Microsatellite Instability and Immune Response: From Microenvironment Features to Therapeutic Actionability—Lessons from Colorectal Cancer Luana Greco, Federica Rubbino, Arianna Dal Buono, Luigi Laghi Genes, 2023 Microsatellite instability (MSI) can be found in 15–20% of all colorectal cancers (CRC) and is the key feature of a defective DNA mismatch repair (MMR) system. Currently, MSI has been established as a unique and pivotal biomarker in the diagnosis, prognosis, and treatment of CRC. MSI tumors display a strong lymphocytic activation and a shift toward a tumoral microenvironment restraining metastatic potential and ensuing in a high responsiveness to immunotherapy of MSI CRC. Indeed, neoplastic cells with an MMR defect overexpress several immune checkpoint proteins, such as programmed death-1 (PD-1) and programmed death-ligand 1(PD-L1), that can be pharmacologically targeted, allowing for the revival the cytotoxic immune response toward the tumor. This review aims to illustrate the role of MSI in the tumor biology of colorectal cancer, focusing on the immune interactions with the microenvironment and their therapeutic implications.
Dysfunctional Extracellular Matrix Remodeling Supports Perianal Fistulizing Crohn′s Disease by a Mechanoregulated Activation of the Epithelial-to-Mesenchymal Transition Giulia Rizzo, Federica Rubbino, Sudharshan Elangovan, Giusy Sammarco, Sara Lovisa, Silvia Restelli, Samuel Elias Pineda Chavez, Luca Massimino, Luigi Lamparelli, Marianna Paulis, Annalisa Maroli, Giulia Roda, Mohammad Shalaby, Michele Carvello, Caterina Foppa, Sheona P. Drummond, Paola Spaggiari, Federica Ungaro, Antonino Spinelli, Alberto Malesci, Alessandro Repici, Anthony J. Day, Alessandro Armuzzi, Silvio Danese, Stefania Vetrano Cellular and Molecular Gastroenterology and Hepatology, 2023 BACKGROUND AND AIMS: Perianal fistula represents one of the most disabling manifestations of Crohn's disease (CD) due to complete destruction of the affected mucosa, which is replaced by granulation tissue and associated with changes in tissue organization. To date, the molecular mechanisms underlying perianal fistula formation are not well defined. Here, we dissected the tissue changes in the fistula area and addressed whether a dysregulation of extracellular matrix (ECM) homeostasis can support fistula formation. METHODS: Surgical specimens from perianal fistula tissue and the surrounding region of fistulizing CD were analyzed histologically and by RNA sequencing. Genes significantly modulated were validated by real-time polymerase chain reaction, Western blot, and immunofluorescence assays. The effect of the protein product of TNF-stimulated gene-6 (TSG-6) on cell morphology, phenotype, and ECM organization was investigated with endogenous lentivirus-induced overexpression of TSG-6 in Caco-2 cells and with exogenous addition of recombinant human TSG-6 protein to primary fibroblasts from region surrounding fistula. Proliferative and migratory assays were performed. RESULTS: A markedly different organization of ECM was found across fistula and surrounding fistula regions with an increased expression of integrins and matrix metalloproteinases and hyaluronan (HA) staining in the fistula, associated with increased newly synthesized collagen fibers and mechanosensitive proteins. Among dysregulated genes associated with ECM, TNFAI6 (gene encoding for TSG-6) was as significantly upregulated in the fistula compared with area surrounding fistula, where it promoted the pathological formation of complexes between heavy chains from inter-alpha-inhibitor and HA responsible for the formation of a crosslinked ECM. There was a positive correlation between TNFAI6 expression and expression of mechanosensitive genes in fistula tissue. The overexpression of TSG-6 in Caco-2 cells promoted migration, epithelial-mesenchymal transition, transcription factor SNAI1, and HA synthase (HAs) levels, while in fibroblasts, isolated from the area surrounding the fistula, it promoted an activated phenotype. Moreover, the enrichment of an HA scaffold with recombinant human TSG-6 protein promoted collagen release and increase of SNAI1, ITGA4, ITGA42B, and PTK2B genes, the latter being involved in the transduction of responses to mechanical stimuli. CONCLUSIONS: By mediating changes in the ECM organization, TSG-6 triggers the epithelial-mesenchymal transition transcription factor SNAI1 through the activation of mechanosensitive proteins. These data point to regulators of ECM as new potential targets for the treatment of CD perianal fistula.
The Effect of Adjuvant Radiotherapy on One- and Two-Stage Prosthetic Breast Reconstruction and on Autologous Reconstruction: A Multicenter Italian Study among 18 Senonetwork Breast Centres Andrea Vittorio Emanuele Lisa, Marzia Salgarello, Alessandra Huscher, Fabio Corsi, Daniele Piovani, Federica Rubbino, Stefania Andreoletti, Giovanni Papa, Francesco Klinger, Corrado Tinterri, Alberto Testori, Marta Scorsetti, Paolo Veronesi, Maria Cristina Leonardi, Mario Rietjens, Umberto Cortinovis, Valeria Summo, Emanuele Rampino Cordaro, Pier Camillo Parodi, Paolo Persichetti, Mauro Barone, Giorgio De Santis, Matteo Murolo, Michele Riccio, Angelica Aquinati, Francesco Cavaliere, Nicola Vaia, Giulia Pagura, Erica Dalla Venezia, Franco Bassetto, Vincenzo Vindigni, Luigi Ciuffreda, Maria Alessandra Bocchiotti, Alberto Sciarillo, Nadia Renzi, Graziano Meneghini, Tajna Kraljic, Andrea Loreti, Lucio Fortunato, Valentina Pino, Valeriano Vinci, Marco Klinger Breast Journal, 2023 Purpose. In modern breast cancer treatment, a growing role has been observed for breast reconstruction together with an increase in clinical indications for postmastectomy radiotherapy (PMRT). Choosing the optimum type of reconstructive technique is a clinical challenge. We therefore conducted a national multicenter study to analyze the impact of PMRT on breast reconstruction. Methods. We conducted a retrospective case-control multicenter study on women undergoing breast reconstruction. Data were collected from 18 Italian Breast Centres and stored in a cumulative database which included the following: autologous reconstruction, direct-to-implant (DTI), and tissue expander/immediate (TE/I). For all patients, we described complications and surgical endpoints to complications such as reconstruction failure, explant, change in type of reconstruction, and reintervention. Results. From 2001 to April 2020, 3116 patients were evaluated. The risk for any complication was significantly increased in patients receiving PMRT (aOR, 1.73; 95% CI, 1.33–2.24; p < 0.001 ). PMRT was associated with a significant increase in the risk of capsular contracture in the DTI and TE/I groups (aOR, 2.24; 95% CI, 1.57–3.20; p < 0.001 ). Comparing type of procedures, the risk of failure (aOR, 1.82; 95% CI, 1.06–3.12, p = 0.030 ), explant (aOR, 3.34; 95% CI, 3.85–7.83, p < 0.001 ), and severe complications (aOR, 2.54; 95% CI, 1.88–3.43, p < 0.001 ) were significantly higher in the group undergoing DTI reconstruction as compared to TE/I reconstruction. Conclusion. Our study confirms that autologous reconstruction is the procedure least impacted by PMRT, while DTI appears to be the most impacted by PMRT, when compared with TE/I which shows a lower rate of explant and reconstruction failure. The trial is registered with NCT04783818, and the date of registration is 1 March, 2021, retrospectively registered.
Epithelial to Mesenchymal Transition as Mechanism of Progression of Pancreatic Cancer: From Mice to Men Luana Greco, Federica Rubbino, Luigi Laghi Cancers, 2022 Owed to its aggressive yet subtle nature, pancreatic cancer remains unnoticed till an advanced stage so that in most cases the diagnosis is made when the cancer has already spread to other organs with deadly efficiency. The progression from primary tumor to metastasis involves an intricate cascade of events comprising the pleiotropic process of epithelial to mesenchymal transition (EMT) facilitating cancer spread. The elucidation of this pivotal phenotypic change in cancer cell morphology, initially heretic, moved from basic studies dissecting the progression of pancreatic cancer in animal models to move towards human disease, although no clinical translation of the concept emerged yet. Despite this transition, a full-blown mesenchymal phenotype may not be accomplished; rather, the plasticity of the program and its dependency on heterotopic signals implies a series of fluctuating modifications of cancer cells encompassing mesenchymal and epithelial features. Despite the evidence supporting the activation of EMT and MET during cancer progression, our understanding of the relationship between tumor microenvironment and EMT is not yet mature for a clinical application. In this review, we attempt to resume the knowledge on EMT and pancreatic cancer, aiming to include the EMT among the hallmarks of cancer that could potentially modify our clinical thinking with the purpose of filling the gap between the results pursued in basic research by animal models and those achieved in translational research by surrogate biomarkers, as well as their application for prognostic and predictive purposes.
GPR120 prevents colorectal adenocarcinoma progression by sustaining the mucosal barrier integrity Federica Rubbino, Valentina Garlatti, Valeria Garzarelli, Luca Massimino, Salvatore Spanò, Paolo Iadarola, Maddalena Cagnone, Martin Giera, Marieke Heijink, Simone Guglielmetti, Vincenzo Arena, Alberto Malesci, Luigi Laghi, Silvio Danese, Stefania Vetrano Scientific Reports, 2022 GPR120 (encoded by FFAR4 gene) is a receptor for long chain fatty acids, activated by ω-3 Polyunsaturated Fatty Acids (PUFAs), and expressed in many cell types. Its role in the context of colorectal cancer (CRC) is still puzzling with many controversial evidences. Here, we explored the involvement of epithelial GPR120 in the CRC development. Both in vitro and in vivo experiments were conducted to mimic the conditional deletion of the receptor from gut epithelium. Intestinal permeability and integrity of mucus layer were assessed by using Evans blue dye and immunofluorescence for MUC-2 protein, respectively. Microbiota composition, presence of lipid mediators and short chain fatty acids were analyzed in the stools of conditional GPR120 and wild type (WT) mice. Incidence and grade of tumors were evaluated in all groups of mice before and after colitis-associated cancer. Finally, GPR120 expression was analyzed in 9 human normal tissues, 9 adenomas, and 17 primary adenocarcinomas. Our work for the first time highlights the role of the receptor in the progression of colorectal cancer. We observed that the loss of epithelial GPR120 in the gut results into increased intestinal permeability, microbiota translocation and dysbiosis, which turns into hyperproliferation of epithelial cells, likely through the activation of β -catenin signaling. Therefore, the loss of GPR120 represents an early event of CRC, but avoid its progression as invasive cancer. these results demonstrate that the epithelial GPR120 receptor is essential to maintain the mucosal barrier integrity and to prevent CRC developing. Therefore, our data pave the way to GPR120 as an useful marker for the phenotypic characterization of CRC lesions and as new potential target for CRC prevention.
Tumor-associated macrophages and risk of recurrence in stage III colorectal cancer Tommaso Cavalleri, Luana Greco, Federica Rubbino, Tsuyoshi Hamada, Maria Quaranta, Fabio Grizzi, Elisabetta Sauta, Vincenzo Craviotto, Paola Bossi, Stefania Vetrano, Lorenza Rimassa, Valter Torri, Riccardo Bellazzi, Alberto Mantovani, Shuji Ogino, Alberto Malesci, Luigi Laghi Journal of Pathology Clinical Research, 2022 Tumor-associated macrophages (TAMs) have a unique favorable effect on the prognosis of colorectal cancer (CRC), although their association with stage-specific outcomes remains unclear. We assessed the densities of CD68+ and CD163+ TAMs at the invasive front of resected CRC stage III CRC from 236 patients, 165 of whom received post-surgical FOLFOX treatment, and their relationship with disease-free survival (DFS). Associations between macrophage mRNAs and clinical outcome were investigated in silico in 59 stage III CRC and FOLFOX-treated patients from The Cancer Genome Atlas (TCGA). Biological interactions of SW480 and HT29 cells and macrophages with FOLFOX were tested in co-culture models. Low TAM densities were associated with shorter DFS among patients receiving FOLFOX (CD68+ , p = 0.0001; CD163+ , p = 0.0008) but not among those who were untreated. By multivariate Cox analysis, only low TAM (CD68+ , p = 0.001; CD163+ , p = 0.002) and nodal status (CD68+ , p = 0.009; CD163+ , p = 0.007) maintained an independent predictive value. In the TCGA cohort, high CD68 mRNA levels were associated with better outcome (p = 0.02). Macrophages enhanced FOLFOX cytotoxicity on CRC cells (p < 0.01), and drugs oriented macrophage polarization from M2- to M1-phenotype. Low TAM densities identify stage III CRC patients at higher risk of recurrence after adjuvant therapy, and macrophages can augment the chemo-sensitivity of micro-metastases.
Journey through crohn’s disease complication: From fistula formation to future therapies Federica Rubbino, Luana Greco, Alessio di Cristofaro, Federica Gaiani, Stefania Vetrano, Luigi Laghi, Stefanos Bonovas, Daniele Piovani Journal of Clinical Medicine, 2021 Crohn’s Disease (CD) is a chronic inflammatory disorder in which up to 50% of patients develop fistula within 20 years after the initial diagnosis, and half of these patients suffer perianal fistulizing disease. The etiopathogenesis of CD-related perianal fistula is still unclear, and its phenotypical and molecular characteristics are even more indefinite. A better understanding would be crucial to develop targeted and more effective therapeutic strategies. At present, the most accredited theory for the formation of CD-related fistula identifies the epithelial-to-mesenchymal transition (EMT) as the driving force. It has been well recognized that CD carries an increased risk of malignancy, particularly mucinous adenocarcinoma is often associated with long-standing fistula in CD patients. Despite the availability of multiple treatment options, perianal fistulizing CD represents a therapeutic challenge and is associated with an important impact on patients’ quality of life. To date, the most effective management is multidisciplinary with the cooperation of gastroenterologists, surgeons, radiologists, and nutritionists and the best recommended treatment is a combination of medical and surgical approaches.
Epithelial to mesenchymal transition: A challenging playground for translational research. current models and focus on TWIST1 relevance and gastrointestinal cancers Luana Greco, Federica Rubbino, Alessandra Morelli, Federica Gaiani, Fabio Grizzi, Gian Luigi de’Angelis, Alberto Malesci, Luigi Laghi International Journal of Molecular Sciences, 2021 Resembling the development of cancer by multistep carcinogenesis, the evolution towards metastasis involves several passages, from local invasion and intravasation, encompassing surviving anoikis into the circulation, landing at distant sites and therein establishing colonization, possibly followed by the outgrowth of macroscopic lesions. Within this cascade, epithelial to mesenchymal transition (EMT) works as a pleiotropic program enabling cancer cells to overcome local, systemic, and distant barriers against diffusion by replacing traits and functions of the epithelial signature with mesenchymal-like ones. Along the transition, a full-blown mesenchymal phenotype may not be accomplished. Rather, the plasticity of the program and its dependency on heterotopic signals implies a pendulum with oscillations towards its reversal, that is mesenchymal to epithelial transition. Cells in intermixed E⇔M states can also display stemness, enabling their replication together with the epithelial reversion next to successful distant colonization. If we aim to include the EMT among the hallmarks of cancer that could modify clinical practice, the gap between the results pursued in basic research by animal models and those achieved in translational research by surrogate biomarkers needs to be filled. We review the knowledge on EMT, derived from models and mechanistic studies as well as from translational studies, with an emphasis on gastrointestinal cancers (GI).
Unveiling role of sphingosine-1-phosphate receptor 2 as a brake of epithelial stem cell proliferation and a tumor suppressor in colorectal cancer Luciana Petti, Giulia Rizzo, Federica Rubbino, Sudharshan Elangovan, Piergiuseppe Colombo, Silvia Restelli, Andrea Piontini, Vincenzo Arena, Michele Carvello, Barbara Romano, Tommaso Cavalleri, Achille Anselmo, Federica Ungaro, Silvia D’Alessio, Antonino Spinelli, Sanja Stifter, Fabio Grizzi, Alessandro Sgambato, Silvio Danese, Luigi Laghi, Alberto Malesci, Stefania Vetrano Journal of Experimental and Clinical Cancer Research, 2020 BackgroundSphingosine-1-phosphate receptor 2 (S1PR2) mediates pleiotropic functions encompassing cell proliferation, survival, and migration, which become collectively de-regulated in cancer. Information on whetherS1PR2participates in colorectal carcinogenesis/cancer is scanty, and we set out to fill the gap.MethodsWe screened expression changes of S1PR2 in human CRC and matched normal mucosa specimens [N = 76]. We compared CRC arising in inflammation-driven and genetically engineered models in wild-type (S1PR2+/+) and S1PR2 deficient (S1PR2−/−) mice. We reconstituted S1PR2 expression in RKO cells and assessed their growth in xenografts. Functionally, we mimicked the ablation of S1PR2 in normal mucosa by treating S1PR2+/+organoids with JTE013 and characterized intestinal epithelial stem cells isolated from S1PR2−/−Lgr5-EGFP- mice.ResultsS1PR2 expression was lost in 33% of CRC; in 55%, it was significantly decreased, only 12% retaining expression comparable to normal mucosa. Both colitis-induced and genetic Apc+/minmouse models of CRC showed a higher incidence in size and number of carcinomas and/or high-grade adenomas, with increased cell proliferation in S1PR2−/−mice compared to S1PR2+/+controls. Loss of S1PR2 impaired mucosal regeneration, ultimately promoting the expansion of intestinal stem cells. Whereas its overexpression attenuated cell cycle progression, it reduced the phosphorylation of AKT and augmented the levels of PTEN.ConclusionsIn normal colonic crypts, S1PR2 gains expression along with intestinal epithelial cells differentiation, but not in intestinal stem cells, and contrasts intestinal tumorigenesis by promoting epithelial differentiation, preventing the expansion of stem cells and braking their malignant transformation. Targeting of S1PR2 may be of therapeutic benefit for CRC expressing high Lgr5.Graphical Abstract. Schematic drawing of the role of S1PR2 in normal mucosa and colorectal cancer. In the normal mucosa, S1PR2 is highly expressed by differentiated cells at the upper region of both colon and intestinal crypts (S1PR2 ON), but not by the undifferentiated stem cell at the base of the crypts (S1PR2 OFF), in which acts as a negative proliferative regulator promoting epithelial differentiation. Its loss leads to the expansion of stem cells and reduced levels of PTEN and Axin-2, two negative regulators respectively of PI3K/AKT and Wnt signaling that control β-catenin signaling. The translocation of β-catenin into the nucleus promotes the transcription of target genes involved in the proliferation and malignant transformation. Thereby, S1PR2 works in the intestine as a tumor suppressor
Activation of the VEGFC/VEGFR3 pathway induces tumor immune escape in colorectal cancer Carlotta Tacconi, Federica Ungaro, Carmen Correale, Vincenzo Arena, Luca Massimino, Michael Detmar, Antonino Spinelli, Michele Carvello, Massimiliano Mazzone, Ana I. Oliveira, Federica Rubbino, Valentina Garlatti, Salvatore Spanò, Enrico Lugli, Federico S. Colombo, Alberto Malesci, Laurent Peyrin-Biroulet, Stefania Vetrano, Silvio Danese, Silvia D’Alessio Cancer Research, 2019 Colorectal cancer (CRC) is a major cause of cancer-related death in Western countries and is associated with increased numbers of lymphatic vessels (LVs) and tumor-associated macrophages (TAMs). The VEGFC/VEGFR3 pathway is regarded as the principal inducer of lymphangiogenesis and it contributes to metastases; however, no data are available regarding its role during primary CRC development. We found that both VEGFC and VEGFR3 were upregulated in human non-metastatic CRC, with VEGFR3 expressed on both LVs and TAMs. With the use of three different preclinical models of CRC, we also discovered that the VEGFC/VEGFR3 axis can shape both lymphatic endothelial cells (LECs) and TAMs to synergistically inhibit anti-tumor immunity and promote primary CRC growth. Therefore, VEGFR3-directed therapy could be envisioned for the treatment of non-metastatic CRC.
Lymphatic endothelium contributes to colorectal cancer growth via the soluble matrisome component GDF11 Federica Ungaro, Piergiuseppe Colombo, Luca Massimino, Giovanni Stefano Ugolini, Carmen Correale, Marco Rasponi, Valentina Garlatti, Federica Rubbino, Carlotta Tacconi, Paola Spaggiari, Antonino Spinelli, Michele Carvello, Matteo Sacchi, Salvatore Spanò, Stefania Vetrano, Alberto Malesci, Laurent Peyrin‐Biroulet, Silvio Danese, Silvia D'Alessio International Journal of Cancer, 2019
