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BACKGROUND: The main drawback of BRAF/MEK inhibitors (BRAF/MEKi)-based targeted therapy in the management of BRAF-mutated cutaneous metastatic melanoma (MM) is the development of therapeutic resistance. We aimed to assess in this context the role of mTORC2, a signaling complex defined by the presence of the essential RICTOR subunit, regarded as an oncogenic driver in several tumor types, including MM. METHODS: After analyzing The Cancer Genome Atlas MM patients' database to explore both overall survival and molecular signatures as a function of intra-tumor RICTOR levels, we investigated the effects of RICTOR downregulation in BRAFV600E MM cell lines on their response to BRAF/MEKi. We performed proteomic screening to identify proteins modulated by changes in RICTOR expression, and Seahorse analysis to evaluate the effects of RICTOR depletion on mitochondrial respiration. The combination of BRAFi with drugs targeting proteins and processes emerged in the proteomic screening was carried out on RICTOR-deficient cells in vitro and in a xenograft setting in vivo. RESULTS: Low RICTOR levels in BRAF-mutated MM correlate with a worse clinical outcome. Gene Set Enrichment Analysis of low-RICTOR tumors display gene signatures suggestive of activation of the mitochondrial Electron Transport Chain (ETC) energy production. RICTOR-deficient BRAFV600E cells are intrinsically tolerant to BRAF/MEKi and anticipate the onset of resistance to BRAFi upon prolonged drug exposure. Moreover, in drug-naïve cells we observed a decline in RICTOR expression shortly after BRAFi exposure. In RICTOR-depleted cells, both mitochondrial respiration and expression of nicotinamide phosphoribosyltransferase (NAMPT) are enhanced, and their pharmacological inhibition restores sensitivity to BRAFi. CONCLUSIONS: Our work unveils an unforeseen tumor-suppressing role for mTORC2 in the early adaptation phase of BRAFV600E melanoma cells to targeted therapy and identifies the NAMPT-ETC axis as a potential therapeutic vulnerability of low RICTOR tumors. Importantly, our findings indicate that the evaluation of intra-tumor RICTOR levels has a prognostic value in metastatic melanoma and may help to guide therapeutic strategies in a personalized manner.
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Resistencia a Medicamentos Antineoplásicos , Alvo Mecanístico do Complexo 2 de Rapamicina , Melanoma , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas B-raf , Proteína Companheira de mTOR Insensível à Rapamicina , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Melanoma/genética , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melanoma/patologia , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteômica/métodos , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Ensaios Antitumorais Modelo de Xenoenxerto , MAP Quinase Quinase Quinases/antagonistas & inibidoresRESUMO
Epigenetic regulators play key roles in cancer and are increasingly being targeted for treatment. However, for many, little is known about mechanisms of resistance to the inhibition of these regulators. We have generated a model of resistance to inhibitors of protein arginine methyltransferase 5 (PRMT5). This study was conducted in KrasG12D;Tp53-null lung adenocarcinoma (LUAD) cell lines. Resistance to PRMT5 inhibitors (PRMT5i) arose rapidly, and barcoding experiments showed that this resulted from a drug-induced transcriptional state switch, not selection of a preexisting population. This resistant state is both stable and conserved across variants arising from distinct LUAD lines. Moreover, it brought with it vulnerabilities to other chemotherapeutics, especially the taxane paclitaxel. This paclitaxel sensitivity depended on the presence of stathmin 2 (STMN2), a microtubule regulator that is specifically expressed in the resistant state. Remarkably, STMN2 was also essential for resistance to PRMT5 inhibition. Thus, a single gene is required for both acquisition of resistance to PRMT5i and collateral sensitivity to paclitaxel in our LUAD cells. Accordingly, the combination of PRMT5i and paclitaxel yielded potent and synergistic killing of the murine LUAD cells. Importantly, the synergy between PRMT5i and paclitaxel also extended to human cancer cell lines. Finally, analysis of The Cancer Genome Atlas patient data showed that high STMN2 levels correlate with complete regression of tumors in response to taxane treatment. Collectively, this study reveals a recurring mechanism of PRMT5i resistance in LUAD and identifies collateral sensitivities that have potential clinical relevance.
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Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Paclitaxel/farmacologia , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sinergismo Farmacológico , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Camundongos , Mutação , Estatmina/genética , Estatmina/metabolismoRESUMO
BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD) biosynthesis, is up-regulated in several cancers, including metastatic melanoma (MM). The BRAF oncogene is mutated in different cancer types, among which MM and thyroid carcinoma (THCA) are prominent. Drugs targeting mutant BRAF are effective, especially in MM patients, even though resistance rapidly develops. Previous data have linked NAMPT over-expression to the acquisition of BRAF resistance, paving the way for therapeutic strategies targeting the two pathways. METHODS: Exploiting the TCGA database and a collection of MM and THCA tissue microarrays we studied the association between BRAF mutations and NAMPT expression. BRAF wild-type (wt) cell lines were genetically engineered to over-express the BRAF V600E construct to demonstrate a direct relationship between over-activation of the BRAF pathway and NAMPT expression. Responses of different cell line models to NAMPT (i)nhibitors were studied using dose-response proliferation assays. Analysis of NAMPT copy number variation was performed in the TCGA dataset. Lastly, growth and colony forming assays were used to study the tumorigenic functions of NAMPT itself. RESULTS: The first finding of this work is that tumor samples carrying BRAF-mutations over-express NAMPT, as demonstrated by analyzing the TCGA dataset, and MM and THC tissue microarrays. Importantly, BRAF wt MM and THCA cell lines modified to over-express the BRAF V600E construct up-regulated NAMPT, confirming a transcriptional regulation of NAMPT following BRAF oncogenic signaling activation. Treatment of BRAF-mutated cell lines with two different NAMPTi was followed by significant reduction of tumor growth, indicating NAMPT addiction in these cells. Lastly, we found that several tumors over-expressing the enzyme, display NAMPT gene amplification. Over-expression of NAMPT in BRAF wt MM cell line and in fibroblasts resulted in increased growth capacity, arguing in favor of oncogenic properties of NAMPT. CONCLUSIONS: Overall, the association between BRAF mutations and NAMPT expression identifies a subset of tumors more sensitive to NAMPT inhibition opening the way for novel combination therapies including NAMPTi with BRAFi/MEKi, to postpone and/or overcome drug resistance. Lastly, the over-expression of NAMPT in several tumors could be a key and broad event in tumorigenesis, substantiated by the finding of NAMPT gene amplification.
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Melanoma , Proteínas Proto-Oncogênicas B-raf , Carcinogênese/genética , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Humanos , Melanoma/patologia , Mutação/genética , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Oncogenes , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
BACKGROUND: Nutrient deprivation, hypoxia, radiotherapy and chemotherapy induce endoplasmic reticulum (ER) stress, which activates the so-called unfolded protein response (UPR). Extensive and acute ER stress directs the UPR towards activation of death-triggering pathways. Cancer cells are selected to resist mild and prolonged ER stress by activating pro-survival UPR. We recently found that drug-resistant tumor cells are simultaneously resistant to ER stress-triggered cell death. It is not known if cancer cells adapted to ER stressing conditions acquire a chemoresistant phenotype. METHODS: To investigate this issue, we generated human cancer cells clones with acquired resistance to ER stress from ER stress-sensitive and chemosensitive cells. RESULTS: ER stress-resistant cells were cross-resistant to multiple chemotherapeutic drugs: such multidrug resistance (MDR) was due to the overexpression of the plasma-membrane transporter MDR related protein 1 (MRP1). Gene profiling analysis unveiled that cells with acquired resistance to ER stress and chemotherapy share higher expression of the UPR sensor protein kinase RNA-like endoplasmic reticulum kinase (PERK), which mediated the erythroid-derived 2-like 2 (Nrf2)-driven transcription of MRP1. Disrupting PERK/Nrf2 axis reversed at the same time resistance to ER stress and chemotherapy. The inducible silencing of PERK reduced tumor growth and restored chemosensitivity in resistant tumor xenografts. CONCLUSIONS: Our work demonstrates for the first time that the adaptation to ER stress in cancer cells produces a MDR phenotype. The PERK/Nrf2/MRP1 axis is responsible for the resistance to ER stress and chemotherapy, and may represent a good therapeutic target in aggressive and resistant tumors.
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Neoplasias do Colo/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Fator 2 Relacionado a NF-E2/genética , eIF-2 Quinase/genética , Animais , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Doxorrubicina/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células HT29 , Humanos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética , Ensaios Antitumorais Modelo de Xenoenxerto , eIF-2 Quinase/antagonistas & inibidoresRESUMO
Frontline treatment and resultant cure rates in patients with advanced ovarian cancer have changed little over the past several decades. Here, we outline a multidisciplinary approach aimed at gaining novel therapeutic insights by focusing on the poorly understood minimal residual disease phase of ovarian cancer that leads to eventual incurable recurrences.
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Neoplasias Ovarianas , Humanos , Feminino , Neoplasia Residual , Neoplasias Ovarianas/tratamento farmacológico , Carcinoma Epitelial do Ovário/terapiaRESUMO
The p140Cap adaptor protein is a tumor suppressor in breast cancer associated with a favorable prognosis. Here we highlight a function of p140Cap in orchestrating local and systemic tumor-extrinsic events that eventually result in inhibition of the polymorphonuclear myeloid-derived suppressor cell function in creating an immunosuppressive tumor-promoting environment in the primary tumor, and premetastatic niches at distant sites. Integrative transcriptomic and preclinical studies unravel that p140Cap controls an epistatic axis where, through the upstream inhibition of ß-Catenin, it restricts tumorigenicity and self-renewal of tumor-initiating cells limiting the release of the inflammatory cytokine G-CSF, required for polymorphonuclear myeloid-derived suppressor cells to exert their local and systemic tumor conducive function. Mechanistically, p140Cap inhibition of ß-Catenin depends on its ability to localize in and stabilize the ß-Catenin destruction complex, promoting enhanced ß-Catenin inactivation. Clinical studies in women show that low p140Cap expression correlates with reduced presence of tumor-infiltrating lymphocytes and more aggressive tumor types in a large cohort of real-life female breast cancer patients, highlighting the potential of p140Cap as a biomarker for therapeutic intervention targeting the ß-Catenin/ Tumor-initiating cells /G-CSF/ polymorphonuclear myeloid-derived suppressor cell axis to restore an efficient anti-tumor immune response.
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Neoplasias da Mama , Feminino , Humanos , beta Catenina/metabolismo , Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Imunidade , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismoRESUMO
The alterations of metabolic pathways in cancer have been investigated for many years, beginning long before the discovery of the role of oncogenes and tumor suppressors, and the last few years have witnessed renewed interest in this topic. Large-scale molecular and clinical data on tens of thousands of samples allow us to tackle the problem from a general point of view. Here, we show that transcriptomic profiles of tumors can be exploited to define metabolic cancer subtypes, which can be systematically investigated for associations with other molecular and clinical data. We find thousands of significant associations between metabolic subtypes and molecular features such as somatic mutations, structural variants, epigenetic modifications, protein abundance and activation, and with clinical/phenotypic data, including survival probability, tumor grade, and histological types, which we make available to the community in a dedicated web resource. Our work provides a methodological framework and a rich database of statistical associations, which will contribute to the understanding of the role of metabolic alterations in cancer and to the development of precision therapeutic strategies.
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Cancer is partly a developmental disease, with malignancies named based on cell or tissue of origin. However, a systematic atlas of tumor origins is lacking. Here we map the single-cell organogenesis of 56 developmental trajectories to the transcriptomes of over 10,000 tumors across 33 cancer types. We deconvolute tumor transcriptomes into signals for individual developmental trajectories. Using these signals as inputs, we construct a developmental multilayer perceptron (D-MLP) classifier that outputs cancer origin. D-MLP (ROC-AUC: 0.974 for top prediction) outperforms benchmark classifiers. We analyze tumors from patients with cancer of unknown primary (CUP), selecting the most difficult cases in which extensive multimodal workup yielded no definitive tumor type. Interestingly, CUPs form groups distinguished by developmental trajectories, and classification reveals diagnosis for patient tumors. Our results provide an atlas of tumor developmental origins, provide a tool for diagnostic pathology, and suggest developmental classification may be a useful approach for patient tumors. SIGNIFICANCE: Here we map the developmental trajectories of tumors. We deconvolute tumor transcriptomes into signals for mammalian developmental programs and use this information to construct a deep learning classifier that outputs tumor type. We apply the classifier to CUP and reveal the developmental origins of patient tumors. See related commentary by Wang, p. 2498. This article is highlighted in the In This Issue feature, p. 2483.
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Neoplasias Primárias Desconhecidas , Humanos , Neoplasias Primárias Desconhecidas/diagnóstico , OncogenesRESUMO
Serine-threonine protein kinase B-RAF (BRAF)-mutated metastatic melanoma (MM) is a highly aggressive type of skin cancer. Treatment of MM patients using BRAF/MEK inhibitors (BRAFi/MEKi) eventually leads to drug resistance, limiting any clinical benefit. Herein, we demonstrated that the nicotinamide adenine dinucleotide (NAD)-biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) is a driving factor in BRAFi resistance development. Using stable and inducible NAMPT over-expression systems, we showed that forced NAMPT expression in MM BRAF-mutated cell lines led to increased energy production, MAPK activation, colony-formation capacity, and enhance tumorigenicity in vivo. Moreover, NAMPT over-expressing cells switched toward an invasive/mesenchymal phenotype, up-regulating expression of ZEB1 and TWIST, two transcription factors driving the epithelial to mesenchymal transition (EMT) process. Consistently, within the NAMPT-overexpressing cell line variants, we observed an increased percentage of a rare, drug-effluxing stem cell-like side population (SP) of cells, paralleled by up-regulation of ABCC1/MRP1 expression and CD133-positive cells. The direct correlation between NAMPT expression and gene set enrichments involving metastasis, invasiveness and mesenchymal/stemness properties were verified also in melanoma patients by analyzing The Cancer Genome Atlas (TCGA) datasets. On the other hand, CRISPR/Cas9 full knock-out NAMPT BRAFi-resistant MM cells are not viable, while inducible partial silencing drastically reduces tumor growth and aggressiveness. Overall, this work revealed that NAMPT over-expression is both necessary and sufficient to recapitulate the BRAFi-resistant phenotype plasticity.
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Breast cancer is a heterogeneous disease whose clinical management is very challenging. Although specific molecular features characterize breast cancer subtypes with different prognosis, the identification of specific markers predicting disease outcome within the single subtypes still lags behind. Both the non-canonical Wingless-type MMTV Integration site (WNT) and the Signal Transducer and Activator of Transcription (STAT)3 pathways are often constitutively activated in breast tumors, and both can induce the small GTPase Ras Homolog Family Member U RhoU. Here we show that RhoU transcription can be triggered by both canonical and non-canonical WNT ligands via the activation of c-JUN N-terminal kinase (JNK) and the recruitment of the Specificity Protein 1 (SP1) transcription factor to the RhoU promoter, identifying for the first time SP1 as a JNK-dependent mediator of WNT signaling. RhoU down-regulation by silencing or treatment with JNK, SP1 or STAT3 inhibitors leads to impaired migration and invasion in basal-like MDA-MB-231 and BT-549 cells, suggesting that STAT3 and SP1 can cooperate to induce high RhoU expression and enhance breast cancer cells migration. Moreover, in vivo concomitant binding of STAT3 and SP1 defines a subclass of genes belonging to the non-canonical WNT and the Interleukin (IL)-6/STAT3 pathways and contributing to breast cancer aggressiveness, suggesting the relevance of developing novel targeted therapies combining inhibitors of the STAT3 and WNT pathways or of their downstream mediators.
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This corrects the article DOI: 10.1038/ncomms14797.
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Tumor growth and metastatic dissemination are complex multistep processes. They clearly depend on the intrinsic behavior of cancer cells, but are remarkably influenced by a variety of stromal cells present in the tumor microenvironment, which include those implicated in tumor angiogenesis, as well as bone marrow-derived cells recruited from the circulation. Moreover, multiple molecular signals exchanged between cancer cells and non-neoplastic stromal cells control tumor growth and metastasis; notably, members of the semaphorin family are emerging players in this scenario.In vivo tumor models represent the best setting for studying metastatic tumor progression, as they allow recapitulating the contribution of multiple cell types and signaling molecules in a complex tissue context, subject to pathophysiological local and systemic responses, such as metabolic changes, hypoxia, necrosis, fibrosis, inflammation, and cytokine release. Here, we describe some experimental approaches based on murine models to study the role of semaphorin signaling in tumor growth and metastatic progression in vivo.
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Modelos Animais de Doenças , Neoplasias/patologia , Semaforinas/metabolismo , Transdução de Sinais , Animais , Camundongos , Metástase NeoplásicaRESUMO
NF-κB is a transcription factor involved in the regulation of multiple physiological and pathological cellular processes, including inflammation, cell survival, proliferation, and cancer cell metastasis. NF-κB is frequently hyperactivated in several cancers, including triple-negative breast cancer. Here we show that NF-κB activation in breast cancer cells depends on the presence of the CHORDC1 gene product Morgana, a previously unknown component of the IKK complex and essential for IκBα substrate recognition. Morgana silencing blocks metastasis formation in breast cancer mouse models and this phenotype is reverted by IκBα downregulation. High Morgana expression levels in cancer cells decrease recruitment of natural killer cells in the first phases of tumor growth and induce the expression of cytokines able to attract neutrophils in the primary tumor, as well as in the pre-metastatic lungs, fueling cancer metastasis. In accordance, high Morgana levels positively correlate with NF-κB target gene expression and poor prognosis in human patients.
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Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Animais , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Quinase I-kappa B/genética , Camundongos , Camundongos Endogâmicos BALB C , Chaperonas Moleculares , NF-kappa B/genética , Metástase Neoplásica , Proteínas de Ligação a Fosfato , Transdução de SinaisRESUMO
The docking protein p140Cap negatively regulates tumour cell features. Its relevance on breast cancer patient survival, as well as its ability to counteract relevant cancer signalling pathways, are not fully understood. Here we report that in patients with ERBB2-amplified breast cancer, a p140Cap-positive status associates with a significantly lower probability of developing a distant event, and a clear difference in survival. p140Cap dampens ERBB2-positive tumour cell progression, impairing tumour onset and growth in the NeuT mouse model, and counteracting epithelial mesenchymal transition, resulting in decreased metastasis formation. One major mechanism is the ability of p140Cap to interfere with ERBB2-dependent activation of Rac GTPase-controlled circuitries. Our findings point to a specific role of p140Cap in curbing the aggressiveness of ERBB2-amplified breast cancers and suggest that, due to its ability to impinge on specific molecular pathways, p140Cap may represent a predictive biomarker of response to targeted anti-ERBB2 therapies.
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Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Neoplasias da Mama/metabolismo , Receptor ErbB-2/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Metástase Neoplásica , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Receptor ErbB-2/genética , Proteínas rac de Ligação ao GTP/genéticaRESUMO
The expression of Prostate Specific-Membrane Antigen (PSMA) increases in high-grade prostate carcinoma envisaging a role in growth and progression. We show here that clustering PSMA at LNCaP or PC3-PSMA cell membrane activates AKT and MAPK pathways thus promoting proliferation and survival. PSMA activity was dependent on the assembly of a macromolecular complex including filamin A, beta1 integrin, p130CAS, c-Src and EGFR. Within this complex beta1 integrin became activated thereby inducing a c-Src-dependent EGFR phosphorylation at Y1086 and Y1173 EGF-independent residues. Silencing or blocking experiments with drugs demonstrated that all the complex components were required for full PSMA-dependent promotion of cell growth and/or survival in 3D culture, but that p130CAS and EGFR exerted a major role. All PSMA complex components were found assembled in multiple samples of two high-grade prostate carcinomas and associated with EGFR phosphorylation at Y1086. The expression of p130CAS and pEGFRY1086 was thus analysed by tissue micro array in 16 castration-resistant prostate carcinomas selected from 309 carcinomas and stratified from GS 3+4 to GS 5+5. Patients with Gleason Score ≤5 resulted negative whereas those with GS≥5 expressed p130CAS and pEGFRY1086 in 75% and 60% of the cases, respectively.Collectively, our results demonstrate for the first time that PSMA recruits a functionally active complex which is present in high-grade patients. In addition, two components of this complex, p130CAS and the novel pEGFRY1086, correlate with progression in castration-resistant patients and could be therefore useful in therapeutic or surveillance strategies of these patients.