E-cadherin variants associated with oral facial clefts trigger aberrant cell motility in a REG1A-dependent manner.

BACKGROUND: Germline mutations of E-cadherin contribute to hereditary diffuse gastric cancer (HDGC) and congenital malformations, such as oral facial clefts (OFC). However, the molecular mechanisms through which E-cadherin loss-of-function triggers distinct clinical outcomes remain unknown. We postulate that E-cadherin-mediated disorders result from abnormal interactions with the extracellular matrix and consequent aberrant intracellular signalling, affecting the coordination of cell migration. METHODS: Herein, we developed in vivo and in vitro models of E-cadherin mutants associated with either OFC or HDGC. Using a Drosophila approach, we addressed the impact of the different variants in cell morphology and migration ability. By combining gap closure migration assays and time-lapse microscopy, we further investigated the migration pattern of cells expressing OFC or HDGC variants. The adhesion profile of the variants was evaluated using high-throughput ECM arrays, whereas RNA sequencing technology was explored for identification of genes involved in aberrant cell motility. RESULTS: We have demonstrated that cells expressing OFC variants exhibit an excessive motility performance and irregular leading edges, which prevent the coordinated movement of the epithelial monolayer. Importantly, we found that OFC variants promote cell adhesion to a wider variety of extracellular matrices than HDGC variants, suggesting higher plasticity in response to different microenvironments. We unveiled a distinct transcriptomic profile in the OFC setting and pinpointed REG1A as a putative regulator of this outcome. Consistent with this, specific RNAi-mediated inhibition of REG1A shifted the migration pattern of OFC expressing cells, leading to slower wound closure with coordinated leading edges. CONCLUSIONS: We provide evidence that E-cadherin variants associated with OFC activate aberrant signalling pathways that support dynamic rearrangements of cells towards improved adaptability to the microenvironment. This proficiency results in abnormal tissue shaping and movement, possibly underlying the development of orofacial malformations.

The ECM and tissue architecture are major determinants of early invasion mediated by E-cadherin dysfunction.

Germline mutations of E-cadherin cause Hereditary Diffuse Gastric Cancer (HDGC), a highly invasive cancer syndrome characterised by the occurrence of diffuse-type gastric carcinoma and lobular breast cancer. In this disease, E-cadherin-defective cells are detected invading the adjacent stroma since very early stages. Although E-cadherin loss is well established as a triggering event, other determinants of the invasive process persist largely unknown. Herein, we develop an experimental strategy that comprises in vitro extrusion assays using E-cadherin mutants associated to HDGC, as well as mathematical models epitomising epithelial dynamics and its interaction with the extracellular matrix (ECM). In vitro, we verify that E-cadherin dysfunctional cells detach from the epithelial monolayer and extrude basally into the ECM. Through phase-field modelling we demonstrate that, aside from loss of cell-cell adhesion, increased ECM attachment further raises basal extrusion efficiency. Importantly, by combining phase-field and vertex model simulations, we show that the cylindrical structure of gastric glands strongly promotes the cell's invasive ability. Moreover, we validate our findings using a dissipative particle dynamics simulation of epithelial extrusion. Overall, we provide the first evidence that cancer cell invasion is the outcome of defective cell-cell linkages, abnormal interplay with the ECM, and a favourable 3D tissue structure.

Activation of Laminin γ2 by Helicobacter pylori Promotes Invasion and Survival of Gastric Cancer Cells With E-Cadherin Defects.

BACKGROUND: Helicobacter pylori infection induces cellular phenotypes relevant for cancer progression, namely cell motility and invasion. We hypothesized that the extracellular matrix (ECM) could be involved in these deleterious effects. METHODS: Microarrays were used to uncover ECM interactors in cells infected with H. pylori. LAMC2, encoding laminin γ2, was selected as a candidate gene and its expression was assessed in vitro and in vivo. The role of LAMC2 was investigated by small interference RNA (siRNA) combined with a set of functional assays. Laminin γ2 and E-cadherin expression patterns were evaluated in gastric cancer cases. RESULTS: Laminin γ2 was found significantly overexpressed in gastric cancer cells infected with H. pylori. This finding was validated in vitro by infection with clinical isolates and in vivo by using gastric biopsies of infected and noninfected individuals. We showed that laminin γ2 overexpression is dependent on the bacterial type IV secretion system and on the CagA. Functionally, laminin γ2 promotes cell invasion and resistance to apoptosis, through modulation of Src, JNK, and AKT activity. These effects were abrogated in cells with functional E-cadherin. CONCLUSIONS: These data highlight laminin γ2 and its downstream effectors as potential therapeutic targets, and the value of H. pylori eradication to delay gastric cancer onset and progression.

Differential Impacts on Tensional Homeostasis of Gastric Cancer Cells Due to Distinct Domain Variants of E-Cadherin.

In epithelia, breakdown of tensional homeostasis is closely associated with E-cadherin dysfunction and disruption of tissue function and integrity. In this study, we investigated the effect of E-cadherin mutations affecting distinct protein domains on tensional homeostasis of gastric cancer cells. We used micropattern traction microscopy to measure temporal fluctuations of cellular traction forces in AGS cells transfected with the wild-type E-cadherin or with variants affecting the extracellular, the juxtamembrane, and the intracellular domains of the protein. We focused on the dynamic aspect of tensional homeostasis, namely the ability of cells to maintain a consistent level of tension, with low temporal variability around a set point. Cells were cultured on hydrogels micropatterned with different extracellular matrix (ECM) proteins to test whether the ECM adhesion impacts cell behavior. A combination of Fibronectin and Vitronectin was used as a substrate that promotes the adhesive ability of E-cadherin dysfunctional cells, whereas Collagen VI was used to test an unfavorable ECM condition. Our results showed that mutations affecting distinct E-cadherin domains influenced differently cell tensional homeostasis, and pinpointed the juxtamembrane and intracellular regions of E-cadherin as the key players in this process. Furthermore, Fibronectin and Vitronectin might modulate cancer cell behavior towards tensional homeostasis.

Proteomic Identification of a Gastric Tumor ECM Signature Associated With Cancer Progression.

The extracellular matrix (ECM) plays an undisputable role in tissue homeostasis and its deregulation leads to altered mechanical and biochemical cues that impact cancer development and progression. Herein, we undertook a novel approach to address the role of gastric ECM in tumorigenesis, which remained largely unexplored. By combining decellularization techniques with a high-throughput quantitative proteomics approach, we have performed an extensive characterization of human gastric mucosa, uncovering its composition and distribution among tumor, normal adjacent and normal distant mucosa. Our results revealed a common ECM signature composed of 142 proteins and indicated that gastric carcinogenesis encompasses ECM remodeling through alterations in the abundance of 24 components, mainly basement membrane proteins. Indeed, we could only identify one de novo tumor-specific protein, the collagen alpha-1(X) chain (COL10A1). Functional analysis of the data demonstrated that gastric ECM remodeling favors tumor progression by activating ECM receptors and cellular processes involved in angiogenesis and cell-extrinsic metabolic regulation. By analyzing mRNA expression in an independent GC cohort available at the TGCA, we validated the expression profile of 12 differentially expressed ECM proteins. Importantly, the expression of COL1A2, LOX and LTBP2 significantly correlated with high tumor stage, with LOX and LTBP2 further impacting patient overall survival. These findings contribute for a better understanding of GC biology and highlight the role of core ECM components in gastric carcinogenesis and their clinical relevance as biomarkers of disease prognosis.

Integrin ß1 orchestrates the abnormal cell-matrix attachment and invasive behaviour of E-cadherin dysfunctional cells.

BACKGROUND: Tumour progression relies on the ability of cancer cells to penetrate and invade neighbouring tissues. E-cadherin loss is associated with increased cell invasion in gastric carcinoma, and germline mutations of the E-cadherin gene are causative of hereditary diffuse gastric cancer. Although E-cadherin dysfunction impacts cell-cell adhesion, cell dissemination also requires an imbalance of adhesion to the extracellular matrix (ECM). METHODS: To identify ECM components and receptors relevant for adhesion of E-cadherin dysfunctional cells, we implemented a novel ECM microarray platform coupled with molecular interaction networks. The functional role of putative candidates was determined by combining micropattern traction microscopy, protein modulation and in vivo approaches, as well as transcriptomic data of 262 gastric carcinoma samples, retrieved from the cancer genome atlas (TCGA). RESULTS: Here, we show that E-cadherin mutations induce an abnormal interplay of cells with specific components of the ECM, which encompasses increased traction forces and Integrin ß1 activation. Integrin ß1 synergizes with E-cadherin dysfunction, promoting cell scattering and invasion. The significance of the E-cadherin-Integrin ß1 crosstalk was validated in Drosophila models and found to be consistent with evidence from human gastric carcinomas, where increased tumour grade and poor survival are associated with low E-cadherin and high Integrin ß1 levels. CONCLUSIONS: Integrin ß1 is a key mediator of invasion in carcinomas with E-cadherin impairment and should be regarded as a biomarker of poor prognosis in gastric cancer.

The Porto European Cancer Research Summit 2021.

Key stakeholders from the cancer research continuum met in May 2021 at the European Cancer Research Summit in Porto to discuss priorities and specific action points required for the successful implementation of the European Cancer Mission and Europe's Beating Cancer Plan (EBCP). Speakers presented a unified view about the need to establish high-quality, networked infrastructures to decrease cancer incidence, increase the cure rate, improve patient's survival and quality of life, and deal with research and care inequalities across the European Union (EU). These infrastructures, featuring Comprehensive Cancer Centres (CCCs) as key components, will integrate care, prevention and research across the entire cancer continuum to support the development of personalized/precision cancer medicine in Europe. The three pillars of the recommended European infrastructures - namely translational research, clinical/prevention trials and outcomes research - were pondered at length. Speakers addressing the future needs of translational research focused on the prospects of multiomics assisted preclinical research, progress in Molecular and Digital Pathology, immunotherapy, liquid biopsy and science data. The clinical/prevention trial session presented the requirements for next-generation, multicentric trials entailing unified strategies for patient stratification, imaging, and biospecimen acquisition and storage. The third session highlighted the need for establishing outcomes research infrastructures to cover primary prevention, early detection, clinical effectiveness of innovations, health-related quality-of-life assessment, survivorship research and health economics. An important outcome of the Summit was the presentation of the Porto Declaration, which called for a collective and committed action throughout Europe to develop the cancer research infrastructures indispensable for fostering innovation and decreasing inequalities within and between member states. Moreover, the Summit guidelines will assist decision making in the context of a unique EU-wide cancer initiative that, if expertly implemented, will decrease the cancer death toll and improve the quality of life of those confronted with cancer, and this is carried out at an affordable cost.

Germline CDH1 G212E Missense Variant: Combining Clinical, In Vitro and In Vivo Strategies to Unravel Disease Burden.

E-cadherin, encoded by CDH1, is an essential molecule for epithelial homeostasis, whose loss or aberrant expression results in disturbed cell-cell adhesion, increased cell invasion and metastasis. Carriers of CDH1 germline mutations have a high risk of developing diffuse gastric cancer and lobular breast cancer, associated with the cancer syndrome Hereditary Diffuse Gastric Cancer (HDGC). The ubiquitous availability of cancer panels has led to the identification of an increasing amount of "incidental" CDH1 genetic variants that pose a serious clinical challenge. This has sparked intensive research aiming at an accurate classification of the variants and consequent validation of their clinical relevance. The present study addressed the significance of a novel CDH1 variant, G212E, identified in an unusually large pedigree displaying strong aggregation of diffuse gastric cancer. We undertook a comprehensive pipeline encompassing family data, in silico predictions, in vitro assays and in vivo strategies, which validated the deleterious phenotype induced by this genetic alteration. In particular, we demonstrated that the G212E variant affects the stability and localization, as well as the adhesive and anti-invasive functions of E-cadherin, triggering epithelial disruption and disorganization. Our findings illustrate the clinical implication of a complementary approach for effective variant categorization and patient management.

A machine learning approach for single cell interphase cell cycle staging.

The cell nucleus is a tightly regulated organelle and its architectural structure is dynamically orchestrated to maintain normal cell function. Indeed, fluctuations in nuclear size and shape are known to occur during the cell cycle and alterations in nuclear morphology are also hallmarks of many diseases including cancer. Regrettably, automated reliable tools for cell cycle staging at single cell level using in situ images are still limited. It is therefore urgent to establish accurate strategies combining bioimaging with high-content image analysis for a bona fide classification. In this study we developed a supervised machine learning method for interphase cell cycle staging of individual adherent cells using in situ fluorescence images of nuclei stained with DAPI. A Support Vector Machine (SVM) classifier operated over normalized nuclear features using more than 3500 DAPI stained nuclei. Molecular ground truth labels were obtained by automatic image processing using fluorescent ubiquitination-based cell cycle indicator (Fucci) technology. An average F1-Score of 87.7% was achieved with this framework. Furthermore, the method was validated on distinct cell types reaching recall values higher than 89%. Our method is a robust approach to identify cells in G1 or S/G2 at the individual level, with implications in research and clinical applications.

Hereditary diffuse gastric cancer: updated clinical practice guidelines.

Hereditary diffuse gastric cancer (HDGC) is an autosomal dominant cancer syndrome that is characterised by a high prevalence of diffuse gastric cancer and lobular breast cancer. It is largely caused by inactivating germline mutations in the tumour suppressor gene CDH1, although pathogenic variants in CTNNA1 occur in a minority of families with HDGC. In this Policy Review, we present updated clinical practice guidelines for HDGC from the International Gastric Cancer Linkage Consortium (IGCLC), which recognise the emerging evidence of variability in gastric cancer risk between families with HDGC, the growing capability of endoscopic and histological surveillance in HDGC, and increased experience of managing long-term sequelae of total gastrectomy in young patients. To redress the balance between the accessibility, cost, and acceptance of genetic testing and the increased identification of pathogenic variant carriers, the HDGC genetic testing criteria have been relaxed, mainly through less restrictive age limits. Prophylactic total gastrectomy remains the recommended option for gastric cancer risk management in pathogenic CDH1 variant carriers. However, there is increasing confidence from the IGCLC that endoscopic surveillance in expert centres can be safely offered to patients who wish to postpone surgery, or to those whose risk of developing gastric cancer is not well defined.

Hereditary Gastric and Breast Cancer Syndromes Related to CDH1 Germline Mutation: A Multidisciplinary Clinical Review.

E-cadherin (CDH1 gene) germline mutations are associated with the development of diffuse gastric cancer in the context of the so-called hereditary diffuse gastric syndrome, and with an inherited predisposition of lobular breast carcinoma. In 2019, the international gastric cancer linkage consortium revised the clinical criteria and established guidelines for the genetic screening of CDH1 germline syndromes. Nevertheless, the introduction of multigene panel testing in clinical practice has led to an increased identification of E-cadherin mutations in individuals without a positive family history of gastric or breast cancers. This observation motivated us to review and present a novel multidisciplinary clinical approach (nutritional, surgical, and image screening) for single subjects who present germline CDH1 mutations but do not fulfil the classic clinical criteria, namely those identified as-(1) incidental finding and (2) individuals with lobular breast cancer without family history of gastric cancer (GC).

E-cadherin deregulation in breast cancer.

E-cadherin protein (CDH1 gene) integrity is fundamental to the process of epithelial polarization and differentiation. Deregulation of the E-cadherin function plays a crucial role in breast cancer metastases, with worse prognosis and shorter overall survival. In this narrative review, we describe the inactivating mechanisms underlying CDH1 gene activity and its possible translation to clinical practice as a prognostic biomarker and as a potential targeted therapy.

The Extracellular Matrix: An Accomplice in Gastric Cancer Development and Progression.

The extracellular matrix (ECM) is a dynamic and highly organized tissue structure, providing support and maintaining normal epithelial architecture. In the last decade, increasing evidence has emerged demonstrating that alterations in ECM composition and assembly strongly affect cellular function and behavior. Even though the detailed mechanisms underlying cell-ECM crosstalk are yet to unravel, it is well established that ECM deregulation accompanies the development of many pathological conditions, such as gastric cancer. Notably, gastric cancer remains a worldwide concern, representing the third most frequent cause of cancer-associated deaths. Despite increased surveillance protocols, patients are usually diagnosed at advanced disease stages, urging the identification of novel diagnostic biomarkers and efficient therapeutic strategies. In this review, we provide a comprehensive overview regarding expression patterns of ECM components and cognate receptors described in normal gastric epithelium, pre-malignant lesions, and gastric carcinomas. Important insights are also discussed for the use of ECM-associated molecules as predictive biomarkers of the disease or as potential targets in gastric cancer.

Helicobacter Pylori Targets the EPHA2 Receptor Tyrosine Kinase in Gastric Cells Modulating Key Cellular Functions.

Helicobacter pylori, a stomach-colonizing Gram-negative bacterium, is the main etiological factor of various gastroduodenal diseases, including gastric adenocarcinoma. By establishing a life-long infection of the gastric mucosa, H. pylori continuously activates host-signaling pathways, in particular those associated with receptor tyrosine kinases. Using two different gastric epithelial cell lines, we show that H. pylori targets the receptor tyrosine kinase EPHA2. For long periods of time post-infection, H. pylori induces EPHA2 protein downregulation without affecting its mRNA levels, an effect preceded by receptor activation via phosphorylation. EPHA2 receptor downregulation occurs via the lysosomal degradation pathway and is independent of the H.pylori virulence factors CagA, VacA, and T4SS. Using small interfering RNA, we show that EPHA2 knockdown affects cell-cell and cell-matrix adhesion, invasion, and angiogenesis, which are critical cellular processes in early gastric lesions and carcinogenesis mediated by the bacteria. This work contributes to the unraveling of the underlying mechanisms of H. pylori-host interactions and associated diseases. Additionally, it raises awareness for potential interference between H. pylori infection and the efficacy of gastric cancer therapies targeting receptors tyrosine kinases, given that infection affects the steady-state levels and dynamics of some receptor tyrosine kinases (RTKs) and their signaling pathways.

Targeting the Tumor Microenvironment: An Unexplored Strategy for Mutant KRAS Tumors.

Current evidence strongly suggests that cancer cells depend on the microenvironment in order to thrive. In fact, signals from the surrounding tumor microenvironment are crucial for cancer cells´ aggressiveness, altering their expression profile and favoring their metastatic potential. As such, targeting the tumor microenvironment to impair cancer progression became an attractive therapeutic option. Interestingly, it has been shown that oncogenic KRAS signaling promotes a pro-tumorigenic microenvironment, and the associated crosstalk alters the expression profile of cancer cells. These findings award KRAS a key role in controlling the interactions between cancer cells and the microenvironment, granting cancer a poor prognosis. Given the lack of effective approaches to target KRAS itself or its downstream effectors in the clinic, exploring such interactions may open new perspectives on possible therapeutic strategies to hinder mutant KRAS tumors. This review highlights those communications and their implications for the development of effective therapies or to provide insights regarding response to existing regimens.

S100P is a molecular determinant of E-cadherin function in gastric cancer.

BACKGROUND: E-cadherin has been awarded a key role in the aetiology of both sporadic and hereditary forms of gastric cancer. In this study, we aimed to identify molecular interactors that influence the expression and function of E-cadherin associated to cancer. METHODS: A data mining approach was used to predict stomach-specific candidate genes, uncovering S100P as a key candidate. The role of S100P was evaluated through in vitro functional assays and its expression was studied in a gastric cancer tissue microarray (TMA). RESULTS: S100P was found to contribute to a cancer pathway dependent on the context of E-cadherin function. In particular, we demonstrated that S100P acts as an E-cadherin positive regulator in a wild-type E-cadherin context, and its inhibition results in decreased E-cadherin expression and function. In contrast, S100P is likely to be a pro-survival factor in gastric cancer cells with loss of functional E-cadherin, contributing to an oncogenic molecular program. Moreover, expression analysis in a gastric cancer TMA revealed that S100P expression impacts negatively among patients bearing Ecad- tumours, despite not being significantly associated with overall survival on its own. CONCLUSIONS: We propose that S100P has a dual role in gastric cancer, acting as an oncogenic factor in the context of E-cadherin loss and as a tumour suppressor in a functional E-cadherin setting. The discovery of antagonist effects of S100P in different E-cadherin contexts will aid in the stratification of gastric cancer patients who may benefit from S100P-targeted therapies.

Clinical spectrum and pleiotropic nature of CDH1 germline mutations.

CDH1 encodes E-cadherin, a key protein in adherens junctions. Given that E-cadherin is involved in major cellular processes such as embryogenesis and maintenance of tissue architecture, it is no surprise that deleterious effects arise from its loss of function. E-cadherin is recognised as a tumour suppressor gene, and it is well established that CDH1 genetic alterations cause diffuse gastric cancer and lobular breast cancer-the foremost manifestations of the hereditary diffuse gastric cancer syndrome. However, in the last decade, evidence has emerged demonstrating that CDH1 mutations can be associated with lobular breast cancer and/or several congenital abnormalities, without any personal or family history of diffuse gastric cancer. To date, no genotype-phenotype correlations have been observed. Remarkably, there are reports of mutations affecting the same nucleotide but inducing distinct clinical outcomes. In this review, we bring together a comprehensive analysis of CDH1-associated disorders and germline alterations found in each trait, providing important insights into the biological mechanisms underlying E-cadherin's pleiotropic effects. Ultimately, this knowledge will impact genetic counselling and will be relevant to the assessment of risk of cancer development or congenital malformations in CDH1 mutation carriers.

SRC inhibition prevents P-cadherin mediated signaling and function in basal-like breast cancer cells.

BACKGROUND: Basal-like breast cancer (BLBC) is a poor prognosis subgroup of triple-negative carcinomas that still lack specific target therapies and accurate biomarkers for treatment selection. P-cadherin is frequently overexpressed in these tumors, promoting cell invasion, stem cell activity and tumorigenesis by the activation of Src-Family kinase (SRC) signaling. Therefore, our aim was to evaluate if the treatment of BLBC cells with dasatinib, the FDA approved SRC inhibitor, would impact on P-cadherin induced tumor aggressive behavior. METHODS: P-cadherin and SRC expression was evaluated in a series of invasive Breast Cancer and contingency tables and chi-square tests were performed. Cell-cell adhesion measurements were performed by Atomic Force Microscopy, where frequency histograms and Gaussian curves were applied. 2D and 3D cell migration and invasion, proteases secretion and self-renew potential were evaluated in vitro. Student's t-tests were used to determine statistically significant differences. The cadherin/catenin complex interactions were evaluated by in situ proximity-ligation assay, and statistically significant results were determined by using Mann-Whitney test with a Bonferroni correction. In vivo xenograft mouse models were used to evaluate the impact of dasatinib on tumor growth and survival. ANOVA test was used to evaluate the differences in tumor size, considering a confidence interval of 95%. Survival curves were estimated by the Kaplan-Meier's method, using the log-rank test to assess significant differences for mice overall survival. RESULTS: Our data demonstrated that P-cadherin overexpression is significantly associated with SRC activation in breast cancer cells, which was also validated in a large series of primary tumor samples. SRC activity suppression with dasatinib significantly prevented the in vitro functional effects of P-cadherin overexpressing cells, as well as their in vivo tumorigenic and metastatic ability, by increasing mice overall survival. Mechanistically, SRC inhibition affects P-cadherin downstream signaling, rescues the E-cadherin/p120-catenin complex to the cell membrane, recovering cell-cell adhesion function. CONCLUSIONS: In conclusion our findings show that targeting P-cadherin/SRC signaling and functional activity may open novel therapeutic opportunities for highly aggressive and poor prognostic basal-like breast cancer.

E-cadherin signal sequence disruption: a novel mechanism underlying hereditary cancer.

The aim of this study was to uncover the pathogenic relevance and the underlying molecular mechanism of a novel CDH1 variant found in a Hereditary Diffuse Gastric Cancer family (p.L13_L15del), which affects the signal peptide of E-cadherin without changing the remaining predicted sequence. We verified that p.L13_L15del cells yield low levels of E-cadherin, decreased cell adhesion and enhanced cell invasion. Further, we demonstrated that the disruption of the highly conserved hydrophobic core of the signal peptide hampers the binding of cellular components crucial for E-cadherin translation and translocation into the endoplasmic reticulum, constituting a new molecular basis for the loss of a tumour suppressor gene causative of hereditary cancer.