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Recently, MET exon 14 deletion (METex14del) has been postulated to be one potential mechanism for MET protein overexpression. We screened for the presence of METex14del transcript by multiplexed fusion transcript analysis using nCounter assay followed by confirmation with quantitative reverse transcription PCR with correlation to MET protein expression by immunohistochemistry (IHC) and MET amplification by fluorescence in situ hybridization (FISH). We extracted RNAs from 230 patients enrolled onto the prospective molecular profiling clinical trial (NEXT-1) (NCT02141152) between November 2013 and August 2014. Thirteen METex14del cases were identified including 3 gastric cancer, 4 colon cancer, 5 non-small cell lung cancer, and one adenocarcinoma of unknown primary. Of these 13 METex14del cases, 11 were MET IHC 3+ and 2 were 2+. Only one out of the 13 METex14del cases was MET amplified (MET/CEP ratio > 2.0). Growths of two (gastric, colon) METex14del+ patient tumor derived cell lines were profoundly inhibited by both MET tyrosine kinase inhibitors and a monoclonal antibody targeting MET. In conclusion, METex14del is a unique molecular aberration present in gastrointestinal (GI) malignancies corresponding with overexpression of MET protein but rarely with MET amplification. Substantial growth inhibition of METex14del+ patient tumor derived cell lines by several MET targeting drugs strongly suggests METex14del is a potential actionable driver mutation in GI malignancies.

Met is a receptor tyrosine kinase that promotes cancer progression. In addition, Met has been implicated in resistance of tumors to various targeted therapies such as epidermal growth factor receptor inhibitors in lung cancers, and has been prioritized as a key molecular target for cancer therapy. However, the underlying mechanism of resistance to Met-targeting drugs is poorly understood. Here, we describe screening of 1310 genes to search for key regulators related to drug resistance to an anti-Met therapeutic antibody (SAIT301) by using a small interfering RNA-based synthetic lethal screening method. We found that knockdown of 69 genes in Met-amplified MKN45 cells sensitized the antitumor activity of SAIT301. Pathway analysis of these 69 genes implicated fibroblast growth factor receptor (FGFR) as a key regulator for antiproliferative effects of Met-targeting drugs. Inhibition of FGFR3 increased target cell apoptosis through the suppression of Bcl-xL expression, followed by reduced cancer cell growth in the presence of Met-targeting drugs. Treatment of cells with the FGFR inhibitors substantially restored the efficacy of SAIT301 in SAIT301-resistant cells and enhanced the efficacy in SAIT301-sensitive cells. In addition to FGFR3, integrin β3 is another potential target for combination treatment with SAIT301. Suppression of integrin β3 decreased AKT phosphorylation in SAIT301-resistant cells and restored SAIT301 responsiveness in HCC1954 cells, which are resistant to SAIT301. Gene expression analysis using CCLE database shows that cancer cells with high levels of FGFR and integrin β3 are resistant to crizotinib treatment, suggesting that FGFR and integrin β3 could be used as predictive markers for Met-targeted therapy and provide a potential therapeutic option to overcome acquired and innate resistance for the Met-targeting drugs.

c-Met, the high affinity receptor for hepatocyte growth factor (HGF), is one of the most frequently activated tyrosine kinases in many human cancers and a target for cancer therapy. However, inhibitory targeting of c-Met with antibodies has proven difficult, because most antibodies have intrinsic agonist activity. Therefore, the strategy for reducing the agonism is critical for successful development of cancer therapies based on anti-c-Met antibodies. Here we developed a mechanism-based assay method for rapid screening of anti-c-Met antibodies, involving the determination of Akt phosphorylation and c-Met degradation for agonism and efficacy, respectively. Using the method, we identified an antibody, F46, that binds to human c-Met with high affinity (Kd = 2.56 nM) and specificity, and induces the degradation of c-Met in multiple cancer cells (including MKN45, a gastric cancer cell line) with minimal activation of c-Met signaling. F46 induced c-Met internalization in both HGF-dependent and HGF-independent cells, suggesting that the degradation of c-Met results from antibody-mediated receptor internalization. Further-more, F46 competed with HGF for binding to c-Met, resulting in the inhibition of both HGF-mediated invasion and angiogenesis. Consistently, F46 inhibited the proliferation of MKN45 cells, in which c-Met is constitutively activated in an HGF-independent manner. Xenograft analysis revealed that F46 markedly inhibits the growth of subcutaneously implanted gastric and lung tumors. These results indicate that F46, identified by a novel mechanism-based assay, induces c-Met degradation with minimal agonism, implicating a potential role of F46 in therapy of human cancers.

Defining the functional modules within transcriptional regulatory factors that govern switching between repression and activation events is a central issue in biology. Recently, we have reported the dynamic role of a β-catenin–reptin chromatin remodelling complex in regulating a metastasis suppressor gene KAI1 (ref.1), which is capable of inhibiting the progression of tumour metastasis2,3,4,5. Here, we identify signalling factors that confer repressive function on reptin and hence repress the expression of KAI1. Biochemical purification of a reptin-containing complex has revealed the presence of specific desumoylating enzymes that reverse the sumoylation of reptin that underlies its function as a repressor. Desumoylation of reptin alters the repressive function of reptin and its association with HDAC1. Furthermore, the sumoylation status of reptin modulates the invasive activity of cancer cells with metastatic potential. These data clearly define a functional model and provide a novel link for SUMO modification in cancer metastasis.

The Met receptor tyrosine kinase is an attractive target for cancer therapy as it promotes invasive tumor growth. SAIT301 is a novel anti-Met antibody, which induces LRIG1-mediated Met degradation and inhibits tumor growth. However, detailed downstream mechanism by which LRIG1 mediates target protein down-regulation is unknown. In the present study, we discovered that SAIT301 induces ubiquitination of LRIG1, which in turn promotes recruitment of Met and LRIG1 complex to the lysosome through its interaction with Hrs, resulting in concomitant degradation of both LRIG1 and Met. We also identified USP8 as a LRIG1-specific deubiquitinating enzyme, reporting the interaction between USP8 and LRIG1 for the first time. SAIT301 triggers degradation of LRIG1 by inhibiting the interaction of LRIG1 and USP8, which regulates ubiquitin modification and stability of LRIG1. In summary, SAIT301 employs ubiquitination of LRIG1 for its highly effective Met degradation. This unique feature of SAIT301 enables it to function as a fully antagonistic antibody without Met activation. We found that USP8 is involved in deubiquitination of LRIG1, influencing the efficiency of Met degradation. The relation of Met, LRIG1 and USP8 strongly supports the potential clinical benefit of a combination treatment of a USP8 inhibitor and a Met inhibitor, such as SAIT301.

Breast cancer metastasis suppressor 1 (BRMS1) suppresses metastasis without affecting primary tumorigenesis. The regulatory mechanism of BRMS1 at the protein level has not been revealed until recently. Here, we found that cullin 3 (Cul3), a component of E3 ubiquitin ligase, is a new binding partner of BRMS1 and the interaction between BRMS1 and Cul3 is mediated by the SPOP adaptor protein. Intriguingly, BRMS1 turns out to be a potent substrate that is ubiquitinated by the Cul3-SPOP complex. Knockdown of SPOP increases the level of BRMS1 protein and represses the expression of BRMS1 repressive target genes such as OPN and uPA in breast cancer cells. These results suggest that the novel regulatory mechanism of BRMS1 by Cul3-SPOP complex is important for breast cancer progression.

Lysine methylation within histones is crucial for transcriptional regulation and thus links chromatin states to biological outcomes. Although recent studies have extended lysine methylation to nonhistone proteins, underlying molecular mechanisms such as the upstream signaling cascade that induces lysine methylation and downstream target genes modulated by this modification have not been elucidated. Here, we show that Reptin, a chromatin-remodeling factor, is methylated at lysine 67 in hypoxic conditions by the methyltransferase G9a. Methylated Reptin binds to the promoters of a subset of hypoxia-responsive genes and negatively regulates transcription of these genes to modulate cellular responses to hypoxia.

KAI1 is a metastasis suppressor gene known to inhibit cancer metastasis without affecting primary tumorigenicity. Although KAI1 expression has been reported to undergo transcriptional regulation, how its expression is up- or down-regulated by specific upstream signaling pathways has not been studied in detail. In this study, we characterized the regulatory elements within the 500 bp upstream region of mouse KAI1 gene and identified a functional hypoxia-response element (HRE) within the promoter region. Hypoxia-dependent induction of KAI1 was directly mediated by hypoxia-inducible factor (HIF)-1α binding on the promoter, which subsequently caused increased recruitment of RNA polymerase II for transcriptional activation. The failure of HIF-1α recruitment to the KAI1 promoter was observed in Hif-1α knockout mouse embryonic fibroblasts. Furthermore, KAI1 protein synthesis was markedly increased in ischemic tissues, suggesting that KAI1 is a hypoxia target gene in vivo.

The Met receptor tyrosine kinase is an attractive target for cancer therapy as it promotes invasive tumor growth. SAIT301 is a novel anti-Met antibody, which induces LRIG1-mediated Met degradation and inhibits tumor growth. However, detailed downstream mechanism by which LRIG1 mediates target protein down-regulation is unknown. In the present study, we discovered that SAIT301 induces ubiquitination of LRIG1, which in turn promotes recruitment of Met and LRIG1 complex to the lysosome through its interaction with Hrs, resulting in concomitant degradation of both LRIG1 and Met. We also identified USP8 as a LRIG1-specific deubiquitinating enzyme, reporting the interaction between USP8 and LRIG1 for the first time. SAIT301 triggers degradation of LRIG1 by inhibiting the interaction of LRIG1 and USP8, which regulates ubiquitin modification and stability of LRIG1. In summary, SAIT301, a Met targeting antibody, employs ubiquitination of LRIG1 for its highly effective Met degradation. This unique feature of SAIT301 enables it to function as a fully antagonistic antibody without Met activation. We found that USP8 is involved in deubiquitination of LRIG1, influencing the efficiency of Met degradation. The relation of Met, LRIG1 and USP8 strongly supports the potential clinical benefit of a combination treatment of a USP8 inhibitor and a Met inhibitor, such as SAIT301. Citation Format: Ji Min Lee, Bogyou Kim, Kyung-Ah Kim. USP8 modulates ubiquitination of LRIG1 for Met degradation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 662. doi:10.1158/1538-7445.AM2015-662

We propose a prototype of silicon-on-glass microchip for protein detection by bead-based affinity chromatography. The microchip has five channels integrated by composing one beads reactor per one channel. Especially, an effective protein analysis mechanism is presented where the three protein-pretreatment processes are simultaneously performed on a single beads reactor: selective detection (purification / sensing), pre-concentration and protein digestion. Since the five channels are closely spaced in parallel on the microchip, it is possible to inspect the five different detection results on real-time in a single microscope image. The microchip is fabricated on silicon-on-glass (SiOG) to make a mechanically strong and vertically transparent structure for efficient fluid interconnection and fluorescence detection, respectively. Within the microchip, the grid-type filter is formed on channel output to physically trap 38 ~ 50 μm diameter microbeads. The dimension of one grid is 30 × 30 μm 2 . The volume flow rate was investigated experimentally on the case of bead-packed chamber, and the resulted value was compared to that of the case of hollow chamber. In this research, we used self-cleavage free aptazymes as detection ligands immobilized on polystyrene microbeads. The target proteins are firstly on-chip concentrated and fluorescence-detected (confocal microscopy), and secondly checked off-chip by using MALDI-TOF. If the two analyses are used cooperatively, it is expected that the accuracy in diagnostic analysis will be enhanced in biosensing system. Especially by using this free aptazymes system, we don't need to consider the requirement of fluorescence tagging and the difficulty of eluting antibody-bound proteins from microbeads without bad effects of harsh elution conditions in protease treatment. We analyzed the on-bead detection of HCV replicase and HCV helicase respectively by measuring fluorescence intensities at different concentrations, and also performed a selectively detection of HCV helicase from protein mixtures.

Defining the molecular strategies that integrate diverse signalling pathways in the expression of specific gene programmes that are critical in homeostasis and disease remains a central issue in biology. This is particularly pertinent in cancer biology because downregulation of tumour metastasis suppressor genes is a common occurrence, and the underlying molecular mechanisms are not well established. Here we report that the downregulation of a metastasis suppressor gene, KAI1, in prostate cancer cells involves the inhibitory actions of beta-catenin, along with a reptin chromatin remodelling complex. This inhibitory function of beta-catenin-reptin requires both increased beta-catenin expression and recruitment of histone deacetylase activity. The coordinated actions of beta-catenin-reptin components that mediate the repressive state serve to antagonize a Tip60 coactivator complex that is required for activation; the balance of these opposing complexes controls the expression of KAI1 and metastatic potential. The molecular mechanisms underlying the antagonistic regulation of beta-catenin-reptin and the Tip60 coactivator complexes for the metastasis suppressor gene, KAI1, are likely to be prototypic of a selective downregulation strategy for many genes, including a subset of NF-kappaB target genes.

Met is a receptor tyrosine kinase that promotes cancer progression. In addition, Met has been implicated in resistance of tumors to various targeted therapies such as EGFR inhibitors in lung cancers, and has been prioritized as a key molecular target for cancer therapy. However, the underlying mechanism of resistance to Met targeting drugs is poorly understood. Here, we describe screening of 1310 genes to search for key regulators related to drug resistance to an anti-Met therapeutic antibody (SAIT301) by employing a siRNA-based synthetic lethal screening method. We found that knockdown of 69 genes in Met-amplified MKN45 cells sensitized the anti-tumor activity of SAIT301. Pathway analysis of these 69 genes implicated FGFR as a key regulator for anti-proliferative effects of Met targeting drugs. Inhibition of FGFR3 increased target cell apoptosis through the suppression of Bcl-xL expression, followed by reduced cancer cell growth in the presence of Met targeting drugs. Treatment of cells with the FGFR inhibitors substantially restored the efficacy of SAIT301 in SAIT301-resistant cells and enhanced the efficacy in SAIT301-sensitive cells. In addition to FGFR3, integrin β3 is another potential target for combination treatment with SAIT301. Suppression of integrin β3 decreased AKT phosphorylation in SAIT301-resistant cells and restores SAIT301 responsiveness in HCC1954 cells, which are resistant to SAIT301. Gene expression analysis from using CCLE DB shows cancer cells with high levels of FGFR and integrin β3 are resistant to crizotinib treatment, suggesting FGFR and integrin β3 could be used as predictive markers for Met targeted therapy and provide a potential therapeutic option to overcome acquired and innate resistance for the Met targeting drugs. Citation Format: Bogyou Kim. Synthetic lethal screening reveals FGFR as one of combinatorial targets to overcome resistance to Met-targeted therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-219. doi:10.1158/1538-7445.AM2014-LB-219

The Met receptor tyrosine kinase, found to be constitutively activated in many tumors, has become a leading target for cancer therapy. Disruptions in Met down-regulation have been associated with aggressive tumor progression with several therapeutic strategies addressing this aspect of Met biology. Cbl E3 ligase-mediated degradation, which attenuates Met signaling via ligand-dependent Met internalization, is a major negative regulator of Met expression. It is believed that one of the mechanisms by which the therapeutic anti-Met antibodies induce cancer cell death in Met over-expressing tumors is via internalization and subsequent degradation of Met from the cell surface. However, a previously reported Met targeting antibody demonstrated intrinsic agonistic activity while capable of inducing Cbl-mediated degradation of Met, suggesting that Cbl-mediated degradation requires receptor activation and impedes therapeutic application. We have developed a potent and selective bivalent Met targeting antibody (SAIT301) that invokes Met degradation using an alternative regulator LRIG1. In this report, we demonstrate that LRIG1 mediates degradation of Met by SAIT301 and this degradation does not require Met activation. Furthermore, SAIT301 was able to down-regulate Met and dramatically inhibit growth of tumors with low or no Cbl expression, as well as tumors with Met exon 14 deletion that prevents Met binding to Cbl. In summary, we demonstrate the enhanced therapeutic potential of a novel tumor-inhibiting anti-Met antibody, SAIT301, which utilizes a Cbl-independent, LRIG1-mediated Met degradation pathway and thereby avoids the agonism that limits the effectiveness of previously reported anti-Met antibodies. Citation Format: Ji Min Lee, Bogyou Kim, Kyung-Ah Kim. Cbl-independent degradation of Met: Ways to avoid agonism of bivalent Met targeting antibody. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1036. doi:10.1158/1538-7445.AM2013-1036