine kinase MET and the DNA damage response. Apart of extending well established concepts over MET biology beyond its function as a growth factor receptor, these observations directly relate to the role of its aberrant activity in resistance to DNA damaging agents, such as ionizing radiation, which are routinely used in cancer therapy and advocate tumor sensitization towards DNA damaging agents in combination with MET targeting.

Biological, diagnostic and therapeutic relevance of the MET receptor signaling in head and neck cancer.

Head and neck cancer constitutes the 6th most common malignancy worldwide and affects the crucial anatomical structures and physiological functions of the upper aerodigestive tract. Classical therapeutic strategies such as surgery and radiotherapy carry substantial toxicity and functional impairment. Moreover, the loco-regional control rates as well as overall survival still need to be improved in subgroups of patients. The scatter-factor/hepatocyte growth factor receptor tyrosine kinase MET is an established effector in the promotion, maintenance and progression of malignant transformation in a wide range of human malignancies, and has been gaining considerable interest in head and neck cancer over the last 15 years. Aberrant MET activation due to overexpression, mutations, tumor-stroma paracrine loops, and cooperative/redundant signaling has been shown to play prominent roles in epithelial-to-mesenchymal transition, angiogenesis, and responses to anti-cancer therapeutic modalities. Accumulating preclinical and translational evidence highly supports the increasing interest of MET as a biomarker for lymph node and distant metastases, as well as a potential marker of stratification for responses to ionizing radiation. The relevance of MET as a therapeutic molecular target in head and neck cancer described in preclinical studies remains largely under-evaluated in clinical trials, and therefore inconclusive. Also in the context of anti-cancer targeted therapy, a large body of preclinical data suggests a central role for MET in treatment resistance towards multiple therapeutic modalities in malignancies of the head and neck region. These findings, as well as the potential use of combination therapies including MET inhibitors in these tumors, need to be further explored.

KRAS and HRAS mutations confer resistance to MET targeting in preclinical models of MET‐expressing tumor cells

The MET receptor tyrosine kinase is often deregulated in human cancers and several MET inhibitors are evaluated in clinical trials. Similarly to EGFR, MET signals through the RAS‐RAF‐ERK/MAPK pathway which plays key roles in cell proliferation and survival. Mutations of genes encoding for RAS proteins, particularly in KRAS, are commonly found in various tumors and are associated with constitutive activation of the MAPK pathway. It was shown for EGFR, that KRAS mutations render upstream EGFR inhibition ineffective in EGFR‐positive colorectal cancers. Currently, there are no clinical studies evaluating MET inhibition impairment due to RAS mutations. To test the impact of RAS mutations on MET targeting, we generated tumor cells responsive to the MET inhibitor EMD1214063 that express KRAS G12V, G12D, G13D and HRAS G12V variants. We demonstrate that these MAPK‐activating RAS mutations differentially interfere with MET‐mediated biological effects of MET inhibition. We report increased residual ERK1/2 phosphorylation indicating that the downstream pathway remains active in presence of MET inhibition. Consequently, RAS variants counteracted MET inhibition‐induced morphological changes as well as anti‐proliferative and anchorage‐independent growth effects. The effect of RAS mutants was reversed when MET inhibition was combined with MEK inhibitors AZD6244 and UO126. In an in vivo mouse xenograft model, MET‐driven tumors harboring mutated RAS displayed resistance to MET inhibition. Taken together, our results demonstrate for the first time in details the role of KRAS and HRAS mutations in resistance to MET inhibition and suggest targeting both MET and MEK as an effective strategy when both oncogenic drivers are expressed.