MET-dependent cancer invasion may be preprogrammed by early alterations of p53-regulated feedforward loop and triggered by stromal cell-derived HGF

MET, a receptor protein tyrosine kinase activated by hepatocyte growth factor (HGF), is a crucial determinant of metastatic progression. Recently, we have identified p53 as an important regulator of MET-dependent cell motility and invasion. This regulation occurs via feedforward loop suppressing MET expression by miR- 34-dependent and -independent mechanisms. Here, by using Dicer conditional knockout, we provide further evidence for microRNA-independent MET regulation by p53. Furthermore, we show that while MET levels increase immediately after p53 inactivation, mutant cells do not contain active phosphorylated MET and remain non-invasive for a long latency period at contrary to cell culture observations. Evaluation of mouse models of ovarian and prostate carcinogenesis indicates that formation of desmoplastic stroma, associated production of HGF by stromal cells and coinciding MET phosphorylation precede cancer invasion. Thus, initiation mutation of p53 is sufficient for preprogramming motile and invasive properties of epithelial cells, but the stromal reaction may represent a critical step for their manifestation during cancer progression.Key words: feedforward loop, HGF, invasion, MET, metastasis, motility, p53 mutation     

Cabozantinib inhibits AXL- and MET-dependent cancer cell migration induced by growth-arrest-specific 6 and hepatocyte growth factor

Cabozantinib is known as an inhibitor of receptor tyrosine kinases mainly targeting AXL receptor tyrosine kinase (AXL), MET proto-oncogene-encoded receptor tyrosine kinase (MET), and vascular endothelial growth factor receptor 2. Growth arrest-specific 6 (GAS6) and hepatocyte growth factor (HGF), the natural ligands of AXL and MET, respectively, are associated with the induction of cancer cell proliferation or metastasis. Currently, it is still unclear how cabozantinib regulates cancer cell migration and invasion by inhibiting AXL and MET. This study was conducted to investigate the mechanism underlying the anti-cancer effects of cabozantinib through regulation of AXL and MET signaling.The results of Boyden chamber assays showed that cancer cell migration was induced by GAS6 and HGF in SKOV3 cells in serum-free medium. Combinatorial treatment with GAS6 and HGF exerted an additive effect on cell migration. Furthermore, we examined the role of AXL and MET signaling in cell migration. Short interfering RNA targeting AXL and MET inhibited GAS6- and HGF-induced migration, respectively. Double knockdown of AXL and MET completely suppressed cell migration induced by combination treatment with GAS6 and HGF compared to AXL or MET inhibition alone. Finally, we investigated the effects of cabozantinib on cell migration and invasion. Cabozantinib inhibited AXL and MET phosphorylation and downregulated the downstream mediators, phosphorylated SRC in the presence of both GAS6 and HGF in SKOV3 cells. The cell migration and invasion induced by combined GAS6 and HGF treatment were suppressed by cabozantinib, but not by capmatinib, a selective MET inhibitor.Our data indicate that the GAS6-AXL and HGF-MET signal pathways markedly contribute to cancer cell migration and invasion in an independent manner, suggesting that simultaneous inhibition of these two pathways contributes to the anti-cancer effects of cabozantinib.     

Hepatic stellate cell promoted hepatoma cell invasion via the HGF/c-Met signaling pathway regulated by p53

The biological behaviors of hepatocellular carcinoma (HCC) are complex mainly due to heterogeneity of progressive genetic and epigenetic mutations as well as tumor environment. Hepatocyte growth factor (HGF)/c-Met signaling pathway is regarded to be a prototypical example for stromal-epithelial interactions during developmental morphogenesis, wound healing, organ regeneration and cancer progression. And p53 plays as an important regulator of Met-dependent cell motility and invasion. Present study showed that 2 HCC cell lines, Hep3B and HepG2, displayed different invasive capacity when treated with HGF which was secreted by hepatic stellate cells (HSCs). We found that HGF promoted Hep3B cells invasion and migration as well as epithelial-mesenchymal transition (EMT) occurrence because Hep3B was p53 deficient, which leaded to the c-Met over-expression. Then we found that HGF/c-Met promoted Hep3B cells invasion and migration by upregulating Snail expression. In conclusion, HGF/c-Met signaling is enhanced by loss of p53 expression, resulting in increased ability of invasion and migration by upregulating the expression of Snail.     

The RON and MET oncogenes are co-expressed in human ovarian carcinomas and cooperate in activating invasiveness

RON is a member of the receptor tyrosine kinase gene family that includes the MET oncogene, whose germline mutations have been causally related to human tumorigenesis. In vitro, RON and MET receptors cross-talk, synergize in intracellular signaling, and cooperate in inducing morphogenic responses. Here we show that the RON and MET oncogenes were expressed in 55% and 56% of human ovarian carcinomas, respectively, and were significantly coexpressed in 42% (P < 0.001). In ovarian carcinoma samples and cell lines we did not find mutations in RON and MET gene kinase domain, nor coexpression of RON and MET receptor ligands (MSP and HGF, respectively). We show that motility and invasiveness of ovarian cancer cells coexpressing MET and RON receptors were elicited by HGF and, to a lesser extent, by MSP. More interestingly, invasion of both reconstituted basement membrane and collagen gel was greatly enhanced by the simultaneous addition of the two ligands. These data suggest that coexpression of the MET and RON receptors confer a selective advantage to ovarian cancer cells and might promote ovarian cancer progression.     

SRC kinase activator CDCP1 promotes hepatocyte growth factor–induced cell migration/invasion of a subset of breast cancer cells

Cancer invasion and metastasis are the major causes of cancer patient mortality. Various growth factors, including hepatocyte growth factor (HGF), are known to promote cancer invasion and metastasis, but the regulatory mechanisms involved are not fully understood. Here, we show that HGF-promoted migration and invasion of breast cancer cells are regulated by CUB domain–containing protein 1 (CDCP1), a transmembrane activator of SRC kinase. In metastatic human breast cancer cell line MDA-MB-231, which highly expresses the HGF receptor MET and CDCP1, we show that CDCP1 knockdown attenuated HGF-induced MET activation, followed by suppression of lamellipodia formation and cell migration/invasion. In contrast, in the low invasive/nonmetastatic breast cancer cell line T47D, which had no detectable MET and CDCP1 expression, ectopic MET expression stimulated the HGF-dependent activation of invasive activity, and concomitant CDCP1 expression activated SRC and further promoted invasive activity. In these cells, CDCP1 expression dramatically activated HGF-induced membrane remodeling, which was accompanied by activation of the small GTPase Rac1. Analysis of guanine nucleotide exchange factors revealed that ARHGEF7 was specifically required for CDCP1-dependent induction of HGF-induced invasive ability. Furthermore, immunofluorescence staining demonstrated that CDCP1 coaccumulated with ARHGEF7. Finally, we confirmed that the CDCP1-SRC axis was also crucial for HGF and ARHGEF7-RAC1 signaling in MDA-MB-231 cells. Altogether, these results demonstrate that the CDCP1-SRC-ARHGEF7-RAC1 pathway plays an important role in the HGF-induced invasion of a subset of breast cancer cells.     

Promise and challenges on the horizon of MET-targeted cancer therapeutics

MET(MNNG HOS transforming gene) is one of the receptor tyrosine kinases whose activities are frequently altered in human cancers, and it is a promising therapeutic target. MET is normally activated by its lone ligand, hepatocyte growth factor(HGF), eliciting its diverse biological activities that are crucial for development and physiology. Alteration of the HGF-MET axis results in inappropriate activation of a cascade of intracellular signaling pathways that contributes to hallmark cancer events including deregulated cell proliferation and survival, angiogenesis, invasion, andmetastasis. Aberrant MET activation results from autocrine or paracrine mechanisms due to overexpression of HGF and/or MET or from a ligand-independent mechanism caused by activating mutations or amplification of MET. The literature provides compelling evidence for the role of MET signaling in cancer development and progression. The finding that cancer cells often use MET activation to escape therapies targeting other pathways strengthens the argument for MET-targeted therapeutics. Diverse strategies have been explored to deactivate MET signaling, and compounds and biologics targeting the MET pathway are in clinical development. Despite promising results from various clinical trials, we are still waiting for true MET-targeted therapeutics in the clinic. This review will explore recent progress and hurdles in the pursuit of METtargeted cancer drugs and discuss the challenges in such development.     

RGS-GAIP-interacting protein controls breast cancer progression

Although the importance of RGS-GAIP-interacting protein (GIPC) in the biology of malignant cells is well known, the molecular mechanism of GIPC in the inhibition of tumor progression has not been identified. This study focused on elucidating the molecular role of GIPC in breast cancer progression. By using a human breast tumor specimen, an in vivo mouse model, and breast cancer cell lines, we showed for the first time that GIPC is involved in breast cancer progression through regulation of breast cancer cell proliferation, survival, and invasion. Furthermore, we found that the Akt/Mdm2/p53 axis, insulin-like growth factor-1 receptor, matrix metalloproteinase-9, and Cdc42 were downstream of GIPC signaling in breast cancer cells. Moreover, we showed that wild-type p53 reduced GIPC-induced breast cancer cell survival, whereas mutant p53 inhibited GIPC-induced cell invasion. Finally, we demonstrated that an N-myristoylated GIPC peptide (CR1023, N-myristoyl-PSQSSSEA) capable of blocking the PDZ domain of GIPC successfully inhibited MDA-MB-231 cell proliferation, survival, and further in vivo tumor growth. Taken together, these findings demonstrate the importance of GIPC in breast tumor progression, which has a potentially significant impact on the development of therapies against many common cancers expressing GIPC, including breast and renal cancer.     

HGF/MET signaling in ovarian cancer

Ovarian cancer is the leading cause of death from gynecological cancers in North America and Europe. Despite its clinical significance, the factors that regulate the development and progression of ovarian cancer are among the least understood of all major human malignancies. A growth factor with pleiotropic effects, which has attracted increasing attention in recent years, is the hepatocyte growth factor (HGF) and its receptor MET. While deregulated HGF/MET signaling is observed in many tumors, the consequences of MET activation are complex and context dependent. Recent observations have demonstrated a cross-talk of other signaling pathways with MET signaling. This review summarizes the key findings and recent advances in our understanding of HGF and MET in the transformation and progression of ovarian cancer. We will begin with a brief discussion on the role of HGF and MET in the physiology of normal ovarian surface epithelium (OSE) and ovarian cancer development. In particular, the coexpression of HGF and MET in OSE of women with hereditary ovarian cancer syndromes emphasizes their importance in neoplastic transformation of OSE. The involvement of HGF in other aspects of tumor progression, such as invasion and metastasis, and novel downstream target genes activated by HGF is summarized next. The therapeutic potential of HGF to treat ovarian cancer and to improve response to conventional chemotherapy is also described. Finally, the most recent progress in drug development and future areas of research in terms of their potential clinical implications are discussed.     

Involvement of stromal p53 in tumor-stroma interactions

p53 is a major tumor-suppressor gene, inactivated by mutations in about half of all human cancer cases, and probably incapacitated by other means in most other cases. Most research regarding the role of p53 in cancer has focused on its ability to elicit apoptosis or growth arrest of cells that are prone to become malignant owing to DNA damage or oncogene activation, i.e. cell-autonomous activities of p53. However, p53 activation within a cell can also exert a variety of effects upon neighboring cells, through secreted factors and paracrine and endocrine mechanisms. Of note, p53 within cancer stromal cells can inhibit tumor growth and malignant progression. Cancer cells that evolve under this inhibitory influence acquire mechanisms to silence stromal p53, either by direct inhibition of p53 within stromal cells, or through pressure for selection of stromal cells with compromised p53 function. Hence, activation of stromal p53 by chemotherapy or radiotherapy might be part of the mechanisms by which these treatments cause cancer regression. However, in certain circumstances, activation of stromal p53 by cytotoxic anti-cancer agents might actually promote treatment resistance, probably through stromal p53-mediated growth arrest of the cancer cells or through protection of the tumor vasculature. Better understanding of the underlying molecular mechanisms is thus required. Hopefully, this will allow their manipulation towards better inhibition of cancer initiation, progression and metastasis.     

Inhibition of transforming growth factor-β signaling potentiates tumor cell invasion into collagen matrix induced by fibroblast-derived hepatocyte growth factor

Interaction between tumor cells and stromal fibroblasts plays essential roles in tumor progression. However, its detailed molecular mechanism remains unclear. To understand the mechanism, we investigated molecules mediating this interaction using the three-dimensional (3D) co-culture system of Panc-1 pancreatic carcinoma cells with normal fibroblasts. When the two kinds of cells were placed on the top of collagen gel, the tumor cells scattered into the fibroblast layer, apparently undergoing epithelial‐mesenchymal transition. When fibroblasts were placed within collagen gel, Panc-1 cells actively invaded into the collagen gel, extending a microtubule-based long protrusion. Although transforming growth factor-β (TGF-β) and hepatocyte growth factor (HGF) individually stimulated the tumor cell invasion into collagen gel without fibroblasts, TGF-β signaling inhibitors (SB431542 and LY2157299) significantly enhanced the Panc-1 cell invasion in the 3D co-culture with fibroblasts. Experiments with HGF/Met signaling inhibitors or with the fibroblast conditioned medium revealed that HGF was a major invasion-promoting factor secreted from fibroblasts and SB431542 increased the HGF secretion by blocking the HGF-suppressing activity of cancer cell-derived TGF-β. These results indicate that HGF and TGF-β are critical regulators for both tumor–stroma interaction and tumor invasion. The results also suggest that TGF-β signaling inhibitors may promote tumor progression under some pathological conditions.     

Competitive enhancement of HGF-induced epithelial scattering by accessory growth factors

HGF signaling induces epithelial cells to disassemble cadherin-based adhesion and increase cell motility and invasion, a process termed epithelial–mesenchymal transition (EMT). EMT plays a major role in cancer metastasis, allowing individual cells to detach from the primary tumor, invade local tissue, and colonize distant tissues with new tumors. While invasion of vascular and lymphatic networks is the predominant route of metastasis, nerves also can act as networks for dissemination of cancer cell to distant sites in a process termed perineual invasion (PNI). Signaling between nerves and invasive cancer cells remains poorly understood, as does cellular decision making that selects the specific route of invasion. Here we examine how HGF signaling contributes to PNI using reductionist culture model systems. We find that TGFβ, produced by PC12 cells, enhances scattering in response to HGF stimulation, increasing both cell–cell junction disassembly and cell migration. Further, gradients of TGFβ induce migratory mesenchymal cells to undergo chemotaxis towards the source of TGFβ. Interestingly, VEGF suppresses TGFβ-induced enhancement of scattering. These results have broad implications for how combinatorial growth factor signaling contributes to cancer metastasis, suggesting that VEGF and TGFβ might modulate HGF signaling to influence route selection during cancer progression.     

Hepatocyte growth factor and Met in tumor biology and therapeutic approach with NK4

Hepatocyte growth factor (HGF) and Met/HGF receptor tyrosine kinase play a role in the progression to invasive and metastatic cancers. A variety of cancer cells secrete molecules that enhance HGF expression in stromal fibroblasts, while fibroblast-derived HGF, in turn, is a potent stimulator of the invasion of cancer cells. In addition to the ligand-dependent activation, Met receptor activation is negatively regulated by cell-cell contact and Ser985 phosphorylation in the juxtamembrane of Met. The loss of intercellular junctions may facilitate an escape from the cell-cell contact-dependent suppression of Met-signaling. Significance of juxtamembrane mutations found in human cancers is assumed to be a loss-of-function in the negative regulation of Met. In attempts to block the malignant behavior of cancers, NK4 was isolated as a competitive antagonist against HGF-Met signaling. Independently on its HGF-antagonist action, NK4 inhibited angiogenesis induced by vascular endothelial cell growth factor and basic fibroblast growth factor, as well as HGF. In experimental models of distinct types of cancers, NK4 inhibited Met activation and this was associated with inhibition of tumor invasion and metastasis. NK4 inhibited tumor angiogenesis, thereby suppressing angiogenesis-dependent tumor growth. Cancer treatment with NK4 suppresses malignant tumors to be "static" in both tumor growth and spreading.     

MET molecular mechanisms and therapies in lung cancer

The MET tyrosine kinase signaling pathway is upregulated in many cancers, including lung cancer. The pathway normally promotes mitosis, cell motility and cell survival; but in cancer it can also promote cell proliferation, invasion, metastasis, and angiogenesis. The activating ligand, hepatocyte growth factor, is normally secreted by fibroblasts and smooth muscle cells, but can also be produced by tumor cells. MET upregulation in lung cancer is caused by overexpression and mutation. These mutations can vary with ethnicity. MET signaling affects cytoskeletal proteins such as paxillin, which participates in cell adhesion, growth and motility. Therapeutic approaches that block MET signaling are being studied, and include the use of: small interference RNA, Geldanamycin, competitive HGF homologues, decoy receptors, and direct MET inhibitors such as K252a, SU11274, PHA665752 and PF2341066. It is hoped that blocking MET signaling may one day become an effective treatment for some lung cancers.     

Hepatocyte growth factor enhances proteolysis and invasiveness of human nasopharyngeal cancer cells through activation of PI3K and JNK

The hepatocyte growth factor (HGF) receptor, Met, is frequently overexpressed in nasopharyngeal cancer (NPC). Here, we showed for the first time that human NPC cells with high Met expression were more sensitive to the cell motility and invasion effect of HGF. The downregulation of Met by small interfering RNA decreased tumor cell invasion/migration. HGF significantly increased matrix metalloproteinase-9 production. This was inhibited by blocking phosphatidylinositide 3-kinase (PI3K) and c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase signaling pathways. We also demonstrated that PI3K induced activation of JNK, with Akt as a potential point of this cross-talk. These results provide new insights into the molecular mechanism responsible for NPC progression and metastasis.     

Regulation of invadopodia by the tumor microenvironment

The tumor microenvironment consists of stromal cells, extracellular matrix (ECM), and signaling molecules that communicate with cancer cells. As tumors grow and develop, the tumor microenvironment changes. In addition, the tumor microenvironment is not only influenced by signals from tumor cells, but also stromal components contribute to tumor progression and metastasis by affecting cancer cell function. One of the mechanisms that cancer cells use to invade and metastasize is mediated by actin-rich, proteolytic structures called invadopodia. Here, we discuss how signals from the tumor environment, including growth factors, hypoxia, pH, metabolism, and stromal cell interactions, affect the formation and function of invadopodia to regulate cancer cell invasion and metastasis. Understanding how the tumor microenvironment affects invadopodia biology could aid in the development of effective therapeutics to target cancer cell invasion and metastasis.     

Regulation of invadopodia by the tumor microenvironment

The tumor microenvironment consists of stromal cells, extracellular matrix (ECM), and signaling molecules that communicate with cancer cells. As tumors grow and develop, the tumor microenvironment changes. In addition, the tumor microenvironment is not only influenced by signals from tumor cells, but also stromal components contribute to tumor progression and metastasis by affecting cancer cell function. One of the mechanisms that cancer cells use to invade and metastasize is mediated by actin-rich, proteolytic structures called invadopodia. Here, we discuss how signals from the tumor environment, including growth factors, hypoxia, pH, metabolism, and stromal cell interactions, affect the formation and function of invadopodia to regulate cancer cell invasion and metastasis. Understanding how the tumor microenvironment affects invadopodia biology could aid in the development of effective therapeutics to target cancer cell invasion and metastasis.