Germline mutations in the spindle assembly checkpoint genes BUB1 and BUB3 are infrequent in familial colorectal cancer and polyposis

Germline mutations in BUB1 and BUB3 have been reported to increase the risk of developing colorectal cancer (CRC) at young age, in presence of variegated aneuploidy and reminiscent dysmorphic traits of mosaic variegated aneuploidy syndrome. We performed a mutational analysis of BUB1 and BUB3 in 456 uncharacterized mismatch repair-proficient hereditary non-polyposis CRC families and 88 polyposis cases. Four novel or rare germline variants, one splice-site and three missense, were identified in four families. Neither variegated aneuploidy nor dysmorphic traits were observed in carriers. Evident functional effects in the heterozygous form were observed for c.1965-1G>A, but not for c.2296G>A (p.E766K), in spite of the positive co-segregation in the family. BUB1 c.2473C>T (p.P825S) and BUB3 c.77C>T (p.T26I) remained as variants of uncertain significance. As of today, the rarity of functionally relevant mutations identified in familial and/or early onset series does not support the inclusion of BUB1 and BUB3 testing in routine genetic diagnostics of familial CRC

HOPE (SOLTI-1903) breast cancer study: real-world, patient-centric, clinical practice study to assess the impact of genomic data on next treatment decision-choice in patients with locally advanced or metastatic breast cancer

Background Metastatic breast cancer (mBC) causes nearly all BC-related deaths. Next-generation sequencing (NGS) technologies allow for the application of personalized medicine using targeted therapies that could improve patients' outcomes. However, NGS is not routinely used in the clinical practice and its cost induces access-inequity among patients. We hypothesized that promoting active patient participation in the management of their disease offering access to NGS testing and to the subsequent medical interpretation and recommendations provided by a multidisciplinary molecular advisory board (MAB) could contribute to progressively overcome this challenge. We designed HOPE (SOLTI-1903) breast cancer trial, a study where patients voluntarily lead their inclusion through a digital tool (DT). The main objectives of HOPE study are to empower mBC patients, gather real-world data on the use of molecular information in the management of mBC and to generate evidence to assess the clinical utility for healthcare systems.Trial design After self-registration through the DT, the study team validates eligibility criteria and assists patients with mBC in the subsequent steps. Patients get access to the information sheet and sign the informed consent form through an advanced digital signature. Afterwards, they provide the most recent (preferably) metastatic archival tumor sample for DNA-sequencing and a blood sample obtained at the time of disease progression for ctDNA analysis. Paired results are reviewed by the MAB, considering patient's medical history. The MAB provides a further interpretation of molecular results and potential treatment recommendations, including ongoing clinical trials and further (germline) genetic testing. Participants self-document their treatment and disease evolution for the next 2 years. Patients are encouraged to involve their physicians in the study. HOPE also includes a patient empowerment program with educational workshops and videos about mBC and precision medicine in oncology. The primary endpoint of the study was to describe the feasibility of a patient-centric precision oncology program in mBC patients when a comprehensive genomic profile is available to decide on a subsequent line of treatment

New roles for Snail1 -expressing CAF during primary tumor progression and secondary niche colonization

Snail1 is the master regulator of the Epithelial-to-Mesenchymal Transition (EMT) and is also crucial for fibroblast activation upon TGFβ signaling. In cancer, Snail1 expression in primary tumors correlates with the appearance of metastasis. We have previously shown that Snail1-expressing cancer-associated fibroblasts (CAF) enhance metastasis. Here we demonstrate that Snail1-expressing CAF attenuate the anti-tumor effector immune response. We observed that Snail1-expressing CAF determine macrophages to present a pro-tumor phenotype in vitro and in the in vivo model of breast cancer MMTV-PyMT what enhanced tumor progression. Moreover, in the context of metastasis, we show that Snail1-dependent TGFβ-induced activation of liver fibroblasts is determinant for colorectal cancer colonization of the organ. Snail1-expressing CAF determine cancer cell evasion of the adaptive anti-tumor effector immunity. In consequence, the absence of Snail1 prevents CAF activation in newly formed metastases and allows immune rejection. These new roles for Snail1-expressing CAF during primary tumor progression and secondary niche colonization increase the relevance of Snail1 protein in oncology.Snail1 és el principal regulador de la Transició Epiteli-Mesènquima (EMT, per les sigles en anglès) i també resulta crucial per a l’activació dels fibroblasts en presència de TGFβ. En càncer, l’expressió de Snail1 en tumors primerencs correlaciona amb l’aparició de metàstasis. Prèviament, el nostre grup va demostrar que els fibroblasts actius associats a tumors (CAF) que expressen Snail1 desencadenen metàstasis. Aquí demostrem que els CAF que expressen Snail1 atenuen la resposta immunitària efectora anti-tumoral. Hem observant que els CAF que expressen Snail1 determinen un fenotip pro-tumoral en macròfags in vitro i també in vivo fent servir el model de càncer de mama MMTV-PyMT on la progressió tumoral es veia accentuada. En el context metastàtic, mostrem que l’activació dels fibroblasts del fetge induïda per TGFβ és determinant per a la colonització d’aquest òrgan per part del càncer colorectal. Els CAF que expressen Snail1 determinen que les cèl·lules del càncer evadeixin la resposta immunitària anti-tumoral. En conseqüència i malgrat la senyalització per TGFβ, l’absència de Snail1 anul·là la presència de CAF actius en les noves metàstasis i foren rebutjades. Aquestes noves funcions dels CAF que expressen Snail1 durant la progressió del tumor primari i la colonització d’un nínxol secundari incrementen la rellevància de la proteïna Snail1 en oncologia.Programa de doctorat en Biomedicin

New roles for Snail1 -expressing CAF during primary tumor progression and secondary niche colonization

Snail1 is the master regulator of the Epithelial-to-Mesenchymal Transition (EMT) and is also crucial for fibroblast activation upon TGFβ signaling. In cancer, Snail1 expression in primary tumors correlates with the appearance of metastasis. We have previously shown that Snail1-expressing cancer-associated fibroblasts (CAF) enhance metastasis. Here we demonstrate that Snail1-expressing CAF attenuate the anti-tumor effector immune response. We observed that Snail1-expressing CAF determine macrophages to present a pro-tumor phenotype in vitro and in the in vivo model of breast cancer MMTV-PyMT what enhanced tumor progression. Moreover, in the context of metastasis, we show that Snail1-dependent TGFβ-induced activation of liver fibroblasts is determinant for colorectal cancer colonization of the organ. Snail1-expressing CAF determine cancer cell evasion of the adaptive anti-tumor effector immunity. In consequence, the absence of Snail1 prevents CAF activation in newly formed metastases and allows immune rejection. These new roles for Snail1-expressing CAF during primary tumor progression and secondary niche colonization increase the relevance of Snail1 protein in oncology.Snail1 és el principal regulador de la Transició Epiteli-Mesènquima (EMT, per les sigles en anglès) i també resulta crucial per a l’activació dels fibroblasts en presència de TGFβ. En càncer, l’expressió de Snail1 en tumors primerencs correlaciona amb l’aparició de metàstasis. Prèviament, el nostre grup va demostrar que els fibroblasts actius associats a tumors (CAF) que expressen Snail1 desencadenen metàstasis. Aquí demostrem que els CAF que expressen Snail1 atenuen la resposta immunitària efectora anti-tumoral. Hem observant que els CAF que expressen Snail1 determinen un fenotip pro-tumoral en macròfags in vitro i també in vivo fent servir el model de càncer de mama MMTV-PyMT on la progressió tumoral es veia accentuada. En el context metastàtic, mostrem que l’activació dels fibroblasts del fetge induïda per TGFβ és determinant per a la colonització d’aquest òrgan per part del càncer colorectal. Els CAF que expressen Snail1 determinen que les cèl·lules del càncer evadeixin la resposta immunitària anti-tumoral. En conseqüència i malgrat la senyalització per TGFβ, l’absència de Snail1 anul·là la presència de CAF actius en les noves metàstasis i foren rebutjades. Aquestes noves funcions dels CAF que expressen Snail1 durant la progressió del tumor primari i la colonització d’un nínxol secundari incrementen la rellevància de la proteïna Snail1 en oncologia

Analyzing the role of cancer‐associated fibroblast activation on macrophage polarization

Snail1 is a transcriptional factor required for cancer‐associated fibroblast (CAF) activation, and mainly detected in CAFs in human tumors. In the mouse mammary tumor virus‐polyoma middle tumor‐antigen (MMTV‐PyMT) model of murine mammary gland tumors, Snai1 gene deletion, besides increasing tumor‐free lifespan, altered macrophage differentiation, with fewer expressing low levels of MHC class II. Snail1 was not expressed in macrophages, and in vitro polarization with interleukin‐4 (IL4) or interferon‐γ (IFNγ) was not altered by Snai1 gene depletion. We verified that CAF activation modified polarization of naïve bone‐marrow‐derived macrophages (BMDMΦs). When BMDMΦs were incubated with Snail1‐expressing (active) CAFs or with conditioned medium derived from these cells, they exhibited a lower cytotoxic capability than when incubated with Snail1‐deleted (inactive) CAFs. Gene expression analysis of BMDMΦs polarized by conditioned medium from wild‐type or Snai1‐deleted CAFs revealed that active CAFs differentially stimulated a complex combination of genes comprising genes that are normally induced by IL4, downregulated by IFNγ, or not altered during the two canonical differentiations. Levels of RNAs relating to this CAF‐induced alternative polarization were sensitive to inhibitors of factors specifically released by active CAFs, such as prostaglandin E2 and TGFβ. Finally, CAF‐polarized macrophages promoted the activation of the immunosuppressive regulatory T cells (T‐regs). Our results imply that an active CAF‐rich tumor microenvironment induces the polarization of macrophages to an immunosuppressive phenotype, preventing the macrophage cytotoxic activity on tumor cells and enhancing the activation of T‐reg cells

Analyzing the role of cancer-associated fibroblast activation on macrophage polarization

Snail1 is a transcriptional factor required for cancer-associated fibroblast (CAF) activation, and mainly detected in CAFs in human tumors. In the mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT) model of murine mammary gland tumors, Snai1 gene deletion, besides increasing tumor-free lifespan, altered macrophage differentiation, with fewer expressing low levels of MHC class II. Snail1 was not expressed in macrophages, and in vitro polarization with interleukin-4 (IL4) or interferon-γ (IFNγ) was not altered by Snai1 gene depletion. We verified that CAF activation modified polarization of naïve bone-marrow-derived macrophages (BMDMΦs). When BMDMΦs were incubated with Snail1-expressing (active) CAFs or with conditioned medium derived from these cells, they exhibited a lower cytotoxic capability than when incubated with Snail1-deleted (inactive) CAFs. Gene expression analysis of BMDMΦs polarized by conditioned medium from wild-type or Snai1-deleted CAFs revealed that active CAFs differentially stimulated a complex combination of genes comprising genes that are normally induced by IL4, downregulated by IFNγ, or not altered during the two canonical differentiations. Levels of RNAs relating to this CAF-induced alternative polarization were sensitive to inhibitors of factors specifically released by active CAFs, such as prostaglandin E2 and TGFβ. Finally, CAF-polarized macrophages promoted the activation of the immunosuppressive regulatory T cells (T-regs). Our results imply that an active CAF-rich tumor microenvironment induces the polarization of macrophages to an immunosuppressive phenotype, preventing the macrophage cytotoxic activity on tumor cells and enhancing the activation of T-reg cells.This study was funded by the grant PID2019-104698RB-I00 funded by MCIN/AEI/10.13039/501100011033 to AGdH. We also acknowledge support from the Instituto Carlos III/FEDER (PIE15/00008; PT17/0015/0011). MB-O and RO-S were recipients of FPI predoctoral fellowships from Ministerio de Educación. The present addresses for MB-O, RM and RO-S are, respectively, Werfen, Mycosynvac, and SOLTI, Barcelona, Spain

TGFβ-activated USP27X deubiquitinase regulates cell migration and chemoresistance via stabilization of snail1

In cancer cells, epithelial-to-mesenchymal transition (EMT) is controlled by Snail1, a transcriptional factor also required for the activation of cancer-associated fibroblasts (CAF). Snail1 is short-lived in normal epithelial cells as a consequence of its coordinated and continuous ubiquitination by several F-box-specific E3 ligases, but its degradation is prevented in cancer cells and in activated fibroblasts. Here, we performed an siRNA screen and identified USP27X as a deubiquitinase that increases Snail1 stability. Expression of USP27X in breast and pancreatic cancer cell lines and tumors positively correlated with Snail1 expression levels. Accordingly, downregulation of USP27X decreased Snail1 protein in several tumor cell lines. USP27X depletion impaired Snail1-dependent cell migration and invasion and metastasis formation and increased cellular sensitivity to cisplatin. USP27X was upregulated by TGFβ during EMT and was required for TGFβ-induced expression of Snail1 and other mesenchymal markers in epithelial cells and CAF. In agreement with this, depletion of USP27X prevented TGFβ-induced EMT and fibroblast activation. Collectively, these results indicate that USP27X is an essential protein controlling Snail1 expression and function and may serve as a target for inhibition of Snail1-dependent tumoral invasion and chemoresistance. SIGNIFICANCE: These findings show that inhibition of USP27X destabilizes Snail1 to impair EMT and renders tumor cells sensitive to chemotherapy, thus opening new strategies for the inhibition of Snail1 expression and its protumoral actions.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/1/33/F1.large.jpg.This study was funded by grants awarded by Ministerio de Economía y Competitividad (MINECO) and Fondo Europeo de Desarrollo Regional-FEDER to A. García de Herreros (SAF2013-48849-C2-1-R and SAF2016-76461-R) and to V.M. Díaz (SAF2013-48849-C2-2-R). Research at the A. García de Herreros lab is supported by funds from the Fundación Científica de la Asociación Española contra el Cáncer and from the Instituto de Salud Carlos III (PIE15/00008). Research at the B.S. Atanassov lab is supported by the Rosswell Park Cancer Institute and NCI grant P30CA016056. Research at the J. Arribas lab is supported by funds from the Breast Cancer Research Foundation (BCRF-17-008) and Instituto de Salud Carlos III (PI16/00253)

TGFβ-activated USP27X deubiquitinase regulates cell migration and chemoresistance via stabilization of snail1

In cancer cells, epithelial-to-mesenchymal transition (EMT) is controlled by Snail1, a transcriptional factor also required for the activation of cancer-associated fibroblasts (CAF). Snail1 is short-lived in normal epithelial cells as a consequence of its coordinated and continuous ubiquitination by several F-box-specific E3 ligases, but its degradation is prevented in cancer cells and in activated fibroblasts. Here, we performed an siRNA screen and identified USP27X as a deubiquitinase that increases Snail1 stability. Expression of USP27X in breast and pancreatic cancer cell lines and tumors positively correlated with Snail1 expression levels. Accordingly, downregulation of USP27X decreased Snail1 protein in several tumor cell lines. USP27X depletion impaired Snail1-dependent cell migration and invasion and metastasis formation and increased cellular sensitivity to cisplatin. USP27X was upregulated by TGFβ during EMT and was required for TGFβ-induced expression of Snail1 and other mesenchymal markers in epithelial cells and CAF. In agreement with this, depletion of USP27X prevented TGFβ-induced EMT and fibroblast activation. Collectively, these results indicate that USP27X is an essential protein controlling Snail1 expression and function and may serve as a target for inhibition of Snail1-dependent tumoral invasion and chemoresistance. SIGNIFICANCE: These findings show that inhibition of USP27X destabilizes Snail1 to impair EMT and renders tumor cells sensitive to chemotherapy, thus opening new strategies for the inhibition of Snail1 expression and its protumoral actions.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/1/33/F1.large.jpg.This study was funded by grants awarded by Ministerio de Economía y Competitividad (MINECO) and Fondo Europeo de Desarrollo Regional-FEDER to A. García de Herreros (SAF2013-48849-C2-1-R and SAF2016-76461-R) and to V.M. Díaz (SAF2013-48849-C2-2-R). Research at the A. García de Herreros lab is supported by funds from the Fundación Científica de la Asociación Española contra el Cáncer and from the Instituto de Salud Carlos III (PIE15/00008). Research at the B.S. Atanassov lab is supported by the Rosswell Park Cancer Institute and NCI grant P30CA016056. Research at the J. Arribas lab is supported by funds from the Breast Cancer Research Foundation (BCRF-17-008) and Instituto de Salud Carlos III (PI16/00253)

Germline mutations in the spindle assembly checkpoint genes BUB1 and BUB3 are infrequent in familial colorectal cancer and polyposis

Germline mutations in BUB1 and BUB3 have been reported to increase the risk of developing colorectal cancer (CRC) at young age, in presence of variegated aneuploidy and reminiscent dysmorphic traits of mosaic variegated aneuploidy syndrome. We performed a mutational analysis of BUB1 and BUB3 in 456 uncharacterized mismatch repair-proficient hereditary non-polyposis CRC families and 88 polyposis cases. Four novel or rare germline variants, one splice-site and three missense, were identified in four families. Neither variegated aneuploidy nor dysmorphic traits were observed in carriers. Evident functional effects in the heterozygous form were observed for c.1965-1G>A, but not for c.2296G>A (p.E766K), in spite of the positive co-segregation in the family. BUB1 c.2473C>T (p.P825S) and BUB3 c.77C>T (p.T26I) remained as variants of uncertain significance. As of today, the rarity of functionally relevant mutations identified in familial and/or early onset series does not support the inclusion of BUB1 and BUB3 testing in routine genetic diagnostics of familial CRC

Germline mutations in the spindle assembly checkpoint genes BUB1 and BUB3 are infrequent in familial colorectal cancer and polyposis

Germline mutations in BUB1 and BUB3 have been reported to increase the risk of developing colorectal cancer (CRC) at young age, in presence of variegated aneuploidy and reminiscent dysmorphic traits of mosaic variegated aneuploidy syndrome. We performed a mutational analysis of BUB1 and BUB3 in 456 uncharacterized mismatch repair-proficient hereditary non-polyposis CRC families and 88 polyposis cases. Four novel or rare germline variants, one splice-site and three missense, were identified in four families. Neither variegated aneuploidy nor dysmorphic traits were observed in carriers. Evident functional effects in the heterozygous form were observed for c.1965-1G>A, but not for c.2296G>A (p.E766K), in spite of the positive co-segregation in the family. BUB1 c.2473C>T (p.P825S) and BUB3 c.77C>T (p.T26I) remained as variants of uncertain significance. As of today, the rarity of functionally relevant mutations identified in familial and/or early onset series does not support the inclusion of BUB1 and BUB3 testing in routine genetic diagnostics of familial CRC