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Overexpression of RasG12V in primary cells induces a permanent growth arrest called oncogene-induced senescence (OIS) that serves as a fail-safe mechanism against malignant transformation. We have performed a genome-wide small interfering RNA (siRNA) screen and a microRNA (miRNA) screen to identify mediators of OIS and show that siRNA-mediated knockdown of p21Waf1/Cip1 rescues from RasG12V-induced senescence in human mammary epithelial cells (HMECs). Moreover, we isolated a total of 28 miRNAs that prevented RasG12V-induced growth arrest, among which all of the miR-106b family members were present. In addition, we obtained a number of hits, miR-130b, miR-302a, miR-302b, miR302c, miR-302d, miR-512-3p and miR-515-3p with seed sequences very similar to miR-106b family members. We show that overexpression of all these miRNAs rescues HMECs from RasG12V-induced senescence by prevention of RasG12V-induced upregulation of p21Waf1/Cip1. Our results establish an important role for the cell cycle inhibitor p21Waf1/Cip1 in growth control of HMECs and extend the repertoire of miRNAs that modulate the activity of this tumour suppressor. [ABSTRACT FROM AUTHOR]

Overexpression of RasG12V in primary cells induces a permanent growth arrest called oncogene-induced senescence (OIS) that serves as a fail-safe mechanism against malignant transformation. We have performed a genome-wide small interfering RNA (siRNA) screen and a microRNA (miRNA) screen to identify mediators of OIS and show that siRNA-mediated knockdown of p21Waf1/Cip1 rescues from RasG12V-induced senescence in human mammary epithelial cells (HMECs). Moreover, we isolated a total of 28 miRNAs that prevented RasG12V-induced growth arrest, among which all of the miR-106b family members were present. In addition, we obtained a number of hits, miR-130b, miR-302a, miR-302b, miR302c, miR-302d, miR-512-3p and miR-515-3p with seed sequences very similar to miR-106b family members. We show that overexpression of all these miRNAs rescues HMECs from RasG12V-induced senescence by prevention of RasG12V-induced upregulation of p21Waf1/Cip1. Our results establish an important role for the cell cycle inhibitor p21Waf1/Cip1 in growth control of HMECs and extend the repertoire of miRNAs that modulate the activity of this tumour suppressor. [ABSTRACT FROM AUTHOR]

The overexpression of the immunoinhibitory receptor programmed death-1 (PD1) on T-cells is involved in immune evasion in cancer. The use of anti-PD-1/PDL-1 strategy has deeply changed the therapies of cancers and patient survival. However, their efficacy diverges greatly along with tumor type and patient populations. Thereby, novel treatments are needed to interfere with the anti-tumoral immune responses and propose an adjunct therapy. In the current study, we found that the antifungal drug Sulconazole (SCZ) inhibits PD-1 expression on activated PBMCs and T cells at the RNA and protein levels. SCZ repressed NF-κB and calcium signaling, both, involved in the induction of PD-1. Further analysis revealed cancer cells treatment with SCZ inhibited their proliferation, and migration and ability to mediate tumor growth in zebrafish embryos. SCZ found also to inhibit calcium mobilization in cancer cells. These results suggest the SCZ therapeutic potential used alone or as adjunct strategy to prevent T-cell exhaustion and promotes cancer cell malignant phenotype repression in order to improve tumor eradication., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Pernot, Tomé, Galeano-Otero, Evrard, Badiola, Delom, Fessart, Smani, Siegfried, Villoutreix and Khatib.)

The AGR2 and AGR3 genes have been shown by numerous groups to be functionally associated with adenocarcinoma progression and metastasis. In this paper, we explore the data available in databases concerning genomic and transcriptomic features of these two genes: the NCBI dbSNP database was used to explore the presence and roles of constitutional SNPs, and the NCI, Cancer Cell Line Encyclopedia (CCLE) and TCGA databases were used to explore somatic mutations and copy number variations (CNVs), as well as mRNA expression of these genes in human cancer cell lines and tumours. Relationships of AGR2 / 3 expression with whole-genome mRNA expression and cancer features (i.e. mutations and CNVs of oncogenes and tumour suppressor genes (TSG)) were established using the CCLE and TCGA databases. In addition, the CCLE data concerning CRISPR gene extinction screens (Achilles project) of these two genes and a panel of oncogenes and TSG were explored. We observed that no functional polymorphism or recurrent mutation could be detected in AGR2 or AGR3 . The expression of these genes was positively correlated with the expression of epithelial genes and inversely correlated with that of mesenchymal genes. It was also significantly associated with several cancer features, such as TP53 or SMAD4 mutations, depending on the gene and the cancer type. In addition, the CRISPR screens revealed the absence of cell fitness modification upon gene extinction, in contrast with oncogenes (cell fitness decrease) and TSG (cell fitness increase). Overall, these explorations revealed that AGR2 and AGR3 proteins appear as common non-genetic evolutionary factors in the process of human tumorigenesis.

Epithelial ovarian cancer (EOC) is the most common gynecologic disorder. Even with the recent progresses made towards the use of new therapeutics, it still represents the most lethal gynecologic malignancy in women from developed countries.The discovery of the anterior gradient proteins AGR2 and AGR3, which are highly related members belonging to the protein disulfide isomerase (PDI) family, attracted researchers' attention due to their putative involvement in adenocarcinoma development. This review compiles the current knowledge on the role of the AGR family and the expression of its members in EOC and discusses the potential clinical relevance of AGR2 and AGR3 for EOC diagnosis, prognosis, and therapeutics.A better understanding of the role of the AGR family may thus provide new handling avenues for EOC patients., (© 2021. The Author(s).)

The human Anterior GRadient 2 (AGR2) protein is an Endoplasmic Reticulum (ER)-resident protein which belongs to the Protein-Disulfide Isomerase (PDI) superfamily and is involved to productive protein folding in the ER. As such AGR2, often found overexpressed in adenocarcinomas, contributes to tumour development by enhancing ER proteostasis. We previously demonstrated that AGR2 is secreted (extracellular AGR2 (eAGR2)) in the tumour microenvironment and plays extracellular roles independent of its ER functions. Herein, we show that eAGR2 triggers cell proliferation and characterize the underlying molecular mechanisms. We demonstrate that eAGR2 enhances tumour cell growth by repressing the tumour suppressor p21 CIP1 . Our findings shed light on a novel mechanism through which eAGR2 behaves as a growth factor in the tumour microenvironment, independently of its ER function, thus promoting tumour cell growth through repression of p21 CIP1 . Our results provide a rationale for targeting eAGR2/p21 CIP1 -based signalling as a potential therapeutic target to impede tumour growth., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Preclinical lung cancer models are essential for a basic understanding of lung cancer biology and its translation into efficient treatment options for affected patients. Lung cancer cell lines and xenografts derived directly from human lung tumors have proven highly valuable in fundamental oncology research and anticancer drug discovery. Both models inherently comprise advantages and caveats that have to be accounted for. Recently, we have enabled reliable in vitro culture techniques from lung cancer biopsies as Patients Lung Derived Tumoroids (PLDTs). This breakthrough provides the possibility of high-throughput drug screening covering the spectrum of lung cancer phenotypes seen clinically. We have adapted and optimized our in vitro three-dimensional model as a preclinical lung cancer model to recapitulate the tumor microenvironment (TME) using matrix reconstitution. Hence, we developed directly PLDTs to screen for chemotherapeutics and radiation treatment. This original model will enable precision medicine to become a reality, allowing a given patient sample to be screened for effective ex vivo therapeutics, aiming at tailoring of treatments specific to that individual. Hence, this tool can enhance clinical outcomes and avoid morbidity due to ineffective therapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Cytochrome P450 1B1 (CYP1B1) is mostly expressed in tumours and displays unusual properties. Its two polymorphic forms were differently associated with anticancer drug sensitivity. We decipher here the role of this polymorphism in anticancer drug efficacy in vitro, in vivo and in the clinical setting., Methods: From head-and-neck squamous cell carcinoma cell lines not expressing CYP1B1, we generated isogenic derivatives expressing the two forms. Proliferation, invasiveness, stem cell characteristics, sensitivity to anticancer agents and transcriptome were analysed. Tumour growth and chemosensitivity were studied in vivo. A prospective clinical trial on 121 patients with advanced head-and-neck cancers was conducted, and a validation-retrospective study was conducted., Results: Cell lines expressing the variant form displayed high rates of in vitro proliferation and invasiveness, stemness features and resistance to DNA-damaging agents. In vivo, tumours expressing the variant CYP1B1 had higher growth rates and were markedly drug-resistant. In the clinical study, overall survival was significantly associated with the genotypes, wild-type patients presenting a longer median survival (13.5 months) than the variant patients (6.3 months) (p = 0.0166)., Conclusions: This frequent CYP1B1 polymorphism is crucial for cancer cell proliferation, migration, resistance to chemotherapy and stemness properties, and strongly influences head-and-neck cancer patients' survival.

The anterior gradient-2 (AGR2) is an endoplasmic reticulum (ER)-resident protein belonging to the protein disulfide isomerase family that mediates the formation of disulfide bonds and assists the protein quality control in the ER. In addition to its role in proteostasis, extracellular AGR2 is responsible for various cellular effects in many types of cancer, including cell proliferation, survival, and metastasis. Various OMICs approaches have been used to identify AGR2 binding partners and to investigate the functions of AGR2 in the ER and outside the cell. Emerging data showed that AGR2 exists not only as monomer, but it can also form homodimeric structure and thus interact with different partners, yielding different biological outcomes. In this review, we summarize the AGR2 "interactome" and discuss the pathological and physiological role of such AGR2 interactions.

Human anterior gradient proteins AGR2 and AGR3 are overexpressed in a variety of adenocarcinomas and are often secreted in cancer patients' specimens, which suggests a role for AGR proteins in intra and extracellular compartments. Although these proteins exhibit high sequence homology, AGR2 is predominantly described as a pro-oncogene and a potential prognostic biomarker. However, little is known about the function of AGR3. Therefore, the aim of the present study was to investigate the role of AGR3 in breast cancer. The results demonstrated that breast cancer cells secrete AGR3. Furthermore, it was revealed that extracellular AGR3 (eAGR3) regulates tumor cell adhesion and migration. The current study indicated that the pharmacological and genetic perturbation of Src kinase signaling, through treatment with Dasatinib (protein kinase inhibitor) or investigating cells that express a dominant-negative form of Src, significantly abrogated eAGR3-mediated breast cancer cell migration. Therefore, the results indicated that eAGR3 may control tumor cell migration via activation of Src kinases. The results of the present study indicated that eAGR3 may serve as a microenvironmental signaling molecule in tumor-associated processes., (Copyright: © Obacz et al.)

Proprotein convertases (PC) activate precursor proteins that play crucial roles in various cancers. In this study, we investigated whether PC enzyme activity is required for expression of the checkpoint protein programmed cell death protein 1 (PD-1) on cytotoxic T lymphocytes (CTL) in colon cancer. Although altered expression of the PC secretory pathway was observed in human colon cancers, only furin showed highly diffuse expression throughout the tumors. Inhibition of PCs in T cells using the general protein-based inhibitor α1-PDX or the pharmacologic inhibitor Decanoyl-Arg-Val-Lys-Arg-chloromethylketone repressed PD-1 and exhausted CTLs via induction of T-cell proliferation and apoptosis inhibition, which improved CTL efficacy against microsatellite instable and microsatellite stable colon cancer cells. In vivo , inhibition of PCs enhanced CTL infiltration in colorectal tumors and increased tumor clearance in syngeneic mice compared with immunodeficient mice. Inhibition of PCs repressed PD-1 expression by blocking proteolytic maturation of the Notch precursor, inhibiting calcium/NFAT and NF-κB signaling, and enhancing ERK activation. These findings define a key role for PCs in regulating PD-1 expression and suggest targeting PCs as an adjunct approach to colorectal tumor immunotherapy. SIGNIFICANCE: Protein convertase enzymatic activity is required for PD-1 expression on T cells, and inhibition of protein convertase improves T-cell targeting of microsatellite instable and stable colorectal cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/19/5008/F1.large.jpg., (©2019 American Association for Cancer Research.)

The extracellular matrix (ECM) plays an instrumental role in determining the spatial orientation of epithelial polarity and the formation of lumens in glandular tissues during morphogenesis. Here, we show that the Endoplasmic Reticulum (ER)-resident protein anterior gradient-2 (AGR2), a soluble protein-disulfide isomerase involved in ER protein folding and quality control, is secreted and interacts with the ECM. Extracellular AGR2 (eAGR2) is a microenvironmental regulator of epithelial tissue architecture, which plays a role in the preneoplastic phenotype and contributes to epithelial tumorigenicity. Indeed, eAGR2, is secreted as a functionally active protein independently of its thioredoxin-like domain (CXXS) and of its ER-retention domain (KTEL), and is sufficient, by itself, to promote the acquisition of invasive and metastatic features. Therefore, we conclude that eAGR2 plays an extracellular role independent of its ER function and we elucidate this gain-of-function as a novel and unexpected critical ECM microenvironmental pro-oncogenic regulator of epithelial morphogenesis and tumorigenesis.

In the present study, we have developed an in vitro three-dimensional model to differentiate normal lung cells from lung cancer cells in order to study the mechanisms resulting in lung cancer. Using a reconstituted laminin-rich basement membrane (Matrigel), we were able to culture normal human bronchial epithelial cells and a subset of malignant cells. The two cell types can be readily distinguished by the ability of normal cells to express a structurally and functionally differentiated phenotype within Matrigel. Human bronchial epithelial cells embedded in Matrigel as single cells were able to form multi-cellular spherical colonies with a final size close to that of true acini in situ. Sections of mature spheres revealed a central lumen surrounded by polarised epithelial cells. In contrast, none of malignant cells tested, cell lines and lung biopsies responded to basement membrane by lumen formation. These results demonstrated that this in vitro glandular tumour model can be useful for studies of bronchial oncogene. Indeed, these findings may provide the basis for a rapid assay to discriminate normal human bronchial epithelial cells from their malignant counterparts. In conclusion, the three-dimensional tumour bronchial epithelial acinar-like sphere represents a novel in vitro model to further investigate pathophysiological functions resulting in lung cancer.

The process by which G protein-coupled receptors (GPCRs) are internalized through the clathrin-coated vesicles involves interactions of multifunctional adaptor proteins. These interactions are tightly controlled by phosphorylation and dephosphorylation mechanisms resulting in the regulation of receptor endocytosis. However, the identities of the kinases involved in this process remained largely unknown until recently. This paper discusses advances in our knowledge of the important role played by protein phosphorylation in the regulation of the endocytic machinery and how phosphorylation controls the coated vesicle cycle.

In a genome-wide siRNA analysis of p16(INK4a) (p16) modulators, we identify the Hedgehog (Hh) pathway component SUFU and formally demonstrate that Hh signaling promotes mitogenesis by suppression of p16. A fragment of the Hh-responsive GLI2 transcription factor directly binds and inhibits the p16 promoter and senescence is associated with the loss of nuclear GLI2. Hh components partially reside in the primary cilium (PC), and the small fraction of cells in mass culture that elaborate a PC have the lowest expression of p16. Suppression of p16 is effected by both PC-dependent and -independent routes, and ablation of p16 renders cells insensitive to an Hh inhibitor and increases PC formation. These results directly link a well-established developmental mitogenic pathway with a key tumor suppressor and contribute to the molecular understanding of replicative senescence, Hh-mediated oncogenesis, and potentially the role of p16 in aging., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The most widely studied pathway underlying agonist-promoted internalization of G protein-coupled receptors (GPCRs) involves beta-arrestin and clathrin-coated pits. However, both beta-arrestin- and clathrin-independent processes have also been reported. Classically, the endocytic routes are characterized using pharmacological inhibitors and various dominant negative mutants, resulting sometimes in conflicting results and interpretational difficulties. Here, taking advantage of the fact that beta-arrestin binding to the beta2 subunit of the clathrin adaptor AP-2 (beta2-adaptin) is needed for the beta-arrestin-mediated targeting of GPCRs to clathrin-coated pits, we developed a bioluminescence resonance energy transfer-based approach directly assessing the molecular steps involved in the endocytosis of GPCRs in living cells. For 10 of the 12 receptors tested, including some that were previously suggested to internalize via clathrin-independent pathways, agonist stimulation promoted beta-arrestin 1 and 2 interaction with beta2-adaptin, indicating a beta-arrestin- and clathrin-dependent endocytic process. Detailed analyses of beta-arrestin interactions with both the receptor and beta2-adaptin also allowed us to demonstrate that recruitment of beta-arrestins to the receptor and the ensuing conformational changes are the leading events preceding AP-2 engagement and subsequent clathrin-mediated endocytosis. Among the receptors tested, only the endothelin A and B receptors failed to promote interaction between beta-arrestins and beta2-adaptin. However, both receptors recruited beta-arrestins upon agonist stimulation, suggesting a beta-arrestin-dependent but clathrin-independent route of internalization for these two receptors. In addition to providing a new tool to dissect the molecular events involved in GPCR endocytosis, the bioluminescence resonance energy transfer-based beta-arrestin/beta2-adaptin interaction assay represents a novel biosensor to assess receptor activation.

Beta-arrestins are known to act as endocytic adaptors by recruiting the clathrin adaptor protein 2 (AP-2) complex to G-protein-coupled receptors (GPCRs), linking them to clathrin-coated pits (CCPs) for internalization. They also act as signaling molecules connecting GPCRs to different downstream effectors. We have previously shown that stimulation of the angiotensin II (Ang II) type 1 receptor (AGTR1, hereafter referred to as AT1R), a member of the GPCR family, promotes the formation of a complex between beta-arrestin, the kinase Src and AP-2. Here, we report that formation of such a complex is involved in the AT1R-mediated tyrosine phosphorylation of beta2-adaptin, the subunit of AP-2 involved in binding beta-arrestin. We identify a crucial tyrosine residue in the ear domain of beta2-adaptin and show in vitro that the phosphorylation of this site regulates the interaction between beta-arrestin and beta2-adaptin. Using fluorescently tagged proteins combined with resonance energy transfer and image cross-correlation spectroscopy approaches, we show in live cells that beta2-adaptin phosphorylation is an important regulatory process for the dissociation of beta-arrestin-AP-2 complexes in CCPs. Finally, we show that beta2-adaptin phosphorylation is involved in the early steps of receptor internalization. Our findings not only unveil beta2-adaptin as a new Src target during AT1R internalization, but also support the role of receptor-mediated signaling in the control of clathrin-dependent endocytosis of receptors.

Beta-arrestins are multifunctional adapters involved in the internalization and signaling of G protein-coupled receptors (GPCRs). They target receptors to clathrin-coated pits (CCPs) through binding with clathrin and clathrin adapter 2 (AP-2) complex. They also act as transducers of signaling by recruiting c-Src kinase to certain GPCRs. Here we sought to determine whether c-Src regulates the recruitment of AP-2 to beta-arrestin and the angiotensin II (Ang II) type 1 receptor (AT1R) during internalization. We show that the agonist stimulation of native AT1R in vascular smooth muscle cells (VSMCs) induces the formation of an endogenous complex containing c-Src, beta-arrestins and AP-2. In vitro studies using coimmunoprecipitation experiments and a yeast three-hybrid assay reveal that c-Src stabilizes the agonist-independent association between beta-arrestin2 and the beta-subunit of AP-2 independently of the kinase activity of c-Src. However, although c-Src expression promoted the rapid dissociation of AP-2 from both beta-arrestin and AT1R after receptor stimulation, a kinase-inactive mutant of c-Src failed to induce the dissociation of AP-2 from the agonist-occupied receptor. Thus, the consequence of c-Src in regulating the dissociation of AP-2 from the receptor was also examined on the internalization of AT1R by depleting c-Src in human embryonic kidney (HEK) 293 cells using a small interfering RNA strategy. Experiments in c-Src depleted cells reveal that AT1R remained mostly colocalized with AP-2 at the plasma membrane after Ang II stimulation, consistent with the observed delay in receptor internalization. Moreover, coimmunoprecipitation experiments in c-Src depleted HEK 293 cells and VSMCs showed an increased association of AP-2 to the agonist-occupied AT1R and beta-arrestin, respectively. Together, our results support a role for c-Src in regulating the dissociation of AP-2 from agonist-occupied AT1R and beta-arrestin during the clathrin-mediated internalization of receptors and suggest a novel function for c-Src kinase in the internalization of AT1R.