Regeneration of Non-Alcoholic Fatty Liver Cells Using Chimeric FGF21/HGFR: A Novel Therapeutic Approach.

Non-alcoholic fatty liver disease (NAFLD) has emerged as a significant liver ailment attributed to factors like obesity and diabetes. While ongoing research explores treatments for NAFLD, further investigation is imperative to address this escalating health concern. NAFLD manifests as hepatic steatosis, precipitating insulin resistance and metabolic syndrome. This study aims to validate the regenerative potential of chimeric fibroblast growth factor 21 (FGF21) and Hepatocyte Growth Factor Receptor (HGFR) in NAFLD-afflicted liver cells. AML12, a murine hepatocyte cell line, was utilized to gauge the regenerative effects of chimeric FGF21/HGFR expression. Polysaccharide accumulation was affirmed through Periodic acid-Schiff (PAS) staining, while LDL uptake was microscopically observed with labeled LDL. The expression of FGF21/HGFR and NAFLD markers was analyzed by mRNA analysis with RT-PCR, which showed a decreased expression in acetyl-CoA carboxylase 1 (ACC1) and sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) with increased expression of hepatocellular growth factor (HGF), hepatocellular nuclear factor 4 alpha (HNF4A), and albumin (ALB). These findings affirm the hepato-regenerative properties of chimeric FGF21/HGFR within AML12 cells, opening novel avenues for therapeutic exploration in NAFLD.

Genetic deletion or tyrosine phosphatase inhibition of PTPRZ1 activates c-Met to up-regulate angiogenesis and lung adenocarcinoma growth.

Protein tyrosine phosphatase receptor zeta 1 (PTPRZ1) is a transmembrane tyrosine phosphatase (TP) expressed in endothelial cells and required for stimulation of cell migration by vascular endothelial growth factor A165 (VEGFA165 ) and pleiotrophin (PTN). It is also over or under-expressed in various tumor types. In this study, we used genetically engineered Ptprz1-/- and Ptprz1+/+ mice to study mechanistic aspects of PTPRZ1 involvement in angiogenesis and investigate its role in lung adenocarcinoma (LUAD) growth. Ptprz1-/- lung microvascular endothelial cells (LMVEC) have increased angiogenic features compared with Ptprz1+/+ LMVEC, in line with the increased lung angiogenesis and the enhanced chemically induced LUAD growth in Ptprz1-/- compared with Ptprz1+/+ mice. In LUAD cells isolated from the lungs of urethane-treated mice, PTPRZ1 TP inhibition also enhanced proliferation and migration. Expression of beta 3 (ß3 ) integrin is decreased in Ptprz1-/- LMVEC, linked to enhanced VEGF receptor 2 (VEGFR2), c-Met tyrosine kinase (TK) and Akt kinase activities. However, only c-Met and Akt seem responsible for the enhanced endothelial cell activation in vitro and LUAD growth and angiogenesis in vivo in Ptprz1-/- mice. A selective PTPRZ1 TP inhibitor, VEGFA165 and PTN also activate c-Met and Akt in a PTPRZ1-dependent manner in endothelial cells, and their stimulatory effects are abolished by the c-Met TK inhibitor (TKI) crizotinib. Altogether, our data suggest that low PTPRZ1 expression is linked to worse LUAD prognosis and response to c-Met TKIs and uncover for the first time the role of PTPRZ1 in mediating c-Met activation by VEGFA and PTN.

Exhaled phospholipid transfer protein and hepatocyte growth factor receptor in lung adenocarcinoma.

BACKGROUND: Screening decreases mortality among lung cancer patients but is not widely implemented, thus there is an unmet need for an easily accessible non-invasive method to enable early diagnosis. Particles in exhaled air offer a promising such diagnostic tool. We investigated the validity of a particles in exhaled air device (PExA) to measure the particle flow rate (PFR) and collect exhaled breath particles (EBP) to diagnose primary lung adenocarcinoma (LUAD). METHODS: Seventeen patients listed for resection of LUAD stages IA-IIIA and 18 non-cancer surgical control patients were enrolled. EBP were collected before and after surgery for LUAD, and once for controls. Proteomic analysis was carried out using a proximity extension assay technology. Results were validated in both plasma from the same cohort and with microarray data from healthy lung tissue and LUAD tissue in the GSE10072 dataset. RESULTS: Of the 92 proteins analyzed, levels of five proteins in EBP were significantly higher in the LUAD patients compared to controls. Levels of phospholipid transfer protein (PLTP) and hepatocyte growth factor receptor (MET) decreased in LUAD patients after surgery compared to control patients. PFR was significantly higher in the LUAD cohort at all timepoints compared to the control group. MET in plasma correlated significantly with MET in EBP. CONCLUSION: Collection of EBP and measuring of PFR has never been performed in patients with LUAD. In the present study PFR alone could distinguish between LUAD and patients without LUAD. PLTP and MET were identified as potential biomarkers to evaluate successful tumor excision.

Thiosemicarbazones suppress expression of the c-Met oncogene by mechanisms involving lysosomal degradation and intracellular shedding.

Considering the role of proto-oncogene c-Met (c-Met) in oncogenesis, we examined the effects of the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), and two NDRG1-inducing thiosemicarbazone-based agents, Dp44mT and DpC, on c-Met expression in DU145 and Huh7 cells. NDRG1 silencing without Dp44mT and DpC up-regulated c-Met expression, demonstrating that NDRG1 modulates c-Met levels. Dp44mT and DpC up-regulated NDRG1 by an iron-dependent mechanism and decreased c-Met levels, c-Met phosphorylation, and phosphorylation of its downstream effector, GRB2-associated binding protein 1 (GAB1). However, incubation with Dp44mT and DpC after NDRG1 silencing or silencing of the receptor tyrosine kinase inhibitor, mitogen-inducible gene 6 (MIG6), decreased c-Met and its phosphorylation, suggesting NDRG1- and MIG6-independent mechanism(s). Lysosomal inhibitors rescued the Dp44mT- and DpC-mediated c-Met down-regulation in DU145 cells. Confocal microscopy revealed that lysosomotropic agents and the thiosemicarbazones significantly increased co-localization between c-Met and lysosomal-associated membrane protein 2 (LAMP2). Moreover, generation of c-Met C-terminal fragment (CTF) and its intracellular domain (ICD) suggested metalloprotease-mediated cleavage. In fact, Dp44mT increased c-Met CTF while decreasing the ICD. Dp44mT and a γ-secretase inhibitor increased cellular c-Met CTF levels, suggesting that Dp44mT induces c-Met CTF levels by increasing metalloprotease activity. The broad metalloprotease inhibitors, EDTA and batimastat, partially prevented Dp44mT-mediated down-regulation of c-Met. In contrast, the ADAM inhibitor, TIMP metallopeptidase inhibitor 3 (TIMP-3), had no such effect, suggesting c-Met cleavage by another metalloprotease. Notably, Dp44mT did not induce extracellular c-Met shedding that could decrease c-Met levels. In summary, the thiosemicarbazones Dp44mT and DpC effectively inhibit oncogenic c-Met through lysosomal degradation and metalloprotease-mediated cleavage.

H2O2 induces nuclear transport of the receptor tyrosine kinase c-MET in breast cancer cells via a membrane-bound retrograde trafficking mechanism.

Reactive oxygen species (ROS) are cellular by-products produced from metabolism and also anticancer agents, such as ionizing irradiation and chemotherapy drugs. The ROS H2O2 has high rates of production in cancer cells because of their rapid proliferation. ROS oxidize DNA, protein, and lipids, causing oxidative stress in cancer cells and making them vulnerable to other stresses. Therefore, cancer cell survival relies on maintaining ROS-induced stress at tolerable levels. Hepatocyte growth factor receptor (c-MET) is a receptor tyrosine kinase overexpressed in malignant cancer types, including breast cancer. Full-length c-MET triggers a signal transduction cascade from the plasma membrane that, through downstream signaling proteins, up-regulates cell proliferation and migration. Recently, c-MET was shown to interact and phosphorylate poly(ADP-ribose) polymerase 1 in the nucleus and to induce poly(ADP-ribose) polymerase inhibitor resistance. However, it remains unclear how c-MET moves from the cell membrane to the nucleus. Here, we demonstrate that H2O2 induces retrograde transport of membrane-associated full-length c-MET into the nucleus of human MCF10A and MCF12A or primary breast cancer cells. We further show that knocking down either coatomer protein complex subunit γ1 (COPG1) or Sec61 translocon ß subunit (SEC61ß) attenuates the accumulation of full-length nuclear c-MET. However, a c-MET kinase inhibitor did not block nuclear c-MET transport. Moreover, nuclear c-MET interacted with KU proteins in breast cancer cells, suggesting a role of full-length nuclear c-MET in ROS-induced DNA damage repair. We conclude that a membrane-bound retrograde vesicle transport mechanism facilitates membrane-to-nucleus transport of c-MET in breast cancer cells.

[Effect of silencing hepatocyte growth factor receptor c-Met expression on biological characteristics of colon cancer cells].

Objective: To investigate the effect of silencing hepatocyte growth factor receptor (c-Met) expression on the biological characteristics of HCT116 colon cancer cells. Methods: Cellular model of c-Met transient transfection was established by using small interfering RNA (siRNA), the expression of c-Met in colon cancer cells was detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and western blot. The apoptosis assay, cell invasion assay, cell migration and other experiments were conducted to observe the effects of silencing c-Met on the biological characteristics of colon cancer cells. Results: RT-qPCR results showed that the relative expression levels of c-Met mRNA in siRNA-Met group, blank control group and siRNA negative control (siRNA-NC) group were 0.32±0.26, 1.01±0.03 and 1.05±0.23, respectively, and the difference was statistically significant (P<0.05). Western blot analysis showed that the expression level of c-Met protein in the siRNA-Met group was 0.24±0.03, significantly lower than 1.23±0.06 in the blank control group and 1.18±0.11 in the siRNA-NC group (P<0.05). The cell counting kit-8 (CCK8) results showed that the 72-hour absorbance (A) values of the siRNA-Met group, blank control group and the siRNA-NC group were 1.13±0.05, 1.48±0.08 and 1.53±0.07, respectively, and the difference was statistically significant (P<0.01). Cell cycle results showed that the proportion of cells in G(2)/M phase was (14.65±1.41)% in siRNA-Met group , (5.07±0.70)% in blank control group and (5.63±0.71)% in siRNA-NC group, and the difference was statistically significant (P<0.05). The expression levels of cell cycle regulatory proteins Cdc25c and cyclin B1 in siRNA-Met group were significantly decreased. The apoptotic rate in siRNA-Met group was (5.85±0.35)%, significantly higher than (1.00±0.17)% in blank control group and (0.91±1.14)% in siRNA-NC group (P<0.05). The expression level of apoptosis-related protein Bcl-2 in the siRNA-Met group was significantly decreased while Bcl-2 associated X protein (BAX) expression level was significantly increased. The cell scratching result showed that the cell migration abilities of the siRNA-Met group, blank control group and the siRNA-NC group were (51.33±8.62)%, (100.00±3.72)% and (102.33±6.43)%, respectively, and the difference was statistically significant (P<0.05). The number of cell penetrating into the basement membrane of the siRNA-Met group, blank control group and the siRNA-NC group were 47.50±10.60, 100.00±5.33 and 102.50±10.61, respectively, and the difference was statistically significant (P<0.05). The expressions of invasion related proteins including MMP-2 and MMP-9 in siRNA-Met group were decreased significantly. Conclusions: c-Met plays an important role in maintaining the biological characteristics of colon cancer cells. Inhibition of c-Met may have important values in the treatment of colon cancer.

A "Prozone-Like" Effect Influences the Efficacy of the Monoclonal Antibody ABT-700 against the Hepatocyte Growth Factor Receptor.

ABT-700 is a therapeutic antibody against the hepatocyte growth factor receptor (MET). At doses or regimens that lead to exposures exceeding optimum in vivo, the efficacy of ABT-700 is unexpectedly reduced. We hypothesized that this reduction in efficacy was due to a "prozone-like" effect in vivo. A prozone-like effect, which is a reduction in efficacy beyond optimum exposure, is caused due a mechanism similar to the generation of false negative flocculation tests by excessive antibody titres. In vitro, we demonstrate that at higher ABT-700 concentrations, this "prozone-like" effect is mediated by a progressive conversion from bivalent to ineffective monovalent binding of the antibody. In vivo, the efficacy of ABT-700 is dependent on an optimum range of exposure as well. Our data suggest that the "prozone-like" effect is operative and independent of target expression. ABT-700 dose, regimen, exposure, and tumor burden are interdependent variables influencing the "prozone-like" effect and mediating and in vivo efficacy. By optimization of dosage and regimen we demonstrate that the "prozone-like" effect can be alleviated and ABT-700 efficacy at varying tumor loads can be further extended in combination with cisplatin. Our results suggest that optimization of exposure taking tumor burden into account may alleviate "prozone-like" effects without compromising efficacy.

Identification of Tyrosine-Phosphorylated Proteins Upregulated during Epithelial-Mesenchymal Transition Induced with TGF-ß.

The epithelial-to-mesenchymal transition (EMT) is a unique process for the phenotypic changes of tumor cells characterized by a transition from polarized rigid epithelial cells to migrant mesenchymal cells, thus conferring the ability of tumor invasion and metastasis. A major challenge in the treatment of lung adenocarcinoma is to identify early stage patients at a high risk of recurrence or metastasis, thereby permitting the best therapeutic strategy and prognosis. In this study, we used a transforming growth factor-ß (TGF-ß)-induced EMT model to quantitatively identify protein tyrosine phosphorylation during the course of EMT in relation to malignant characteristics of lung adenocarcinoma cells. We performed relative quantitation analysis of tyrosine-phosphorylated peptides in TGF-ß-treated and -untreated lung adenocarcinoma cells and identified tyrosine-phosphorylated proteins that were upregulated in TGF-ß-treated cells. These include tensin-1 (TNS1) phosphorylated on Y1404, hepatocyte growth factor receptor (c-Met) phosphorylated on Y1234, and NT-3 growth factor receptor (TrkC) phosphorylated on Y516. We also found that these protein phosphorylation profiles were specifically observed in tissue samples of patients with poor prognostic lung adenocarcinoma. Tyrosine phosphorylations of these proteins represent possible candidates of prognostic prediction markers for lung adenocarcinoma.

Quantitative analysis of individual hepatocyte growth factor receptor clusters in influenza A virus infected human epithelial cells using localization microscopy.

In this report, we applied a special localization microscopy technique (Spectral Precision Distance/Spatial Position Determination Microscopy/SPDM) to quantitatively analyze the effect of influenza A virus (IAV) infection on the spatial distribution of individual HGFR (Hepatocyte Growth Factor Receptor) proteins on the membrane of human epithelial cells at the single molecule resolution level. We applied this SPDM method to Alexa 488 labeled HGFR proteins with two different ligands. The ligands were either HGF (Hepatocyte Growth Factor), or IAV. In addition, the HGFR distribution in a control group of mock-incubated cells without any ligands was investigated. The spatial distribution of 1×10(6) individual HGFR proteins localized in large regions of interest on membranes of 240 cells was quantitatively analyzed and found to be highly non-random. Between 21% and 24% of the HGFR molecules were located in 44,304 small clusters with an average diameter of 54nm. The mean density of HGFR molecule signals per individual cluster was very similar in control cells, in cells with ligand only, and in IAV infected cells, independent of the incubation time. From the density of HGFR molecule signals in the clusters and the diameter of the clusters, the number of HGFR molecule signals per cluster was estimated to be in the range between 4 and 11 (means 5-6). This suggests that the membrane bound HGFR clusters form small molecular complexes with a maximum diameter of few tens of nm, composed of a relatively low number of HGFR molecules. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking.

Ginsenoside-Rg1 induces angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a.

Therapeutic angiogenesis has been implicated in ischemic diseases and wound healing. Ginsenoside-Rg1 (Rg1), one of the most abundant active components of ginseng, has been demonstrated as an angiogenesis-stimulating compound in different models. There is increasing evidence implicating microRNAs (miRNAs), a group of non-coding RNAs, as important regulators of angiogenesis, but the role of microRNAs in Rg1-induced angiogenesis has not been fully explored. In this report, we found that stimulating endothelial cells with Rg1 could reduce miR-23a expression. In silico experiments predicted hepatocyte growth factor receptor (MET), a well-established mediator of angiogenesis, as the target of miR-23a. Transfection of the miR-23a precursor or inhibitor oligonucleotides validated the inverse relationship of miR-23a and MET expression. Luciferase reporter assays further confirmed the interaction between miR-23a and the MET mRNA 3'-UTR. Intriguingly, ginsenoside-Rg1 was found to increase MET protein expression in a time-dependent manner. We further demonstrated that ginsenoside-Rg1-induced angiogenic activities were indeed mediated through the down-regulation of miR-23a and subsequent up-regulation of MET protein expression, as confirmed by gain- and loss-of-function angiogenic experiments. In summary, our results demonstrated that ginsenoside-Rg1 could induce angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a. This study has broadened our understanding of the non-genomic effects of ginsenoside-Rg1, and provided molecular evidence that warrant further development of natural compound as novel angiogenesis-promoting therapy.

Regulation of MET-mediated proliferation of thyroid carcinoma cells by miR-449b.

Thyroid carcinoma (TC) is a lethal cancer worldwide, whereas its carcinogenesis is not fully understood. Although growing evidence has demonstrated that dysregulation of microRNAs (miRNAs) contributes to the development of various cancers, the role of miRNAs in the pathogenesis of TC is poorly characterized. Here, we analyzed the levels of miR-449b in TC tissues and detected significantly lower miR-449b levels in TC tissues. Moreover, the low miR-449b levels were associated with poor survival. We then overexpressed miR-449b by miRNA mimic transfection and inhibited miR-449b by miRNA antisense transfection. Cell growth was analyzed by CCK-8 assay and MTT assay, and apoptosis and cell proliferation were analyzed by flow cytometry. Overexpression of miR-449b significantly inhibited cell growth, while depletion of miR-449b increased cell growth. Moreover, the effects of miR-449b on cell growth were through modulation of cell proliferation rather than through modulation of cell apoptosis. Targeted genes were predicted by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay, showing that miR-449b binds to the 3'-UTR of MET (hepatocyte growth factor receptor) mRNA, to inhibit its expression in TC cells. MET levels were regulated by miR-449b in TT cells. Together, we show that reduced miR-449b levels in TT tissues may promote TC growth, through MET-mediated cell proliferation.

Heat-shock protein 27 (HSP27, HSPB1) is up-regulated by MET kinase inhibitors and confers resistance to MET-targeted therapy.

The tyrosine kinase encoded by the MET oncogene is activated by gene mutation or amplification in tumors, which in most instances maintain addiction, i.e., dependency, to MET activation. This makes MET an attractive candidate for targeted therapies. Here we show that, in 3/3 MET-addicted human gastric cancer cell lines, MET kinase inhibition resulted in a 3- to 4-fold increased expression of the antiapoptotic small heat-shock protein of 27 kDa (HSP27, HSPB1). HSP27 increase depended on the inhibition of the MEK/ERK pathway and on heat-shock factor 1 (HSF1) and hypoxia-inducible factor-1α (HIF-1α) regulation. Importantly, HSP27-silenced MET-addicted cells underwent 2- and 3-fold more apoptosis following MET inhibition in vitro and in vivo, respectively. Likewise, in human cancer cells susceptible to epidermal growth factor receptor (EGFR) inhibition, EGFR inhibitors induced HSP27 expression and were strengthened by HSP27 suppression. In control cell lines that were not affected by drugs targeting MET or EGFR, these drugs did not induce HSP27 increase. Therefore, in cancer therapies targeting the MET pathway, the induction of HSP27 might limit the efficacy of anti-MET agents. As HSP27 increase also impairs the effectiveness of EGFR inhibitors and is known to protect cells from chemotherapeutics, the induction of HSP27 by targeted agents might strongly affect the success of combination treatments.

Unlocking c-MET: A comprehensive journey into targeted therapies for breast cancer.

Breast cancer is the most common malignancy among women, posing a formidable health challenge worldwide. In this complex landscape, the c-MET (cellular-mesenchymal epithelial transition factor) receptor tyrosine kinase (RTK), also recognized as the hepatocyte growth factor (HGF) receptor (HGFR), emerges as a prominent protagonist, displaying overexpression in nearly 50% of breast cancer cases. Activation of c-MET by its ligand, HGF, secreted by neighboring mesenchymal cells, contributes to a cascade of tumorigenic processes, including cell proliferation, metastasis, angiogenesis, and immunosuppression. While c-MET inhibitors such as crizotinib, capmatinib, tepotinib and cabozantinib have garnered FDA approval for non-small cell lung cancer (NSCLC), their potential within breast cancer therapy is still undetermined. This comprehensive review embarks on a journey through structural biology, multifaceted functions, and intricate signaling pathways orchestrated by c-MET across cancer types. Furthermore, we highlight the pivotal role of c-MET-targeted therapies in breast cancer, offering a clinical perspective on this promising avenue of intervention. In this pursuit, we strive to unravel the potential of c-MET as a beacon of hope in the fight against breast cancer, unveiling new horizons for therapeutic innovation.

Membrane Receptor-Mediated Disruption of Cellular Homeostasis: Changes in Intracellular Signaling Pathways Increase the Toxicity of Ochratoxin A.

Organisms maintain their cellular homeostatic balance by interacting with their environment through the use of their cell surface receptors. Membrane based receptors such as the transforming growth factor ß receptor (TGFR), the prolactin receptor (PRLR), and hepatocyte growth factor receptor (HGFR), along with their associated signaling cascade, play significant roles in retaining cellular homeostasis. While these receptors and related signaling pathways are essential for health of cell and organism, their dysregulation can lead to imbalance in cell function with severe pathological conditions such as cell death or cancer. Ochratoxin A (OTA) can disrupt cellular homeostasis by altering expression levels of these receptors and/or receptor-associated intracellular downstream signaling modulators and/or pattern and levels of their phosphorylation/dephosphorylation. Recent studies have shown that the activity of the TGFR, the PRLR, and HGFR and their associated signaling cascades change upon OTA exposure. A critical evaluation of these findings suggests that while increased activity of the HGFR and TGFR signaling pathways leads to an increase in cell survival and fibrosis, decreased activity of the PRLR signaling pathway leads to tissue damage. This review explores the roles of these receptors in OTA-related pathologies and effects on cellular homeostasis.

Involvement of the AKT Pathway in Resistance to Erlotinib and Cabozantinib in Triple-Negative Breast Cancer Cell Lines.

Resistance to protein tyrosine kinase inhibitors (TKIs) presents a significant challenge in therapeutic target development for cancers such as triple-negative breast cancer (TNBC), where conventional therapies are ineffective at combatting systemic disease. Due to increased expression, the receptor tyrosine kinases EGFR (epidermal growth factor receptor) and c-Met are potential targets for treatment. However, targeted anti-EGFR and anti-c-Met therapies have faced mixed results in clinical trials due to acquired resistance. We hypothesize that adaptive responses in regulatory kinase networks within the EGFR and c-Met signaling axes contribute to the development of acquired erlotinib and cabozantinib resistance. To test this, we developed two separate models for cabozantinib and erlotinib resistance using the MDA-MB-231 and MDA-MB-468 cell lines, respectively. We observed that erlotinib- or cabozantinib-resistant cell lines demonstrate enhanced cell proliferation, migration, invasion, and activation of EGFR or c-Met downstream signaling (respectively). Using a SILAC (Stable Isotope Labeling of Amino acids in Cell Culture)-labeled quantitative mass spectrometry proteomics approach, we assessed the effects of erlotinib or cabozantinib resistance on the phosphoproteome, proteome, and kinome. Using this integrated proteomics approach, we identified several potential kinase mediators of cabozantinib resistance and confirmed the contribution of AKT1 to erlotinib resistance in TNBC-resistant cell lines.

Expression of c-MET in Estrogen Receptor Positive and HER2 Negative Resected Breast Cancer Correlated with a Poor Prognosis.

Introduction: The mesenchymal-epithelial transition factor (c-MET) receptor is overexpressed in about 14−54% of invasive breast cancers, but its prognostic value in clinical practice is still unclear. Methods: In order to investigate the relationship between c-MET expression levels and prognosis, we retrospectively reviewed the clinical features and outcomes of 105 women with estrogen receptor positive HER2 negative (ER+/HER2-) resected breast cancer. We used the Kaplan Meier method to estimate Disease Free Survival (DFS) and Breast Cancer Specific Survival (BCSS) in the subgroups of patients with high (≥50%) and low (<50%) c-MET expression. Univariate and multivariate Cox proportional regression models were performed to assess the prognostic impact of clinicopathological parameters for DFS an BCSS. Results: High c-MET values significantly correlated with tumor size, high Ki67 and low (<20%) progesterone receptor expression. At a median follow up of 60 months, patients with high c-MET tumor had significantly worse (p = 0.00026) and BCSS (p = 0.0013). Univariate analysis showed a significant association between large tumor size, elevated Ki67, c-MET values and increased risk of recurrence or death. The multivariate COX regression model showed that tumor size and high c-MET expression were independent predictors of DFS (p = 0.019 and p = 0.022). Moreover, large tumor size was associated with significantly higher risk of cancer related death at multivariate analysis (p = 0.017), while a trend towards a poorer survival was registered in the high c-MET levels cohort (p = 0.084). Conclusions: In our series, high c-MET expression correlated with poor survival outcomes. Further studies are warranted to validate the clinical relevance and applicability of c-MET as a prognostic factor in ER+/HER2- early BC.

MET Exon 14 Splice-Site Mutations Preferentially Activate KRAS Signaling to Drive Tumourigenesis.

Targeted therapies for MET exon 14-skipping (METΔex14)-driven lung cancers have generated some promising results but response rates remain below that seen for other kinase-driven cancers. One strategy for improving treatment outcomes is to employ rational combination therapies to enhance the suppression of tumour growth and delay or prevent the emergence of resistance. To this end, we profiled the transcriptomes of MET-addicted lung tumours and cell lines and identified the RAS-mitogen-activated protein kinase (MAPK) pathway as a critical effector required for METΔex14-dependent growth. Ectopic expression of MET in an isogenic cell line model showed that overexpression of the mutant MET receptor led to higher levels of MAPK phosphorylation and nuclear import, resulting in increased expression and phosphorylation of nuclear MAPK targets. In comparison, other known MET effectors were unaffected. Inhibition of this pathway by KRAS knockdown in MET-addicted cells in vitro led to decreased viability in only the METΔex14-mutant cells. Conversely, decoupling RAS-MAPK axis, but not other effector pathways, from MET activity via the introduction of constitutively active mutants conferred resistance to MET inhibitors in vitro. Our results suggest that aberrant hyperactivity of the MET receptor caused by the exon 14-skipping mutation does not uniformly upregulate all known downstream effectors, rather gaining a predilection for aberrantly activating and subsequently relying on the RAS-MAPK pathway. These findings provide a rationale for the co-targeting of the RAS-MAPK pathway alongside MET to prolong therapeutic response and circumvent resistance to improve patient survival.

Macrocyclic Peptide-Conjugated Tip for Fast and Selective Molecular Recognition Imaging by High-Speed Atomic Force Microscopy.

Fast and selective recognition of molecules at the nanometer scale without labeling is a much desired but still challenging goal to achieve. Here, we show the use of high-speed atomic force microscopy (HS-AFM) for real-time and real-space recognition of unlabeled membrane receptors using tips conjugated with small synthetic macrocyclic peptides. The single-molecule recognition method is validated by experiments on the human hepatocyte growth factor receptor (hMET), which selectively binds to the macrocyclic peptide aMD4. By testing and comparing aMD4 synthesized with linkers of different lengths and rigidities, we maximize the interaction between the functionalized tip and hMET added to both a mica surface and supported lipid bilayers. Phase contrast imaging by HS-AFM enables us to discriminate nonlabeled hMET against the murine MET homologue, which does not bind to aMD4. Moreover, using ligands and linkers of small size, we achieve minimal deterioration of the spatial resolution in simultaneous topographic imaging. The versatility of macrocyclic peptides in detecting unlimited types of membrane receptors with high selectivity and the fast imaging by HS-AFM broaden the range of future applications of this method for molecular recognition without labeling.

3-phosphoinositide-dependent protein kinase 1 (PDK1) mediates crosstalk between Src and Akt pathways in MET receptor signaling.

The high-affinity tyrosine kinase receptor MET plays a pivotal role in several facets of cell regulation. Although its mitogenic effect is well documented, some aspects of connection patterns between signaling pathways involved in cell cycle progression remain to be deciphered. We have used a tractable heterologous expression system, the Xenopus oocyte, to detect connections between distinct MET signaling cascades involved in G2/M progression. Our results reveal that Src acts as an adapter via its SH2 domain to recruit 3-phosphoinositide-dependent protein kinase 1 (PDK1) to the MET signaling complex leading to Akt phosphorylation. These data define an original crosstalk between Src and Akt signaling pathways that contributes to MET-induced entry into the M phase, and deserves further investigation in pathologies harboring deregulation of this receptor.

Cell of origin in biliary tract cancers and clinical implications.

Biliary tract cancers (BTCs) are aggressive epithelial malignancies that can arise at any point of the biliary tree. Albeit rare, their incidence and mortality rates have been rising steadily over the past 40 years, highlighting the need to improve current diagnostic and therapeutic strategies. BTCs show high inter- and intra-tumour heterogeneity both at the morphological and molecular level. Such complex heterogeneity poses a substantial obstacle to effective interventions. It is widely accepted that the observed heterogeneity may be the result of a complex interplay of different elements, including risk factors, distinct molecular alterations and multiple potential cells of origin. The use of genetic lineage tracing systems in experimental models has identified cholangiocytes, hepatocytes and/or progenitor-like cells as the cells of origin of BTCs. Genomic evidence in support of the distinct cell of origin hypotheses is growing. In this review, we focus on recent advances in the histopathological subtyping of BTCs, discuss current genomic evidence and outline lineage tracing studies that have contributed to the current knowledge surrounding the cell of origin of these tumours.