Down-regualtion of miR-106b induces epithelial-mesenchymal transition but suppresses metastatic colonization by targeting Prrx1 in colorectal cancer

Accumulating evidence identified that epithelial-mesenchymal transition (EMT) is acquired during progression to metastatic, but whether it is an absolute requirement is still controversial. MiR-106b has been confirmed to promote cancer cell proliferation; however few studies are available on its functions in EMT and metastasis in colorectal cancer (CRC). In this study, we found that knocking down miR-106b induced EMT conferring migratory and invasive properties. MiR-106b knockdown induced cytoskeletal reorganization through staining intracellular F-actin. The expression of Rho GTPases (Rac1 and Cdc42) and Tiam1 was significantly enforced after miR-106b down-regulation. However, miR-106b knocking down could suppress metastatic colonization in vivo. Correspondingly, over expression of miR-106b obtained an opposite effect. We identified Prrx1 was a direct target of miR-106b through using target prediction algorithms and dual-Luciferase reporter assay. Moreover, Moreover, we also found TGF-β1 could down-regulate miR-106b, and simultaneously miR-106b also influences the expression of TGF-β1, establishing a negative feedback loop to regulate the expression of Prrx1 together. Taken together, these findings demonstrated that miR-106b knockdown could induce EMT which conferring cells migratory and invasive properties but could not accomplish distant metastatic colonization efficiently.     

Mesenchymal–epithelial transition of pancreatic cancer cells at perineural invasion sites is induced by Schwann cells

Epithelial–mesenchymal transition (EMT) promotes invasion and metastasis of pancreatic ductal adenocarcinoma (PDAC). However, the importance of its reverse process, mesenchymal–epithelial transition (MET), for PDAC remains unclear. We aimed to characterize the histological finding “focal differentiation” in PDAC at perineural invasion sites in the context of MET and to investigate the role of Schwann cells in inducing tumor MET. Tumor differentiation and immunohistochemical expressions of E‐cadherin, SMAD3, and vimentin at perineural invasion sites were examined in 168 PDAC tissues. Four PDAC cell lines were co‐cultured with Schwann cells to investigate cell morphology, motility, or EMT‐related markers using immunocytochemistry and quantitative PCR. Of 168 tumors, 124 (74%) showed focal differentiation with enhanced E‐cadherin membrane expression (P < 0.001) and decreased nuclear accumulation of SMAD3 (P < 0.001). Among 115 PDACs harboring grade 1/2 tumor, tumors with focal differentiation showed worse survival compared to those without focal differentiation (P = 0.019). PDAC cells co‐cultured with Schwann cells demonstrated a sheet‐like appearance, increased E‐cadherin expression, decreased expressions of SMAD3 and vimentin, and reduced cell motility. In conclusion, MET‐like change is induced by Schwann cells, suggesting that Schwann cells contribute to PDAC colonization in pancreatic nerves through activating the MET machinery inside tumor cells in the pancreatic tumor microenvironment.     

Epithelial–mesenchymal plasticity in carcinoma metastasis

Tumor metastasis is a multistep process by which tumor cells disseminate from their primary site and form secondary tumors at a distant site. Metastasis occurs through a series of steps: local invasion, intravasation, transport, extravasation, and colonization. A developmental program termed epithelial–mesenchymal transition (EMT) has been shown to play a critical role in promoting metastasis in epithelium-derived carcinoma. Recent experimental and clinical studies have improved our knowledge of this dynamic program and implicated EMT and its reverse program, mesenchymal–epithelial transition (MET), in the metastatic process. Here, we review the functional requirement of EMT and/or MET during the individual steps of tumor metastasis and discuss the potential of targeting this program when treating metastatic diseases.     

PAK1 mediates pancreatic cancer cell migration and resistance to MET inhibition

Pancreatic adenocarcinoma (PDAC) is a major unmet medical need and a deeper understanding of molecular drivers is needed to advance therapeutic options for patients. We report here that p21‐activated kinase 1 (PAK1) is a central node in PDAC cells downstream of multiple growth factor signalling pathways, including hepatocyte growth factor (HGF) and MET receptor tyrosine kinase. PAK1 inhibition blocks signalling to cytoskeletal effectors and tumour cell motility driven by HGF/MET. MET antagonists, such as onartuzumab and crizotinib, are currently in clinical development. Given that even highly effective therapies have resistance mechanisms, we show that combination with PAK1 inhibition overcomes potential resistance mechanisms mediated either by activation of parallel growth factor pathways or by direct amplification of PAK1. Inhibition of PAK1 attenuated in vivo tumour growth and metastasis in a model of pancreatic adenocarcinoma. In human tissues, PAK1 is highly expressed in a proportion of PDACs (33% IHC score 2 or 3; n = 304) and its expression is significantly associated with MET positivity (p < 0.0001) and linked to a widespread metastatic pattern in patients (p = 0.067). Taken together, our results provide evidence for a functional role of MET/PAK1 signalling in pancreatic adenocarcinoma and support further characterization of therapeutic inhibitors in this indication. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

SPRR2A expression in cholangiocarcinoma increases local tumor invasiveness but prevents metastasis

Cholangiocarcinoma morbidity and mortality is attributable to local invasiveness and regional lymph node and distant organ metastasis. Cholangiocarcinoma progression follows a series of sequential events that resemble wound healing reactions: local invasion resembles the epithelial migration phase involving epithelial–mesenchymal transition (EMT); colonization at distant sites resembles epithelial restitution seen during the reverse process, mesenchymal–epithelial transition (MET). In this study we compare the in vivo local and metastatic growth potential of cholangiocarcinoma cell lines with respect to expression of a novel pSTAT3-dependent, biliary epithelial cell wound healing protein, small proline-rich protein 2A (SPRR2A). SPRR2A has been associated with local aggressiveness, but decreased metastatic capabilities in other cancers. Stable SPRR2A transfection into two cholangiocarcinoma cell lines (SG231 and HuCCT-1), previously shown by us to induce permanent EMT, resulted in local aggressiveness but an inability to form metastases. In contrast, SPRR2A-negative epithelial control cells showed relatively poor local aggressiveness, but readily formed metastatic tumors. Post-intrasplenic injection cell tracking showed that: (a) mesenchymal (SPRR2A+) cells were not trapped in the liver, but were rapidly cleared through mesenteric lymph nodes and did not form metastases; whereas (b) epithelial (SPRR2A?) controls were primarily entrapped within MUC-1-associated liver “micro-infarcts” that later evolved into metastatic colonies. SPRR2A-associated tumor behavior was mimicked by MUC1 shRNA, which induced EMT and, like SPRR2A+ cells, showed reduced metastatic capabilities. Cholangiocarcinoma local invasion involves EMT processes, whereas MET and MUC1 expression promote metastasis. A better understanding of disease progression should help target treatment for this deadly neoplasm.     

Nestin Delineates Pancreatic Cancer Stem Cells in Metastatic Foci of NOD/Shi-scid IL2Rγnull (NOG) Mice

Pancreatic ductal adenocarcinoma (PDAC) is associated with a high incidence of hepatic metastases, as well as occasional pulmonary metastases. To delineate the potential role of cancer stem cells (CSCs) in PDAC metastasis, human PDAC cells were injected into the spleen of mice. The characteristics and expression of markers associated with CSC and epithelial–mesenchymal transition (EMT) of metastatic cells that developed in the liver and lung were then compared with parental cells. The metastatic cells were polygonal, and larger than parental cells. Metastatic cells also exhibited decreased proliferation and increased adhesion to extracellular matrices, as well as enhanced migration and invasion in vitro and increased metastatic capacity in vivo. The CSC markers ALDH1A1, ABCG2, and nestin were expressed at high levels in metastatic cells and exhibited changes consistent with EMT (eg, decreased E-cadherin expression). Moreover, metastatic cells readily formed spheres in culture and exhibited an increased side population by flow analysis. Nestin and ABCG2 were also expressed at high levels in metastatic lesions from PDAC patients, and silencing nestin with shRNA in PDAC cells derived from lung metastases resulted in a marked decrease in the capacity of the cells to form spheres and to yield pulmonary or hepatic metastases. Thus, the metastatic potential of human PDAC cells correlates with CSCs and with EMT characteristics and is dependent on nestin expression.Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a high incidence of distant metastasis.¹ The metastatic process involves a series of complex biological processes, including migration, invasion, adhesion, and survival.² Furthermore, the extracellular matrix, interstitial cells, blood and lymph vessels, nerve fibers, and immune cells contribute to these processes. It is difficult to completely resolve the pathways that promote invasion and metastasis of PDAC cells, but elucidating the mechanisms that contribute to their enhanced metastatic efficiency is important for development of novel therapeutic approaches. Accordingly, a variety of mouse models, ranging from orthotopic models to genetically engineered mice, have been developed to study the metastatic processes that govern PDAC metastasis.Injection of human PDAC cells into the spleen of nude mice or NOD/SCID mice is a model that yields a small number of metastatic nodules in the liver.³ By contrast, in the recently developed NOD/Shi-scid IL2Rγ^null (NOG) mice (which have severe immunodeficiency, including defects in T, B, natural killer, and dendritic cell function) human pancreatic and colorectal cancer cells easily proliferate and metastasize to the liver after intrasplenic injection.⁴ It is not known, however, whether cancer stem cells (CSCs) have a role in the enhanced metastatic propensity of NOG mice. CSCs exhibit pluripotency, have the potential to self-renew, and are crucially important in cancer cell growth, invasion, metastasis, and recurrence.^5–7 CSCs preferentially spread to distant organs and contribute to the formation of lesions that phenocopy the primary tumor.⁸Given these observations, it has been suggested that targeting CSCs could provide a new therapeutic strategy in cancer.⁹ It is therefore important to examine the temporal and spatial alterations of CSCs in metastatic lesions in vivo. Furthermore, epithelial–mesenchymal transition (EMT), a fundamental mechanism controlling multiple events during metastasis,¹⁰ may generate additional CSCs endowed with a more invasive, migratory, and metastatic phenotype.¹¹ We reasoned that NOG mice would be useful for assessing the role of CSCs in the metastatic process in PDAC, because this model avoids the complexity engendered by an active immune system.In the present study, we analyzed pathological features of metastatic tumors originating from human PDAC cells. Metastatic PDAC cells exhibited lower growth rates, but higher migration and metastatic abilities. The metastatic lesions contained more CSCs than did primary tumors, and these cells exhibited a mesenchymal phenotype. Moreover, inhibition of one of the CSC markers, nestin, in metastatic PDAC cells reduced sphere formation in vitro and metastasis in vivo.     

The MET receptor tyrosine kinase contributes to invasive tumour growth in rhabdomyosarcomas

The receptor tyrosine kinase MET and its ligand hepatocyte growth factor (HGF), have been implicated in the genesis of the paediatric tumour rhabdomyosarcoma (RMS). Addition of exogenous HGF to RH30 RMS cells enhanced non-chemotactic migration. Stable transfection of dominant negative MET into RH30 cells attenuated Matrigel invasion and in vivo tumour growth. To assess the role of a putative HGF–MET pathway in human RMS, we measured their expression in a panel of 68 human primary tumours. All tumours expressed MET but with a three orders of magnitude variation of expression and 62% of tumours co-expressed HGF. In contrast with other tumour types, neither high-MET expression nor HGF/MET coexpression correlated with metastatic disease. In a microarray screen, we identified CCN1 as being 7.8-fold up regulated following addition of HGF to RH30 cells and in RMS tumours, CCN1 expression correlated with HGF expression. Surprisingly, we identified MET as a consistent feature of embryonal and not alveolar RMS.     

The MET receptor tyrosine kinase contributes to invasive tumour growth in rhabdomyosarcomas

The receptor tyrosine kinase MET and its ligand hepatocyte growth factor (HGF), have been implicated in the genesis of the paediatric tumour rhabdomyosarcoma (RMS). Addition of exogenous HGF to RH30 RMS cells enhanced non-chemotactic migration. Stable transfection of dominant negative MET into RH30 cells attenuated Matrigel invasion and in vivo tumour growth. To assess the role of a putative HGF-MET pathway in human RMS, we measured their expression in a panel of 68 human primary tumours. All tumours expressed MET but with a three orders of magnitude variation of expression and 62% of tumours co-expressed HGF. In contrast with other tumour types, neither high-MET expression nor HGF/MET coexpression correlated with metastatic disease. In a microarray screen, we identified CCN1 as being 7.8-fold up regulated following addition of HGF to RH30 cells and in RMS tumours, CCN1 expression correlated with HGF expression. Surprisingly, we identified MET as a consistent feature of embryonal and not alveolar RMS.     

Usefulness of immunohistochemical staining for MUC5AC in differentiating primary pancreatic cancer from pancreatic metastasis of breast cancer

Diagnosis of pancreatic ductal adenocarcinoma (PDAC) and its differentiation from metastases to the pancreas from other organs remains challenging. We report a case in which immunohistochemical staining for MUC5AC was useful in distinguishing primary pancreatic cancer from breast cancer metastasis. A 51‐year‐old Japanese woman who underwent curative resection of her breast cancer was referred to our hospital with a pancreatic head tumor. Although we surmised her pancreatic tumor to be metastatic breast cancer based on her past history and imaging studies, she was subsequently diagnosed with PDAC on the basis of immunohistochemical staining for MUC5AC using specimens obtained by endoscopic ultrasound‐fine‐needle aspiration. Thus, MUC5AC may be a useful diagnostic marker for discriminating PDAC from a secondary malignancy.     

Regulation of uveal melanoma interconverted phenotype by hepatocyte growth factor/scatter factor (HGF/SF).

Human uveal melanoma disseminates initially and preferentially to the liver. This study describes the relationship between the expression of the c-met proto-oncogene (receptor for hepatocyte growth factor/scatter factor (HGF/SF)) in interconverted uveal melanoma cells (co-expressing vimentin and keratin intermediate filaments) and the regulation of their motogenic response to HGF/SF, a key step in local invasion and targeted dissemination to the liver. Expression of c-met in uveal melanoma cell lines correlates with both the appearance of an interconverted phenotype and invasive ability (measured in vitro). Using chemotactic checkerboard analysis, the greatest motogenic response to HGF/SF was achieved by invasive, interconverted, c-met-positive uveal melanoma cells. C-met was observed histologically in a uveal melanoma containing interconverted cells but was absent in a tumor composed of non-interconverted cells (vimentin positive/keratin negative). The c-met ligand, HGF/SF, although not expressed by uveal melanoma cell lines, was localized in tissue sections of primary uveal melanomas and metastatic melanoma to the liver. In the primary tumor, staining for HGF/SF was most intense at the level of the choriocapillaris, a finding that is significant because 1) highly remodeled neovascular loops and networks, which appear in tumors likely to disseminate, can be traced to the choriocapillaris and the draining vortex veins and 2) HGF/SF plays a role in tumor angiogenesis. Foci of metastatic melanoma to the liver stain diffusely for HGF/SF. Regulation of the uveal melanoma interconverted phenotype by HGF/SF may play an important role in the dissemination of this tumor.     

The molecular biology of medulloblastoma metastasis

Medulloblastoma (MB) is the most common primary malignant brain tumor of childhood and a significant contributor to pediatric morbidity and death. While metastatic dissemination is the predominant cause of morbidity and mortality for patients with this disease, most research efforts and clinical trials to date have focused on the primary tumor; this is due mostly to the paucity of metastatic tumor samples and lack of robust mouse models of MB dissemination. Most current insights into the molecular drivers of metastasis have been derived from comparative molecular studies of metastatic and non‐metastatic primary tumors. However, small studies on matched primary and metastatic tissues and recently developed mouse models of dissemination have begun to uncover the molecular biology of MB metastasis more directly. With respect to anatomical routes of dissemination, a hematogenous route for MB metastasis has recently been demonstrated, opening new avenues of investigation. The tumor micro‐environment of the primary and metastatic niches has also been increasingly scrutinized in recent years, and further investigation of these tumor compartments is likely to result in a better understanding of the molecular mediators of MB colonization and growth in metastatic compartments.     

Proteome of formalin-fixed paraffin-embedded pancreatic ductal adenocarcinoma and lymph node metastases

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer-related death, largely due to metastatic disease. To better understand PDAC metastatic spread and identify novel therapeutic targets, we analysed the proteome of primary tumours and matched lymph node (LN) metastases. As frozen specimens of metastatic lesions are scarce, we examined formalin-fixed paraffin-embedded (FFPE) tissues. This poses technical challenges because of the cross-linkages induced by fixation. Using laser capture microdissection (PALM system), we isolated malignant epithelia from seven FFPE primary PDAC tumours and matched LN metastases. Following dissection, samples were analysed in duplicate using Multidimensional Protein Identification Technology (MudPIT); this resulted in the identification of 1504 proteins, 854 of which were common to all samples analysed. Comparison of the obtained proteins with data from previous proteomics studies on pancreatic tissue, pancreatic juice, serum, and urine resulted in a less than 30% overlap, indicating that our study has substantially expanded the current database of proteins expressed in this malignancy. Statistical analysis further showed that 115/854 proteins (13.5%) were significantly differentially expressed (g-value ≥ 3.8). Two proteins, S100P and 14-3-3 sigma, with highly significant g-values were confirmed to be significantly differentially expressed (S100P: p = 0.05 and 14-3-3 sigma: p < 0.001) in a larger series of 55 cases of matched primary PDAC and LN metastases using immunohistochemistry. Thus, laser capture microdissection of FFPE tissue coupled with downstream proteomic analysis is a valid approach for the investigation of metastatic PDAC. This is the first study to establish and compare the protein composition of primary PDAC and matched LN metastases, and has resulted in the identification of several potential epithelial-specific therapeutic targets, including 14-3-3 sigma and S100P.     

SURVIVIN as a marker for quiescent-breast cancer stem cells—An intermediate,adherent, pre-requisite phase of breast cancer metastasis

Cancer stem cells drive the metastatic cascade by undergoing epithelial to mesenchymal transition (EMT) and again mesenchymal to epithelial transition (MET). Using multiple breast cancer cell lines including cigarette smoke induced breast cancer cells and tumor derived primary cells from patient sample; we developed a breast cancer metastasis model and reported the existence of an adherent, distinct pre-metastatic phase, quiescent-breast cancer stem cells (Q-BCSCs) prior to attaining an EMT. SURVIVIN was found to be expressed in Q-BCSCs. Time dependant biphasic expression of SURVIVIN in Q-BCSCs reveals that Q-BCSCs is a pre-metastatic phase distinct from both epithelial and mesenchymal counterparts. SURVIVIN favours metastasis and up-regulates WNT/β-CATENIN pathway in a PI3 K/AKT-dependant manner for self-renewal. Knockdown of SURVIVIN in Q-BCSCs lost the metastatic property of cells by inhibiting invasion, EMT-MET, PI3 K/AKT/WNT cascade, and induced apoptosis. Thus, our data suggest the existence of a novel pre-metastatic phase (Q-BCSCs) before EMT and SURVIVIN acts as a marker for Quiescent-BCSCs.     

The differential distributions of ASPM isoforms and their roles in Wnt signaling,cell cycle progression,and pancreatic cancer prognosis

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and treatment-resistant malignancy. The lack of pathway-informed biomarkers hampers the development of rational diagnostics or therapies. Recently, the protein abnormal spindle-like microcephaly-associated (ASPM) was identified as a novel Wnt and stemness regulator in PDAC, while the pathogenic roles of its protein isoforms remain unclarified. We developed novel isoform-specific antibodies and genetic knockdown (KD) of putative ASPM isoforms, whereby we uncovered that the levels of ASPM isoform 1 (iI) and ASPM-iII are variably upregulated in PDAC cells. ASPM isoforms show remarkably different subcellular locations; specifically, ASPM-iI is exclusively localized to the cortical cytoplasm of PDAC cells, while ASPM-iII is predominantly expressed in cell nuclei. Mechanistically, ASPM-iI co-localizes with disheveled-2 and active β-catenin as well as the stemness marker aldehyde dehydrogenase-1 (ALDH-1), and its expression is indispensable for the Wnt activity, stemness, and the tumorigenicity of PDAC cells. By contrast, ASPM-iII selectively regulates the expression level of cyclin E and cell cycle progression in PDAC cells. The expression of ASPM-iI and ASPM-iII displays considerable intratumoral heterogeneity in PDAC tissues and only that of ASPM-iI was prognostically significant; it outperformed ALDH-1 staining and clinico-pathological variables in a multivariant analysis. Collectively, the distinct expression patterns and biological functions of ASPM isoforms may illuminate novel molecular mechanisms and prognosticators in PDAC and may pave the way for the development of therapies targeting this novel oncoprotein. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

MET mutations in cancers of unknown primary origin (CUPs)

Cancer of unknown primary origin (CUP) defines metastatic disease of unknown origin, accounting for 3-5% of all cancers. Growing evidence demonstrates that inappropriate execution of a genetic program named "invasive growth," driven by the MET oncogene, is implicated in the metastatic process. MET activation in cancers is mainly consequent to overexpression, whereas mutations are rarely found. We reasoned that the occurrence of MET somatic mutations might sustain premature occult dissemination of cancer cells, such as that observed in CUPs. We sequenced MET in genomic DNA obtained from 47 early metastatic cancers. By extensive immunohistochemical analysis a primary site was afterward postulated in 24 patients, whereas 23 cases remained of unknown primary (CUPs). MET somatic mutations were found in seven cases, all belonging to the CUP cohort. Mutational incidence (30%) was thus significantly higher than the expected one (4%), in the absence of high mutational background. Several nucleotide changes were novel and clustered either in the kinase domain or in the extracellular semaphorin domain. Mutated receptors were functional and sustained the transformed phenotype, suggesting that MET activating mutations are genetic markers associated with the CUP syndrome.