Browsing by Author "Fernandes, Afonso"
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- Analysis of a bone metastasis gene expression signature in patients with bone metastasis from solid tumorsPublication . Casimiro, Sandra; Vaz Luis, Ines; Fernandes, Afonso; Pires, Ricardo; Pinto, Andreia; Gouveia, António G.; Francisco, António F.; Portela, José; Correia, Lurdes; Costa, LuisBone is a major target for metastases in the most frequent solid tumors, which result in severe complications and are a major cause of pain. A bone metastasis gene expression signature was identified using human breast cancer cells in a mouse model. The bone metastasis-related genes encode secretory and cell surface proteins implicated in bone-homing (CXCR4), angiogenesis (CTGF and FGF5), invasion (MMP-1 and ADAMTS1), and osteoclast recruitment (IL11). This signature superimposes on the 70-gene poor prognosis gene expression signature for breast cancer, and only ADAMTS1, CTGF and IL11 were found to be overexpressed in human primary breast cancers with bone relapse. We analyzed the expression of the six bone metastasis-related genes in bone metastases from patients with different types of solid tumors, to assess its relevance in human clinical samples. MMP-1, CXCR4, FGF5 and CTGF were found to be overexpressed in tumor cells of human bone metastases when compared to a human normal epithelial cell line. All the analyzed genes were overexpressed in the tumor cells of breast cancer bone metastases when compared to normal breast tissue. We did not detect any differences between the expression of these genes in bone metastases from breast cancer or from other types of solid tumors. Importantly, there was a significant correlation between the expressions of IL11/CTGF, IL11/ADAMTS1, CTGF/CXCR4, CTGF/ADAMTS1, and MMP-1/ADAMTS1, supporting the cooperative function of these proteins in the bone microenvironment, and the potential functional role of these genes in the establishment of bone metastases in vivo.
- Genetic and microenvironmental intra-tumor heterogeneity impacts colorectal cancer evolution and metastatic developmentPublication . Sobral, Daniel; Martins, Marta; Kaplan, Shannon; Golkaram, Mahdi; Salmans, Michael; Khan, Nafeesa; Vijayaraghavan, Raakhee; Casimiro, Sandra; Fernandes, Afonso; Borralho, Paula; Ferreira, Cristina; Pinto, Rui; Marques, Catarina; Costa, Ana Lúcia; Zhang, Shile; Pawlowski, Traci; Godsey, Jim; Mansinho, André; Macedo, Daniela; Lobo-Martins, Soraia; Filipe, Pedro; Esteves, Rui; Coutinho, Joao; Costa, Paulo M.; Ramires, Afonso; Aldeia, Fernando; Quintela, António; So, Alex; Liu, Li; Grosso, Ana Rita; Costa, LuisColorectal cancer (CRC) is a highly diverse disease, where different genomic instability pathways shape genetic clonal diversity and tumor microenvironment. Although intra-tumor heterogeneity has been characterized in primary tumors, its origin and consequences in CRC outcome is not fully understood. Therefore, we assessed intra- and inter-tumor heterogeneity of a prospective cohort of 136 CRC samples. We demonstrate that CRC diversity is forged by asynchronous forms of molecular alterations, where mutational and chromosomal instability collectively boost CRC genetic and microenvironment intra-tumor heterogeneity. We were able to depict predictor signatures of cancer-related genes that can foresee heterogeneity levels across the different tumor consensus molecular subtypes (CMS) and primary tumor location. Finally, we show that high genetic and microenvironment heterogeneity are associated with lower metastatic potential, whereas late-emerging copy number variations favor metastasis development and polyclonal seeding. This study provides an exhaustive portrait of the interplay between genetic and microenvironment intra-tumor heterogeneity across CMS subtypes, depicting molecular events with predictive value of CRC progression and metastasis development.
- HERVs establish a distinct molecular subtype in stage II/III colorectal cancer with poor outcomePublication . Golkaram, Mahdi; Salmans, Michael L.; Kaplan, Shannon; Vijayaraghavan, Raakhee; Martins, Marta; Khan, Nafeesa; Garbutt, Cassandra; Wise, Aaron; Yao, Joyee; Casimiro, Sandra; Marques, Catarina; Macedo, Daniela; Costa, Ana Lúcia; Alvim, Cecilia; Mansinho, André; Filipe, Pedro; Marques da Costa, Pedro; Fernandes, Afonso; Borralho, Paula; Ferreira, Cristina; Aldeia, Fernando; Malaquias, João; Godsey, Jim; So, Alex; Pawlowski, Traci; Costa, Luis; Zhang, Shile; Liu, LiColorectal cancer (CRC) is one of the most lethal malignancies. The extreme heterogeneity in survival rate is driving the need for new prognostic biomarkers. Human endogenous retroviruses (hERVs) have been suggested to influence tumor progression, oncogenesis and elicit an immune response. We examined multiple next-generation sequencing (NGS)-derived biomarkers in 114 CRC patients with paired whole-exome and whole-transcriptome sequencing (WES and WTS, respectively). First, we demonstrate that the median expression of hERVs can serve as a potential biomarker for prognosis, relapse, and resistance to chemotherapy in stage II and III CRC. We show that hERV expression and CD8+ tumor-infiltrating T-lymphocytes (TILs) synergistically stratify overall and relapse-free survival (OS and RFS): the median OS of the CD8-/hERV+ subgroup was 29.8 months compared with 37.5 months for other subgroups (HR = 4.4, log-rank P < 0.001). Combing NGS-based biomarkers (hERV/CD8 status) with clinicopathological factors provided a better prediction of patient survival compared to clinicopathological factors alone. Moreover, we explored the association between genomic and transcriptomic features of tumors with high hERV expression and establish this subtype as distinct from previously described consensus molecular subtypes of CRC. Overall, our results underscore a previously unknown role for hERVs in leading to a more aggressive subtype of CRC.
- Metadherin expression and lung relapse in patients with colorectal carcinomaPublication . Casimiro, Sandra; Fernandes, Afonso; Oliveira, Antonio; Franco, Marco; Pires, Ricardo; Peres, Mafalda; Matias, Margarida; Tato-Costa, Joana; Carvalho Guerra, Nuno; Ramos, Madalena; Cruz, Jorge; Costa, LuisColorectal cancer (CRC) is the third most common malignant disease in men and the second in women worldwide. CRC relapse occurs mostly in liver and lungs, decreasing the 5-year survival to 6 %. Metadherin (MTDH) is overexpressed in several types of cancer, has been implicated in proliferation, invasion, metastasis, angiogenesis, and chemoresistance, and is a factor of poor prognosis in CRC. In this work we addressed the prognostic significance of MTDH expression in CRC progression to the lungs. We found that MTDH gene was more frequently amplified (copy number >1.8) in patients with CRC and relapse to the lung, when compared to patients without lung metastases (17.4 vs 100 %; p < 0.001). We observed a correlation between MTDH gene copy number and MTDH expression by IHC (p = 0.0001). Next we also analyzed MTDH expression by IHC in samples from 85 patients diagnosed with CRC, stage II or III, M0, with at least 3 years of follow-up. Kaplan-Meier survival analysis showed that lung relapse-free survival (HR 5.29, 95 % CI 1.90-14.77, p = 0.0004), liver relapse-free survival (HR 8.59, 95 % CI 0.99-74.18, p = 0.003), relapse-free survival (HR 4.85, 95 % CI 1.88-12.45, p = 0.0003) and overall survival (HR 3.75, 95 % CI 1.15-12.18, p = 0.018) were significantly lower in the group with high MTDH expression. Multivariate analysis showed that high MTDH expression was an independent factor for all outcomes. This study demonstrates that high MTDH expression is a biomarker of relapse in CRC, including lung-specific relapse. Determination of MTDH expression in primary CRC may be useful in the earlier detection of lung metastases in patients with high expression and increased risk.
- Predictive and therapeutic implications of a novel PLCγ1/SHP2-driven mechanism of cetuximab resistance in metastatic colorectal cancerPublication . Duarte, Raquel; Rebelo de Almeida, Cátia; Negrão, Magda; Fernandes, Afonso; Borralho, Paula; Sobral, Daniel; Gallego-Paez, Lina M.; Machado, Daniel; Gramaça, João; Vílchez, José; Xavier, Ana T.; Ferreira, Miguel Godinho; Miranda, Ana R.; Mansinho, Helder; Brito, Maria J.; Pacheco, Teresa; Marques, Catarina; Lucia Costa, Ana; Mansinho, André; Fior, Rita; Costa, Luis; Martins, MartaPurpose: Cetuximab is an EGFR-targeted therapy approved for the treatment of RAS wild-type (WT) metastatic colorectal cancer (mCRC). However, about 60% of these patients show innate resistance to cetuximab. To increase cetuximab efficacy, it is crucial to successfully identify responder patients, as well as to develop new therapeutic approaches to overcome cetuximab resistance. Experimental design: We evaluated the value of EGFR effector phospholipase C gamma 1 (PLCγ1) in predicting cetuximab responses, by analyzing progression-free survival (PFS) of a multicentric retrospective cohort of 94 treated patients with mCRC (log-rank test and Cox regression model). Furthermore, we used in vitro and zebrafish xenotransplant models to identify and target the mechanism behind PLCγ1-mediated resistance to cetuximab. Results: In this study, levels of PLCγ1 were found increased in RAS WT tumors and were able to predict cetuximab responses in clinical samples and in vitro and in vivo models. Mechanistically, PLCγ1 expression was found to bypass cetuximab-dependent EGFR inhibition by activating ERK and AKT pathways. This novel resistance mechanism involves a noncatalytic role of PLCγ1 SH2 tandem domains in the propagation of downstream signaling via SH2-containing protein tyrosine phosphatase 2 (SHP2). Accordingly, SHP2 inhibition sensitizes PLCγ1-resistant cells to cetuximab. Conclusions: Our discoveries reveal the potential of PLCγ1 as a predictive biomarker for cetuximab responses and suggest an alternative therapeutic approach to circumvent PLCγ1-mediated resistance to cetuximab in patients with RAS WT mCRC. In this way, this work contributes to the development of novel strategies in the medical management and treatment of patients with mCRC.
- Prognostic significance of AKT/mTOR signaling in advanced neuroendocrine tumors treated with somatostatin analogsPublication . Fernandes, Isabel; Pacheco, Teresa; Costa, Adília; Santos, Ana C.; Fernandes, Ana R.; Santos, Mara; Oliveira, António G.; Casimiro, Sandra; Quintela, António; Fernandes, Afonso; Ramos, Madalena; Costa, LuisIntroduction: Somatostatin analogs (SSAs) are used as part of standard treatment for advanced neuroendocrine tumors (NETs). The mechanisms behind the antiproliferative action of SSAs remain largely unknown, but a connection with the mammalian target of rapamycin (mTOR) signaling pathway has been suggested. Our purpose was to evaluate the activation status of the AKT/mTOR pathway in advanced metastatic NETs and identify biomarkers of response to SSA therapy. Patients and methods: Expression of phosphatase and tensin homolog (PTEN), phosphorylated (p)-AKT(Ser473), and p-S6(Ser240/244) was evaluated using immunohistochemistry in archival paraffin samples from 23 patients. Expression levels were correlated with clinicopathological parameters and progression-free survival under treatment with SSAs. Results: A positive association between p-AKT and p-S6 expression was identified (P = 0.01) and higher expression of both markers was observed in pancreatic NETs. AKT/mTOR activation was observed without the loss of PTEN expression. Tumors showing AKT/mTOR signaling activation progressed faster when treated with SSAs: higher expression of p-AKT or p-S6 predicted a median progression-free survival of 1 month vs 26.5 months for lower expression (P = 0.02). Conclusion: Constitutive activation of the AKT/mTOR pathway was associated with shorter time-to-progression in patients undergoing treatment with SSAs. Larger case series are needed to validate whether p-AKT(Ser473) and p-S6(Ser240/244) can be used as prognostic markers of response to therapy with SSAs.
- Therapy-induced cellular senescence induces epithelial-to-mesenchymal transition and increases invasiveness in rectal cancerPublication . Tato-Costa, Joana; Casimiro, Sandra; Pacheco, Teresa; Pires, Ricardo; Fernandes, Afonso; Alho, Irina; Pereira, Pedro; Costa, Paulo M.; Castelo, Henrique Bicha; Ferreira, João; Costa, LuisIntroduction: DNA damaging agents and ionizing radiation used in the therapy of human cancers can induce senescence of cancer cells. Senescent cells exhibit a secretory phenotype (senescence-associated secretome [SAS]) that can affect cancer cell behavior and, eventually, clinical prognosis. We assessed the effects of the SAS on the induction of epithelial-to-mesenchymal transition (EMT) in vitro and in clinical samples from patients with rectal cancer who had undergone neoadjuvant chemoradiotherapy (CRT). Materials and methods: Colorectal cancer cells (HCT 116) were induced into senescence by exposure to either 5-fluorouracil (5-FU) or doxorubicin. The senescent state was confirmed by staining for senescence-associated β-galactosidase (SA-β-Gal). The paracrine effects of SASs were assessed on proliferating HCT 116 cells. The quantified parameters were cell proliferation, invasive capacity, and induction of EMT. Senescence and EMT in clinical samples were assessed by the expression levels (reverse transcriptase-quantitative polymerase chain reaction) of genes related to senescence and EMT after laser-assisted microdissection of cancer cell clusters that stained either positive or negative for SA-β-Gal. Results: We have shown that cultured colon cancer cells induced into senescence by exposure to 5-FU exhibit a SAS capable of paracrine induction of EMT in colon and rectal cancer cell lines and increased cell invasion in vitro. Using laser-assisted microdissection, we found that in rectal cancer samples from patients treated with neoadjuvant CRT, tumor cell niches enriched for senescent cells bookmark regions of increased mRNA expression levels of EMT-related proteins (Slug, Snail, vimentin) compared with the nearby senescent-null tumor cell niches. Conclusion: We have provided, first-hand, strongly suggestive evidence that senescent cancer cells emerging in the context of neoadjuvant CRT for rectal cancer influenced the tumor microenvironment by promoting EMT by way of short-range interactions.