Desarrollo de un modelo de adhesión celular "in vitro" para el estudio de la adhesión trofoblástica al endometrio humano: análisis de la expresión génica durante la fase inicial de la implantación embrionaria

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular, Fecha de lectura 10-03-2006Bibliogr.: h. 139-14

A prognostic six-gene expression risk-score derived from proteomic profiling of the metastatic colorectal cancer secretome

14 p.-6 fig.-1 tab.The necessity to accurately predict recurrence and clinical outcome in early stage colorectal cancer (CRC) is critical to identify those patients who may benefit from adjuvant chemotherapy. Here, we developed and validated a gene-based risk-score algorithm for patient stratification and personalised treatment in early stage disease based on alterations in the secretion of metastasis-related proteins. A quantitative label-free proteomic analysis of the secretome of highly and poorly metastatic CRC cell lines with different genetic backgrounds revealed 153 differentially secreted proteins (fold-change >5). These changes in the secretome were validated at the transcriptomic level. Starting from 119 up-regulated proteins, a six-gene/protein-based prognostic signature composed of IGFBP3, CD109, LTBP1, PSAP, BMP1, and NPC2 was identified after sequential discovery, training,and validation in four different cohorts. This signature was used to develop a risk-score algorithm, named SEC6,for patient stratification. SEC6 risk-score components showed higher expression in the poor prognosis CRC sub types: consensus molecular subtype 4 (CMS4), CRIS-B, and stem-like. High expression of the signature was also associated with patients showing dMMR, CIMP+ status, and BRAF mutations. In addition, the SEC6 signature was associated with lower overall survival, progression-free interval, and disease-specific survival in stage II and III patients. SEC6-based risk stratification indicated that 5-FU treatment was beneficial for low-risk patients,whereas only aggressive treatments (FOLFOX and FOLFIRI) provided benefits to high-risk patients in stages II and III. In summary, this novel risk-score demonstrates the value of the secretome compartment as a reliable source for the retrieval of biomarkers with high prognostic and chemotherapy-predictive capacity, providing a potential new tool for tailoring decision-making in patient care.This project was supported by grants RTI2018-095055-B-100 and PID2021-122227OB-I00 from the MICYT, IND2019/BMD-17153 from the Comunidad de Madrid and PRB3 (ISCIII-SGEFI/FEDER- PT17/0019/0008) from the ISCIII.Peer reviewe

SOSTDC1 promotes invasion and liver metastasis in colorectal cancer via interaction with ALCAM/CD166

38 p.-7 fig.The mechanistic basis of liver metastasis in colorectal cancer remains poorly understood. We previously reported that the sclerostin domain containing-1 (SOSTDC1) protein is overexpressed in the secretome of metastatic colorectal cancer cells and can inhibit liver homing. Here, we investigated the mechanisms of SOSTDC1 for promoting invasiveness and progression of colorectal cancer liver metastasis. SOSTDC1 inhibition of BMP4 maintains the expression of cancer stem cell traits, including SOX2 and NANOG. Immunoprecipitation and mass spectrometry analyses reveal the association of SOSTDC1 with ALCAM/CD166, which was confirmed by confocal microscopy and competition ELISA. Interaction with ALCAM is mediated by the N-terminal region of SOSTDC1, which contains a sequence similar to the ALCAM-binding motif used by CD6. Knocking down either SOSTDC1 or ALCAM expression, or using blocking antibodies, reduces the invasive activity by inhibiting Src and PI3K/AKT signaling pathways. In addition, ALCAM interacts with the alpha2SS1 and alpha1SS1 integrins, providing a possible link to Src activation. Finally, inoculation of SOSTDC1-silenced metastatic cells increases mouse survival by inhibiting liver metastasis. In conclusion, SOSTDC1 promotes invasion and liver metastasis in colorectal cancer, by overcoming BMP4-specific antimetastatic signals and inducing ALCAM-mediated Src and PI3K/AKT activation. These experiments underscore the potential of SOSTDC1 as a therapeutic target in metastatic colorectal cancer.This research was supported by grants from the Ministerio de Ciencia e Innovación (BIO2015-66489-R, RTI2018-095055-B100), Foundation Ramón Areces and PRB3 (ISCIII-SGEFI/FEDER-PT17/0019/0008).Peer reviewe

An RGD motif present in cadherin 17 induces integrin activation and tumor growth

15 p.-9 fig.Little is known about the mechanism of integrin activation by cadherin 17 (CDH17). Here we observed the presence of a tri-peptide motif, RGD, in domain 6 of the human CDH17 sequence and other cadherins such as cadherin 5 and cadherin 6. The use of CDH17 RAD mutants demonstrated a considerable decrease of proliferation and adhesion in RKO and KM12SM colon cancer cells. Furthermore, RGD peptides inhibited the adhesion of both cell lines to recombinant CDH17 domain 6. The RGD motif added exogenously to the cells provoked a change in β1 integrin to an active, high-affinity conformation and an increase in focal adhesion kinase and ERK1/2 activation. In vivo experiments with Swiss nude mice demonstrated that cancer cells expressing the CDH17 RAD mutant showed a considerable delay in tumor growth and liver homing. CDH17 RGD effects were also active in pancreatic cancer cells. Our results suggest that α2β1 integrin interacts with two different ligands, collagen IV and CDH17, using two different binding sites. In summary, the RGD binding motif constitutes a switch for integrin pathway activation and shows a novel capacity of CDH17 as an integrin ligand. This motif could be targeted to avoid metastatic dissemination in tumors overexpressing CDH17 and other RGD-containing cadherins.This research was supported by grant BIO2012-31023 from the Spanish Ministry of Economy and Competitiveness, by a grant to established research groups (AECC), and by grant S2011/BMD-2344/ (Colomics2) from Comunidad de Madrid and ProteoRed-ISCIII.Peer reviewe

Inhibition of liver metastasis in colorectal cancer by targeting IL-13/IL13Rα2 binding site with specific monoclonal antibodies

17 p.-6 fig.Background: IL13Rα2 is reportedly a promising therapeutic target in different cancers. Still, no specific antagonists have reached the clinics yet. We investigated the use of a IL-13/IL13Rα2 binding motif, called D1, as a new target for the development of therapeutic monoclonal antibodies (mAbs) for colorectal cancer (CRC) metastasis. Methods: IL13Rα2 D1 peptides were prepared and used for immunization and antibody development. Antibodies were tested for inhibition of cellular invasion through Matrigel using CRC cell lines. Effects of the mAbs on cell signaling, receptor internalization and degradation were determined by western blot and flow cytometry. Swiss nude mice were used for survival analysis after treatment with IL13Rα2-specific mAbs and metastasis development. Results: IL13Rα2 D1 peptides were used to generate highly selective mAbs that blocked IL13/IL13Rα2-mediated SRC activation and cell invasion in colorectal cancer cells. Antibodies also provoked a significant reduction in cell adhesion and proliferation of metastatic cancer cells. Treatment with mAbs impaired the FAK, SRC and PI3K/AKT pathway activation. Blocking effectivity was shown to correlate with the cellular IL13Rα2 expression level. Despite mAb 5.5.4 partially blocked IL-13 mediated receptor internalization from the cancer cell surface it still promotes receptor degradation. Compared with other IL13Rα2-specific antibodies, 5.5.4 exhibited a superior efficacy to inhibit metastatic growth in vivo, providing a complete mouse survival in different conditions, including established metastasis. Conclusions: Monoclonal antibody 5.5.4 showed a highly selective blocking capacity for the interaction between IL-13 and IL13Rα2 and caused a complete inhibition of liver metastasis in IL13Rα2-positive colorectal cancer cells. This capacity might be potentially applicable to other IL13Rα2-expressing tumors.This research was funded by grants from the MINECO and MICYT (BIO2015-66489-R and RTI2018-095055-B100), Ramón Areces Foundation, and PRB3 (ISCIII-SGEFI/FEDER- PT17/0019/0008).Peer reviewe

CDH17 RGD antibodies inhibit cancer metastasis [Running title]

37 p.-6 fig.Purpose: New targets are required for the control of advanced metastatic disease. We investigated the use of cadherin RGD motifs, which activate the α2β1integrin pathway, as targets for the development of therapeutic monoclonal antibodies (mAb).Experimental Design: Cadherin 17 (CDH17) fragments and peptides were prepared and used for immunization and antibody development. Antibodies were tested for inhibition of β1 integrin and cell adhesion, proliferation, and invasion assays using cell lines from different cancer types (colorectal, pancreatic, melanoma, and breast cancer). Effects of the mAbs on cell signaling were determined by Western blot analysis. Nude mice were used for survival analysis after treatment with RGD-specific mAbs and metastasis development.Results: Antibodies against full-length CDH17 failed to block the binding to α2β1 integrin. However, CDH17 RGD peptides generated highly selective RGD mAbs that blocked CDH17 and vascular-endothelial (VE)-cadherin-mediated β1 integrin activation in melanoma and breast, pancreatic, and colorectal cancer cells. Antibodies provoked a significant reduction in cell adhesion and proliferation of metastatic cancer cells. Treatment with mAbs impaired the integrin signaling pathway activation of FAK in colorectal cancer, of JNK and ERK kinases in colorectal and pancreatic cancers, and of JNK, ERK, Src, and AKT in melanoma and breast cancer. In vivo, RGD-specific mAbs increased mouse survival after inoculation of melanoma and colorectal cancer cell lines to cause lung and liver metastasis, respectively.Conclusions: Blocking the interaction between RGD cadherins and α2β1 integrin with highly selective mAbs constitutes a promising therapy against advanced metastatic disease in colon cancer, melanoma, and, potentially, other cancers.This research was supported by grants BIO2015-66489-R from the MINECO, Foundation Ramón Areces and PRB2 (IPT13/0001-ISCIII-SGEFI/FEDER).Peer reviewe

Running title: Integrin crosstalk in ovarian and renal carcinomas

17 p.-7 fig.Cadherin 6 (CDH6) is significantly overexpressed in advanced ovarian and renal cancers. However, the role of CDH6 in cancer metastasis is largely unclear. Here, we investigated the impact of CDH6 expression on integrin‐mediated metastatic progression. CDH6 preferentially bound to αIIbβ3 integrin, a platelet receptor scarcely expressed in cancer cells, and this interaction was mediated through the cadherin RGD motif. Furthermore, CDH6 and CDH17 were found to interact with α2β1 in αIIbβ3low cells. Transient silencing of CDH6, ITGA2B or ITGB3 genes caused a significant loss of proliferation, adhesion, invasion and lung colonization through the downregulation of SRC, FAK, AKT and ERK signaling. Interaction of αIIbβ3 with CDH6, and subsequent αIIbβ3 activation, promoted activation of α2β1 and cell adhesion in ovarian and renal cancer cells. Additionally, monoclonal antibodies specific to the cadherin RGD motif and clinically‐approved αIIbβ3 inhibitors could block pro‐metastatic activity in ovarian and renal tumors. In summary, the interaction between CDH6 and αIIbβ3 regulate α2β1‐mediated adhesion and invasion of ovarian and renal cancer metastatic cells, and constitutes a therapeutic target of broad potential for treating metastatic progression.This research was supported by grants from the Ministerio de Ciencia e Innovación (Spain) (RTC2017-2017-6260-1 and RTI2018-095055-B-100).Peer reviewe

CDH17 as a biomarker and target of chemoresistance to irinotecan

2 p.Introduction Cadherin 17 (CDH17) plays a key role in colorectal cancer (CRC) metastasis. CDH17 binds and activates α2β1 integrin, through its RGD motif, promoting cell adhesion and proliferation. Blocking this interaction using RGD-specific monoclonal antibodies (mAbs) inhibited the metastatic capacity in mouse models. In a previous report, an overexpression of CDH17 in CRC cells treated with irinotecan was observed. Here, our goal was to investigate the participation of CDH17 in chemotherapy resistance mechanisms and to test the efficacy of RGD CDH17 mAbs in irinotecan-resistant cells.Material and Methods We generated irinotecan-resistant CRC cell lines (COLO205,KM12SM,SW620) by increasing the irinotecan dose from 0.05 to 0.5 mM. Cells were analyzed by Western Blot (WB), flow cytometry (FC) and qPCR assays to study expression of CDH17 and other proteins involved in chemoresistance (i.e. MDR1, ABCG2) or epithelial–mesenchymal transition mediators. In addition, cell cycle and the adhesive and proliferative properties of resistant cells were analyzed in the presence or absence of RGD-specific mAbs to test its efficacy. Then, CDH17 was silenced to determine its role in irinotecan chemoresistance. Finally, we performed a label-free proteomic quantification to survey global changes in proteins of wt cells compared to resistant cells.Results and Discussions CRC cell lines resistant to 0.5 μM irinotecan increased their epithelial phenotype with an overexpression of CDH17, ITGA2, CDX2, MDR1, ABCG2 and ALDH1A markers as determined by WB and FC. In agreement with these results, an increase in adhesion was observed in irinotecan-treated cells. Cell cycle in resistant cells showed preferential arrest in G2/M phase, as irinotecan inhibits the action of topoisomerase I. Proteins involved in chemoresistance, tumorigenesis or modulation of tumor response were founded dysregulated after proteomic analysis of resistant cells. Interestingly, resistant cells were more sensitive to the inhibitory effects of RGD-specific mAb. However, CDH17-silencing experiments in resistant and parental cells indicated that CDH17 was not involved in the mechanisms of resistance to irinotecan.Conclusion CDH17 has shown to be a specific marker of irinotecan-resistance and, therefore, a good target for CDH17 RGD-specific mAb. Therefore, patients that develop resistance to irinotecan could benefit from this therapy.Peer reviewe