Novel insights into the role of Discoidin domain receptor 2 (DDR2) in cancer progression: a new avenue of therapeutic intervention.

Discoidin domain receptors (DDRs), including DDR1 and DDR2, are a unique class of receptor tyrosine kinases (RTKs) activated by collagens at the cell-matrix boundary interface. The peculiar mode of activation makes DDRs as key cellular sensors of microenvironmental changes, with a critical role in all physiological and pathological processes governed by collagen remodeling. DDRs are widely expressed in fetal and adult tissues, and experimental and clinical evidence has shown that their expression is deregulated in cancer. Strong findings supporting the role of collagens in tumor progression and metastasis have led to renewed interest in DDRs.  However, despite an increasing number of studies, DDR biology remains poorly understood, particularly the less studied DDR2, whose involvement in cancer progression mechanisms is undoubted. Thus, the understanding of a wider range of DDR2 functions and related molecular mechanisms is expected. To date, several lines of evidence support DDR2 as a promising target in cancer therapy. Its involvement in key functions in the tumor microenvironment makes DDR2 inhibition particularly attractive to achieve simultaneous targeting of tumor and stromal cells, and tumor regression, which is beneficial for improving the response to different types of anti-cancer therapies, including chemo- and immunotherapy. This review summarizes current research on DDR2, focusing on its role in cancer progression through its involvement in tumor and stromal cell functions, and discusses findings that support the rationale for future development of direct clinical strategies targeting DDR2.

Transcription factors in fibroblast plasticity and CAF heterogeneity.

In recent years, research focused on the multifaceted landscape and functions of cancer-associated fibroblasts (CAFs) aimed to reveal their heterogeneity and identify commonalities across diverse tumors for more effective therapeutic targeting of pro-tumoral stromal microenvironment. However, a unified functional categorization of CAF subsets remains elusive, posing challenges for the development of targeted CAF therapies in clinical settings.The CAF phenotype arises from a complex interplay of signals within the tumor microenvironment, where transcription factors serve as central mediators of various cellular pathways. Recent advances in single-cell RNA sequencing technology have emphasized the role of transcription factors in the conversion of normal fibroblasts to distinct CAF subtypes across various cancer types.This review provides a comprehensive overview of the specific roles of transcription factor networks in shaping CAF heterogeneity, plasticity, and functionality. Beginning with their influence on fibroblast homeostasis and reprogramming during wound healing and fibrosis, it delves into the emerging insights into transcription factor regulatory networks. Understanding these mechanisms not only enables a more precise characterization of CAF subsets but also sheds light on the early regulatory processes governing CAF heterogeneity and functionality. Ultimately, this knowledge may unveil novel therapeutic targets for cancer treatment, addressing the existing challenges of stromal-targeted therapies.

hMENA isoforms regulate cancer intrinsic type I IFN signaling and extrinsic mechanisms of resistance to immune checkpoint blockade in NSCLC.

BACKGROUND: Understanding how cancer signaling pathways promote an immunosuppressive program which sustains acquired or primary resistance to immune checkpoint blockade (ICB) is a crucial step in improving immunotherapy efficacy. Among the pathways that can affect ICB response is the interferon (IFN) pathway that may be both detrimental and beneficial. The immune sensor retinoic acid-inducible gene I (RIG-I) induces IFN activation and secretion and is activated by actin cytoskeleton disturbance. The actin cytoskeleton regulatory protein hMENA, along with its isoforms, is a key signaling hub in different solid tumors, and recently its role as a regulator of transcription of genes encoding immunomodulatory secretory proteins has been proposed. When hMENA is expressed in tumor cells with low levels of the epithelial specific hMENA11a isoform, identifies non-small cell lung cancer (NSCLC) patients with poor prognosis. Aim was to identify cancer intrinsic and extrinsic pathways regulated by hMENA11a downregulation as determinants of ICB response in NSCLC. Here, we present a potential novel mechanism of ICB resistance driven by hMENA11a downregulation. METHODS: Effects of hMENA11a downregulation were tested by RNA-Seq, ATAC-Seq, flow cytometry and biochemical assays. ICB-treated patient tumor tissues were profiled by Nanostring IO 360 Panel enriched with hMENA custom probes. OAK and POPLAR datasets were used to validate our discovery cohort. RESULTS: Transcriptomic and biochemical analyses demonstrated that the depletion of hMENA11a induces IFN pathway activation, the production of different inflammatory mediators including IFNß via RIG-I, sustains the increase of tumor PD-L1 levels and activates a paracrine loop between tumor cells and a unique macrophage subset favoring an epithelial-mesenchymal transition (EMT). Notably, when we translated our results in a clinical setting of NSCLC ICB-treated patients, transcriptomic analysis revealed that low expression of hMENA11a, high expression of IFN target genes and high macrophage score identify patients resistant to ICB therapy. CONCLUSIONS: Collectively, these data establish a new function for the actin cytoskeleton regulator hMENA11a in modulating cancer cell intrinsic type I IFN signaling and extrinsic mechanisms that promote protumoral macrophages and favor EMT. These data highlight the role of actin cytoskeleton disturbance in activating immune suppressive pathways that may be involved in resistance to ICB in NSCLC.

Actin Cytoskeleton Dynamics and Type I IFN-Mediated Immune Response: A Dangerous Liaison in Cancer?

Chronic viral infection and cancer are closely inter-related and are both characterized by profound alteration of tissue homeostasis. The actin cytoskeleton dynamics highly participate in tissue homeostasis and act as a sensor leading to an immune-mediated anti-cancer and anti-viral response. Herein we highlight the crucial role of actin cytoskeleton dynamics in participating in a viral mimicry activation with profound effect in anti-tumor immune response. This still poorly explored field understands the cytoskeleton dynamics as a platform of complex signaling pathways which may regulate Type I IFN response in cancer. This emerging network needs to be elucidated to identify more effective anti-cancer strategies and to further advance the immuno-oncology field which has revolutionized the cancer treatment. For a progress to occur in this exciting arena we have to shed light on actin cytoskeleton related pathways and immune response. Herein we summarize the major findings, considering the double sword of the immune response and in particular the role of Type I IFN pathways in resistance to anti-cancer treatment.

Actin Cytoskeleton and Regulation of TGFß Signaling: Exploring Their Links.

Human tissues, to maintain their architecture and function, respond to injuries by activating intricate biochemical and physical mechanisms that regulates intercellular communication crucial in maintaining tissue homeostasis. Coordination of the communication occurs through the activity of different actin cytoskeletal regulators, physically connected to extracellular matrix through integrins, generating a platform of biochemical and biomechanical signaling that is deregulated in cancer. Among the major pathways, a controller of cellular functions is the cytokine transforming growth factor ß (TGFß), which remains a complex and central signaling network still to be interpreted and explained in cancer progression. Here, we discuss the link between actin dynamics and TGFß signaling with the aim of exploring their aberrant interaction in cancer.

3D models in the new era of immune oncology: focus on T cells, CAF and ECM.

Immune checkpoint inhibitor therapy has changed clinical practice for patients with different cancers, since these agents have demonstrated a significant improvement of overall survival and are effective in many patients. However, an intrinsic or acquired resistance frequently occur and biomarkers predictive of responsiveness should help in patient selection and in defining the adequate treatment options. A deep analysis of the complexity of the tumor microenvironment is likely to further advance the field and hopefully identify more effective combined immunotherapeutic strategies. Here we review the current knowledge on tumor microenvironment, focusing on T cells, cancer associated fibroblasts and extracellular matrix. The use of 3D cell culture models to resemble tumor microenvironment landscape and to screen immunomodulatory drugs is also reviewed.

Mesenchymal traits at the convergence of tumor-intrinsic and -extrinsic mechanisms of resistance to immune checkpoint blockers.

Targeting of immune checkpoint blockers (ICBs), such as cytotoxic T-lymphocyte antigen-4 and programmed-death 1/programmed-death ligand 1, has dramatically changed the landscape of cancer treatment. Seeing patients who were refractory to conventional therapy recover after immunotherapy, with high rates of objective durable responses and increased overall survival, has raised great enthusiasm in cancer care and research. However, to date, only a restricted portion of patients benefit from these therapies, due to natural and acquired resistance relying on the ever-evolving cross-talk between tumor and stromal cells. Here, we review the convergence of tumor-intrinsic and -extrinsic cues, both affecting tumor plasticity and tumor stroma leading to an immunosuppressive tumor microenvironment, which may account for the heterogeneous responses and resistance to ICB therapies. A deeper knowledge of the mechanisms and fingerprints involved in natural and acquired resistance is likely to bring clinical benefit to the majority of patients, offering important clues for overcoming drug resistance and boosting the effectiveness of treatment. We discuss the need to define tumor subtypes based on the tumor, immune and stromal gene signature and propose that the better we understand tumor mesenchymal traits, the more we will be able to identify predictive biomarkers of response to ICB treatments.

hMENA(11a), a hMENA isoform sending survival signals.

Human MENA(11a) (hMENA(11a)), an epithelial-associated isoform of the actin binding protein enabled homolog (ENAH, also known as mammalian ENA [MENA]), is upregulated and phosphorylated following the activation of human epidermal growth factor receptor (HER) 1, HER2, and HER3. Here, we reveal a novel role of this isoform in sustaining cell survival and propose hMENA(11a) as a marker of HER3 activation and resistance to phosphatidylinositol-3-kinase inhibition therapies.

The pattern of hMENA isoforms is regulated by TGF-ß1 in pancreatic cancer and may predict patient outcome.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease in need of prognostic markers to address therapeutic choices. We have previously shown that alternative splicing of the actin regulator, hMENA, generates hMENA11a, and hMENAΔv6 isoforms with opposite roles in cell invasion. We examined the expression pattern of hMENA isoforms by immunohistochemistry, using anti-pan hMENA and specific anti-hMENA11a antibodies, in 285 PDACs, 15 PanINs, 10 pancreatitis, and normal pancreas. Pan hMENA immunostaining, absent in normal pancreas and low-grade PanINs, was weak in PanIN-3 and had higher levels in virtually all PDACs with 64% of cases showing strong staining. Conversely, the anti-invasive hMENA11a isoform only showed strong staining in 26% of PDAC. The absence of hMENA11a in a subset (34%) of pan-hMENA-positive tumors significantly correlated with poor outcome. The functional effects of hMENA isoforms were analyzed by loss and gain of function experiments in TGF-ß1-treated PDAC cell lines. hMENA11a knock-down in PDAC cell lines affected cell-cell adhesion but not invasion. TGF-ß1 cooperated with ß-catenin signaling to upregulate hMENA and hMENAΔv6 expression but not hMENA11a In the absence of hMENA11a, the hMENA/hMENAΔv6 up-regulation is crucial for SMAD2-mediated TGF-ß1 signaling and TGF-ß1-induced EMT. Since the hMENA isoform expression pattern correlates with patient outcome, the data suggest that hMENA splicing and related pathways are novel key players in pancreatic tumor microenvironment and may represent promising targets for the development of new prognostic and therapeutic tools in PDAC.

The T-box transcription factor Brachyury promotes epithelial-mesenchymal transition in human tumor cells.

Metastatic disease is responsible for the majority of human cancer deaths. Understanding the molecular mechanisms of metastasis is a major step in designing effective cancer therapeutics. Here we show that the T-box transcription factor Brachyury induces in tumor cells epithelial-mesenchymal transition (EMT), an important step in the progression of primary tumors toward metastasis. Overexpression of Brachyury in human carcinoma cells induced changes characteristic of EMT, including upregulation of mesenchymal markers, downregulation of epithelial markers, and an increase in cell migration and invasion. Brachyury overexpression also repressed E-cadherin transcription, an effect partially mediated by Slug. Conversely, inhibition of Brachyury resulted in downregulation of mesenchymal markers and loss of cell migration and invasion and diminished the ability of human tumor cells to form lung metastases in a xenograft model. Furthermore, we found Brachyury to be overexpressed in various human tumor tissues and tumor cell lines compared with normal tissues. We also determined that the percentage of human lung tumor tissues positive for Brachyury expression increased with the stage of the tumor, indicating a potential association between Brachyury and tumor progression. The selective expression of Brachyury in tumor cells and its role in EMT and cancer progression suggest that Brachyury may be an attractive target for antitumor therapies.

The cooperation between hMena overexpression and HER2 signalling in breast cancer.

hMena and the epithelial specific isoform hMena(11a) are actin cytoskeleton regulatory proteins belonging to the Ena/VASP family. EGF treatment of breast cancer cell lines upregulates hMena/hMena(11a) expression and phosphorylates hMena(11a), suggesting cross-talk between the ErbB receptor family and hMena/hMena(11a) in breast cancer. The aim of this study was to determine whether the hMena/hMena(11a) overexpression cooperates with HER-2 signalling, thereby affecting the HER2 mitogenic activity in breast cancer. In a cohort of breast cancer tissue samples a significant correlation among hMena, HER2 overexpression, the proliferation index (high Ki67), and phosphorylated MAPK and AKT was found and among the molecular subtypes the highest frequency of hMena overexpressing tumors was found in the HER2 subtype. From a clinical viewpoint, concomitant overexpression of HER2 and hMena identifies a subgroup of breast cancer patients showing the worst prognosis, indicating that hMena overexpression adds prognostic information to HER2 overexpressing tumors. To identify a functional link between HER2 and hMena, we show here that HER2 transfection in MCF7 cells increased hMena/hMena(11a) expression and hMena(11a) phosphorylation. On the other hand, hMena/hMena(11a) knock-down reduced HER3, AKT and p44/42 MAPK phosphorylation and inhibited the EGF and NRG1-dependent HER2 phosphorylation and cell proliferation. Of functional significance, hMena/hMena(11a) knock-down reduced the mitogenic activity of EGF and NRG1. Collectively these data provide new insights into the relevance of hMena and hMena(11a) as downstream effectors of the ErbB receptor family which may represent a novel prognostic indicator in breast cancer progression, helping to stratify patients.

Intercalated disk remodeling in delta-sarcoglycan-deficient hamsters fed with an alpha-linolenic acid-enriched diet.

Cardiomyocyte intercalated disks of delta-sarcoglycan-deficient cardiomyopathic hamsters (CMPHs) exhibit a pathological accumulation of the N-cadherin/catenin complex. CMPHs fed with an alpha-linolenic acid (ALA)-enriched diet (CMPH/FS) display an extended longevity compared to those fed with a standard diet (CMPH/PT) owing to, among others, the amelioration of both cardiac tissue structure and myocardial function. The present investigation was aimed at evaluating whether and to what extent the ALA-enriched diet affects the remodeling of CMPH cardiomyocyte intercalated disks and the expression of molecules, including N-cadherin, catenins and connexin 43 (CX43), involved in their organization. Western blot and immunohistochemical analysis demonstrated that the expression of N-cadherin, alpha- and beta-catenin is significantly reduced in cardiomyocyte intercalated disks of CMPH/FS vs. CMPH/PT and is lowered to levels similar to those found in healthy hamsters (GSH/PT), as well as transmission electron microscopy indicated that the cardiomyocyte intercalated disk ultrastructure is also re-established in CMPH/FS. In addition, the delocalization of CX43 as well as the presence of gap junctions were detectable at the lateral plasmamembrane of CMPH/FS cardiomyocytes, while the expression of myocardial CX43 was markedly reduced in both CMPH/PT and CMPH/FS, as compared to GSH/PT. Collectively, the present results demonstrate a substantial effect of an ALA-enriched diet on cardiomyocyte intercalated disk structure and molecular composition and further supports the beneficial effects of omega-3 polyunsaturated fatty acids in the prevention of potentially dangerous arrhythmias in cardiac diseases.

The ribosomal P0 protein induces a spontaneous immune response in patients with head and neck advanced stage carcinoma that is not dependent on its overexpression in carcinomas.

A typical feature in systemic lupus erythemathosus patients is the presence of autoantibodies to the carboxyl-terminal homologous P proteins (P0, P1, P2) domain (C-22 P0 epitope). In this report we provide evidence for the in vivo immunogenicity of the P0 protein in head and neck cancer patients as well as overexpression by immunohistochemistry of the C-22 P0 epitope in invasive carcinomas (55/57). Overexpression of this epitope was also significantly associated with a number of pathological lesions arising in the head and neck stratified epithelium including acanthosis (8/8), benign tumors (11/11), dysplasia (23/25) and in situ carcinomas (9/9). Intermediate cell layer restricted epitope overexpression was observed in well differentiated carcinomas, while undifferentiated tumors showed overexpression throughout the cell layers. Employing recombinant P proteins, sera from 40 of the 57 carcinoma patients and 39 normal donors, were subjected to immunoblot analysis. Immunity to P0 protein (7/40) was associated with malignancy and with advanced disease stage, but it was not dependent on the C-22 P0 epitope overexpression, although it was the preferential autoantibody target in 4 patients. Increased expression of the C-22 P0 epitope on the surface of pharynx cancer cells following cellular stress in vitro, may imply P0 protein presentation as an in vivo autoantibody target in cancer patients. Evidence for immunity to the P0 protein, as well as overexpression in patients with head and neck carcinoma may be relevant for monitoring cancer progression, in planning immunotherapeutic strategies, and contribute to the understanding of immuno-biological behaviour of head and neck carcinomas.

Gene-specific inhibition of breast carcinoma in BALB-neuT mice by active immunization with rat Neu or human ErbB receptors.

Employing the transgenic BALB-neuT mouse tumor model, we explored the in vivo biologic relevance of immunocompetent epitopes shared among the four ErbB receptors. The outcome of neu-mediated tumorigenesis was compared following vaccination with isogeneic normal rat ErbB2/Neu (LTR-Neu) or xenogeneic human ErbB receptors (LTR-EGFR, LTR-ErbB2, LTR-ErbB3 and LTR-ErbB4), each recombinantly expressed in an NIH3T3 murine cell background. Vaccination using rat LTR-Neu at the stage of atypical hyperplasia potently inhibited neu-mediated mammary tumorigenesis. Moreover, all human ErbB receptors specifically interfered with tumor development in BALB-neuT mice. Relative increase in tumor-free survival and reduction in tumor incidence corresponded to structural similarity shared with the etiologic neu oncogene, as rat orthologue LTR-Neu proved most effective followed by the human homologue LTR-ErbB2 and the other three human ErbB receptors. Vaccination resulted in high titer specific serum antibodies, whose tumor-inhibitory effect correlated with cross-reactivity to purified rat Neu extracellular domain in vitro. Furthermore, a T cell response specific for peptide epitopes of rat Neu was elicited in spleen cells of mice immunized with LTR-Neu and was remotely detectable for discrete peptides upon vaccination with LTR-ErbB2 and LTR-EGFR. The most pronounced tumor inhibition by LTR-Neu vaccination was associated with leukocyte infiltrate and tumor necrosis in vivo, while immune sera specifically induced cytotoxicity and apoptosis of BALB-neuT tumor cells in vitro. Our findings indicated that targeted inhibition of neu oncogene-mediated mammary carcinogenesis is conditional upon the immunization schedule and discrete immunogenic epitopes shared to a variable extent by different ErbB receptors.