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John M Carethers - One of the best experts on this subject based on the ideXlab platform.
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Flanking nucleotide specificity for DNA mismatch repair-deficient frameshifts within Activin Receptor 2 (ACVR2).
Mutation research, 2011Co-Authors: Heekyung Chung, Joy Chaudhry, Jenny F Lai, Dennis J Young, John M CarethersAbstract:We previously demonstrated that exonic selectivity for frameshift mutation (exon 10 over exon 3) of ACVR2 in mismatch repair (MMR)-deficient cells is partially determined by 6 nucleotides flanking 5' and 3' of each microsatellite. Substitution of flanking nucleotides surrounding the exon 10 microsatellite with those surrounding the exon 3 microsatellite greatly diminished heteroduplex (A(7)/T(8)) and full (A(7)/T(7)) mutation, while substitution of flanking nucleotides from exon 3 with those from exon 10 enhanced frameshift mutation. We hypothesized that specific individual nucleotide(s) within these flanking sequences control ACVR2 frameshift mutation rates. Only the 3rd nucleotide 5' of the microsatellite, and 3rd, 4th, and 5th nucleotides 3' of the microsatellite were altered from the native flanking sequences and these locations were individually altered (sites A, B, C, and D, respectively). Constructs were cloned +1bp out-of-frame of EGFP, allowing a -1bp frameshift to express EGFP. Plasmids were stably transfected into MMR-deficient cells. Non-fluorescent cells were sorted, cultured for 35 days, and harvested for flow cytometry and DNA-sequencing. Site A (C to T) and B (G to C) in ACVR2 exon 10 decreased both heteroduplex and full mutant as much as the construct containing all 4 alterations. For ACVR2 exon 3, site A (T to C), C (A to G), and D (G to C) are responsible for increased heteroduplex formation, whereas site D is responsible for full mutant formation by ACVR2 exon 10 flanking sequences. Exonic selectivity for frameshift mutation within ACVR2's sequence context is specifically controlled by individual nucleotides flanking each microsatellite.
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224 Specific Nucleotides That Flank Coding Microsatellites of Activin Receptor 2 (ACVR2) Determine Exonic Selectivity and Control Frameshift Mutation Rates in Defective DNA Mismatch Repair (MMR)
Gastroenterology, 2010Co-Authors: Heekyung Chung, Jenny F Lai, Dennis J Young, Joy Holmstrom, John M CarethersAbstract:Carcinoembryonic antigen (CEA) is a tumor marker for the clinical management of colorectal cancer (CRC). The elevated blood levels of CEA are associated with metastasis and poor prognosis in CRC. There is mounting evidence that CEA enhances the metastatic potential of cancer cells. CEA increases the ability of weakly metastatic CRC to colonize the liver and to develop spontaneous hematogeneous liver and lung metastases. CEA expression has also been related with resistance to cytotoxic chemotherapy and to anoikis, a form of apoptosis caused by detachment from cell matrix. Yet the mechanism of CEA mediated metastasis is only partially understood. The TGF-β (transforming growth factor beta) signaling pathway contributes to tumorigenesis by controlling several biological processes, including cell proliferation, differentiation, migration and apoptosis. It has been reported that TGF-β regulates CEA transcription and secretion, however, little is known about the effects of CEA on TGFβ signaling. Aims: Based on the above facts, we focused on the influence of CEA on the TGF-β signaling in both normal cells and colorectal cancer cells. Results: Our preliminary data showed that CEA directly interacted with TGF-β Receptors. Overexpression of CEA blocked TGF-β induced SMAD3 phosphorylation, SMAD3 translocation to nuclear and the downregulation of c-myc transcription. Targeting CEA with anti-CEA antibody rescued TGFβ response in CRC cell lines with elevated CEA expression, thereby restoring the inhibitory effects of TGF-β on the proliferation of these cancer cells. Finally, in animal experiment, we found that CEA enhanced survival of colorectal cancer cell in both local colonization and liver metastasis. Conclusion: Since CEA is a well-characterized tumor-associated antigen that is frequently overexpressed in tumors, specific antibodies targeting CEA have been developed as a novel therapeutic approach for treatment of tumors expressing CEA on their surface. Based on our study, it may be helpful to combine CEA antibody and TGF-β to inhibit cancer cell proliferation and metastasis in some cases.
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841 Mechanism for Selectivity of Specific Exonic Activin Receptor 2 (ACVR2) Frameshift Mutation in Colorectal Cells with Defective DNA Mismatch Repair (MMR)
Gastroenterology, 2009Co-Authors: Heekyung Chung, Dennis J Young, Claudia Lopez, Betty L. Cabrera, Deena Ream-robinson, John M CarethersAbstract:Preoperative Biliary Drainage Versus Direct Operation for Pancreatic Tumors Causing Obstructive Jaundice Niels Anthony Van Der Gaag, Erik Rauws, Casper H. van Eijck, Marco Bruno, Erwin van der Harst, Josephus J. Gerritsen, J. W. M. Greve, Michael F. Gerhards, Ignace H. de Hingh, Jean H. Klinkenbijl, Chung Y. Nio, Steve M. de Castro, Olivier R. Busch, Thomas M. Van Gulik, Patrick M. Bossuyt, Dirk J. Gouma
Heekyung Chung - One of the best experts on this subject based on the ideXlab platform.
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Flanking nucleotide specificity for DNA mismatch repair-deficient frameshifts within Activin Receptor 2 (ACVR2).
Mutation research, 2011Co-Authors: Heekyung Chung, Joy Chaudhry, Jenny F Lai, Dennis J Young, John M CarethersAbstract:We previously demonstrated that exonic selectivity for frameshift mutation (exon 10 over exon 3) of ACVR2 in mismatch repair (MMR)-deficient cells is partially determined by 6 nucleotides flanking 5' and 3' of each microsatellite. Substitution of flanking nucleotides surrounding the exon 10 microsatellite with those surrounding the exon 3 microsatellite greatly diminished heteroduplex (A(7)/T(8)) and full (A(7)/T(7)) mutation, while substitution of flanking nucleotides from exon 3 with those from exon 10 enhanced frameshift mutation. We hypothesized that specific individual nucleotide(s) within these flanking sequences control ACVR2 frameshift mutation rates. Only the 3rd nucleotide 5' of the microsatellite, and 3rd, 4th, and 5th nucleotides 3' of the microsatellite were altered from the native flanking sequences and these locations were individually altered (sites A, B, C, and D, respectively). Constructs were cloned +1bp out-of-frame of EGFP, allowing a -1bp frameshift to express EGFP. Plasmids were stably transfected into MMR-deficient cells. Non-fluorescent cells were sorted, cultured for 35 days, and harvested for flow cytometry and DNA-sequencing. Site A (C to T) and B (G to C) in ACVR2 exon 10 decreased both heteroduplex and full mutant as much as the construct containing all 4 alterations. For ACVR2 exon 3, site A (T to C), C (A to G), and D (G to C) are responsible for increased heteroduplex formation, whereas site D is responsible for full mutant formation by ACVR2 exon 10 flanking sequences. Exonic selectivity for frameshift mutation within ACVR2's sequence context is specifically controlled by individual nucleotides flanking each microsatellite.
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224 Specific Nucleotides That Flank Coding Microsatellites of Activin Receptor 2 (ACVR2) Determine Exonic Selectivity and Control Frameshift Mutation Rates in Defective DNA Mismatch Repair (MMR)
Gastroenterology, 2010Co-Authors: Heekyung Chung, Jenny F Lai, Dennis J Young, Joy Holmstrom, John M CarethersAbstract:Carcinoembryonic antigen (CEA) is a tumor marker for the clinical management of colorectal cancer (CRC). The elevated blood levels of CEA are associated with metastasis and poor prognosis in CRC. There is mounting evidence that CEA enhances the metastatic potential of cancer cells. CEA increases the ability of weakly metastatic CRC to colonize the liver and to develop spontaneous hematogeneous liver and lung metastases. CEA expression has also been related with resistance to cytotoxic chemotherapy and to anoikis, a form of apoptosis caused by detachment from cell matrix. Yet the mechanism of CEA mediated metastasis is only partially understood. The TGF-β (transforming growth factor beta) signaling pathway contributes to tumorigenesis by controlling several biological processes, including cell proliferation, differentiation, migration and apoptosis. It has been reported that TGF-β regulates CEA transcription and secretion, however, little is known about the effects of CEA on TGFβ signaling. Aims: Based on the above facts, we focused on the influence of CEA on the TGF-β signaling in both normal cells and colorectal cancer cells. Results: Our preliminary data showed that CEA directly interacted with TGF-β Receptors. Overexpression of CEA blocked TGF-β induced SMAD3 phosphorylation, SMAD3 translocation to nuclear and the downregulation of c-myc transcription. Targeting CEA with anti-CEA antibody rescued TGFβ response in CRC cell lines with elevated CEA expression, thereby restoring the inhibitory effects of TGF-β on the proliferation of these cancer cells. Finally, in animal experiment, we found that CEA enhanced survival of colorectal cancer cell in both local colonization and liver metastasis. Conclusion: Since CEA is a well-characterized tumor-associated antigen that is frequently overexpressed in tumors, specific antibodies targeting CEA have been developed as a novel therapeutic approach for treatment of tumors expressing CEA on their surface. Based on our study, it may be helpful to combine CEA antibody and TGF-β to inhibit cancer cell proliferation and metastasis in some cases.
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841 Mechanism for Selectivity of Specific Exonic Activin Receptor 2 (ACVR2) Frameshift Mutation in Colorectal Cells with Defective DNA Mismatch Repair (MMR)
Gastroenterology, 2009Co-Authors: Heekyung Chung, Dennis J Young, Claudia Lopez, Betty L. Cabrera, Deena Ream-robinson, John M CarethersAbstract:Preoperative Biliary Drainage Versus Direct Operation for Pancreatic Tumors Causing Obstructive Jaundice Niels Anthony Van Der Gaag, Erik Rauws, Casper H. van Eijck, Marco Bruno, Erwin van der Harst, Josephus J. Gerritsen, J. W. M. Greve, Michael F. Gerhards, Ignace H. de Hingh, Jean H. Klinkenbijl, Chung Y. Nio, Steve M. de Castro, Olivier R. Busch, Thomas M. Van Gulik, Patrick M. Bossuyt, Dirk J. Gouma
Dennis J Young - One of the best experts on this subject based on the ideXlab platform.
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Flanking nucleotide specificity for DNA mismatch repair-deficient frameshifts within Activin Receptor 2 (ACVR2).
Mutation research, 2011Co-Authors: Heekyung Chung, Joy Chaudhry, Jenny F Lai, Dennis J Young, John M CarethersAbstract:We previously demonstrated that exonic selectivity for frameshift mutation (exon 10 over exon 3) of ACVR2 in mismatch repair (MMR)-deficient cells is partially determined by 6 nucleotides flanking 5' and 3' of each microsatellite. Substitution of flanking nucleotides surrounding the exon 10 microsatellite with those surrounding the exon 3 microsatellite greatly diminished heteroduplex (A(7)/T(8)) and full (A(7)/T(7)) mutation, while substitution of flanking nucleotides from exon 3 with those from exon 10 enhanced frameshift mutation. We hypothesized that specific individual nucleotide(s) within these flanking sequences control ACVR2 frameshift mutation rates. Only the 3rd nucleotide 5' of the microsatellite, and 3rd, 4th, and 5th nucleotides 3' of the microsatellite were altered from the native flanking sequences and these locations were individually altered (sites A, B, C, and D, respectively). Constructs were cloned +1bp out-of-frame of EGFP, allowing a -1bp frameshift to express EGFP. Plasmids were stably transfected into MMR-deficient cells. Non-fluorescent cells were sorted, cultured for 35 days, and harvested for flow cytometry and DNA-sequencing. Site A (C to T) and B (G to C) in ACVR2 exon 10 decreased both heteroduplex and full mutant as much as the construct containing all 4 alterations. For ACVR2 exon 3, site A (T to C), C (A to G), and D (G to C) are responsible for increased heteroduplex formation, whereas site D is responsible for full mutant formation by ACVR2 exon 10 flanking sequences. Exonic selectivity for frameshift mutation within ACVR2's sequence context is specifically controlled by individual nucleotides flanking each microsatellite.
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224 Specific Nucleotides That Flank Coding Microsatellites of Activin Receptor 2 (ACVR2) Determine Exonic Selectivity and Control Frameshift Mutation Rates in Defective DNA Mismatch Repair (MMR)
Gastroenterology, 2010Co-Authors: Heekyung Chung, Jenny F Lai, Dennis J Young, Joy Holmstrom, John M CarethersAbstract:Carcinoembryonic antigen (CEA) is a tumor marker for the clinical management of colorectal cancer (CRC). The elevated blood levels of CEA are associated with metastasis and poor prognosis in CRC. There is mounting evidence that CEA enhances the metastatic potential of cancer cells. CEA increases the ability of weakly metastatic CRC to colonize the liver and to develop spontaneous hematogeneous liver and lung metastases. CEA expression has also been related with resistance to cytotoxic chemotherapy and to anoikis, a form of apoptosis caused by detachment from cell matrix. Yet the mechanism of CEA mediated metastasis is only partially understood. The TGF-β (transforming growth factor beta) signaling pathway contributes to tumorigenesis by controlling several biological processes, including cell proliferation, differentiation, migration and apoptosis. It has been reported that TGF-β regulates CEA transcription and secretion, however, little is known about the effects of CEA on TGFβ signaling. Aims: Based on the above facts, we focused on the influence of CEA on the TGF-β signaling in both normal cells and colorectal cancer cells. Results: Our preliminary data showed that CEA directly interacted with TGF-β Receptors. Overexpression of CEA blocked TGF-β induced SMAD3 phosphorylation, SMAD3 translocation to nuclear and the downregulation of c-myc transcription. Targeting CEA with anti-CEA antibody rescued TGFβ response in CRC cell lines with elevated CEA expression, thereby restoring the inhibitory effects of TGF-β on the proliferation of these cancer cells. Finally, in animal experiment, we found that CEA enhanced survival of colorectal cancer cell in both local colonization and liver metastasis. Conclusion: Since CEA is a well-characterized tumor-associated antigen that is frequently overexpressed in tumors, specific antibodies targeting CEA have been developed as a novel therapeutic approach for treatment of tumors expressing CEA on their surface. Based on our study, it may be helpful to combine CEA antibody and TGF-β to inhibit cancer cell proliferation and metastasis in some cases.
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841 Mechanism for Selectivity of Specific Exonic Activin Receptor 2 (ACVR2) Frameshift Mutation in Colorectal Cells with Defective DNA Mismatch Repair (MMR)
Gastroenterology, 2009Co-Authors: Heekyung Chung, Dennis J Young, Claudia Lopez, Betty L. Cabrera, Deena Ream-robinson, John M CarethersAbstract:Preoperative Biliary Drainage Versus Direct Operation for Pancreatic Tumors Causing Obstructive Jaundice Niels Anthony Van Der Gaag, Erik Rauws, Casper H. van Eijck, Marco Bruno, Erwin van der Harst, Josephus J. Gerritsen, J. W. M. Greve, Michael F. Gerhards, Ignace H. de Hingh, Jean H. Klinkenbijl, Chung Y. Nio, Steve M. de Castro, Olivier R. Busch, Thomas M. Van Gulik, Patrick M. Bossuyt, Dirk J. Gouma
Jenny F Lai - One of the best experts on this subject based on the ideXlab platform.
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Flanking nucleotide specificity for DNA mismatch repair-deficient frameshifts within Activin Receptor 2 (ACVR2).
Mutation research, 2011Co-Authors: Heekyung Chung, Joy Chaudhry, Jenny F Lai, Dennis J Young, John M CarethersAbstract:We previously demonstrated that exonic selectivity for frameshift mutation (exon 10 over exon 3) of ACVR2 in mismatch repair (MMR)-deficient cells is partially determined by 6 nucleotides flanking 5' and 3' of each microsatellite. Substitution of flanking nucleotides surrounding the exon 10 microsatellite with those surrounding the exon 3 microsatellite greatly diminished heteroduplex (A(7)/T(8)) and full (A(7)/T(7)) mutation, while substitution of flanking nucleotides from exon 3 with those from exon 10 enhanced frameshift mutation. We hypothesized that specific individual nucleotide(s) within these flanking sequences control ACVR2 frameshift mutation rates. Only the 3rd nucleotide 5' of the microsatellite, and 3rd, 4th, and 5th nucleotides 3' of the microsatellite were altered from the native flanking sequences and these locations were individually altered (sites A, B, C, and D, respectively). Constructs were cloned +1bp out-of-frame of EGFP, allowing a -1bp frameshift to express EGFP. Plasmids were stably transfected into MMR-deficient cells. Non-fluorescent cells were sorted, cultured for 35 days, and harvested for flow cytometry and DNA-sequencing. Site A (C to T) and B (G to C) in ACVR2 exon 10 decreased both heteroduplex and full mutant as much as the construct containing all 4 alterations. For ACVR2 exon 3, site A (T to C), C (A to G), and D (G to C) are responsible for increased heteroduplex formation, whereas site D is responsible for full mutant formation by ACVR2 exon 10 flanking sequences. Exonic selectivity for frameshift mutation within ACVR2's sequence context is specifically controlled by individual nucleotides flanking each microsatellite.
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224 Specific Nucleotides That Flank Coding Microsatellites of Activin Receptor 2 (ACVR2) Determine Exonic Selectivity and Control Frameshift Mutation Rates in Defective DNA Mismatch Repair (MMR)
Gastroenterology, 2010Co-Authors: Heekyung Chung, Jenny F Lai, Dennis J Young, Joy Holmstrom, John M CarethersAbstract:Carcinoembryonic antigen (CEA) is a tumor marker for the clinical management of colorectal cancer (CRC). The elevated blood levels of CEA are associated with metastasis and poor prognosis in CRC. There is mounting evidence that CEA enhances the metastatic potential of cancer cells. CEA increases the ability of weakly metastatic CRC to colonize the liver and to develop spontaneous hematogeneous liver and lung metastases. CEA expression has also been related with resistance to cytotoxic chemotherapy and to anoikis, a form of apoptosis caused by detachment from cell matrix. Yet the mechanism of CEA mediated metastasis is only partially understood. The TGF-β (transforming growth factor beta) signaling pathway contributes to tumorigenesis by controlling several biological processes, including cell proliferation, differentiation, migration and apoptosis. It has been reported that TGF-β regulates CEA transcription and secretion, however, little is known about the effects of CEA on TGFβ signaling. Aims: Based on the above facts, we focused on the influence of CEA on the TGF-β signaling in both normal cells and colorectal cancer cells. Results: Our preliminary data showed that CEA directly interacted with TGF-β Receptors. Overexpression of CEA blocked TGF-β induced SMAD3 phosphorylation, SMAD3 translocation to nuclear and the downregulation of c-myc transcription. Targeting CEA with anti-CEA antibody rescued TGFβ response in CRC cell lines with elevated CEA expression, thereby restoring the inhibitory effects of TGF-β on the proliferation of these cancer cells. Finally, in animal experiment, we found that CEA enhanced survival of colorectal cancer cell in both local colonization and liver metastasis. Conclusion: Since CEA is a well-characterized tumor-associated antigen that is frequently overexpressed in tumors, specific antibodies targeting CEA have been developed as a novel therapeutic approach for treatment of tumors expressing CEA on their surface. Based on our study, it may be helpful to combine CEA antibody and TGF-β to inhibit cancer cell proliferation and metastasis in some cases.
Barbara Jung - One of the best experts on this subject based on the ideXlab platform.
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Activin and TGFβ use diverging mitogenic signaling in advanced colon cancer
Molecular cancer, 2015Co-Authors: Jessica Bauer, Ozkan Ozden, Naomi Akagi, Timothy J. Carroll, Daniel R. Principe, Jonas J. Staudacher, Martina E. Spehlmann, Lars Eckmann, Paul J. Grippo, Barbara JungAbstract:Understanding cell signaling pathways that contribute to metastatic colon cancer is critical to risk stratification in the era of personalized therapeutics. Here, we dissect the unique involvement of mitogenic pathways in a TGFβ or Activin-induced metastatic phenotype of colon cancer. Mitogenic signaling/growth factor Receptor status and p21 localization were correlated in primary colon cancers and intestinal tumors from either AOM/DSS treated ACVR2A (Activin Receptor 2) −/− or wild type mice. Colon cancer cell lines (+/− SMAD4) were interrogated for ligand-induced PI3K and MEK/ERK pathway activation and downstream protein/phospho-isoform expression/association after knockdown and pharmacologic inhibition of pathway members. EMT was assessed using epithelial/mesenchymal markers and migration assays. In primary colon cancers, loss of nuclear p21 correlated with upstream activation of Activin/PI3K while nuclear p21 expression was associated with TGFβ/MEK/ERK pathway activation. Activin, but not TGFβ, led to PI3K activation via interaction of ACVR1B and p85 independent of SMAD4, resulting in p21 downregulation. In contrast, TGFβ increased p21 via MEK/ERK pathway through a SMAD4-dependent mechanism. While Activin induced EMT via PI3K, TGFβ induced EMT via MEK/ERK activation. In vivo, loss of ACVR2A resulted in loss of pAkt, consistent with Activin-dependent PI3K signaling. Although Activin and TGFβ share growth suppressive SMAD signaling in colon cancer, they diverge in their SMAD4-independent pro-migratory signaling utilizing distinct mitogenic signaling pathways that affect EMT. p21 localization in colon cancer may determine a dominant Activin versus TGFβ ligand signaling phenotype warranting further validation as a therapeutic biomarker prior to targeting TGFβ family Receptors.
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Activin and TGFβ use diverging mitogenic signaling in advanced colon cancer
Molecular Cancer, 2015Co-Authors: Jessica Bauer, Ozkan Ozden, Naomi Akagi, Daniel R. Principe, Jonas J. Staudacher, Martina E. Spehlmann, Lars Eckmann, Paul J. Grippo, Timothy Carroll, Barbara JungAbstract:Background Understanding cell signaling pathways that contribute to metastatic colon cancer is critical to risk stratification in the era of personalized therapeutics. Here, we dissect the unique involvement of mitogenic pathways in a TGFβ or Activin-induced metastatic phenotype of colon cancer. Method Mitogenic signaling/growth factor Receptor status and p21 localization were correlated in primary colon cancers and intestinal tumors from either AOM/DSS treated ACVR2A (Activin Receptor 2) −/− or wild type mice. Colon cancer cell lines (+/− SMAD4) were interrogated for ligand-induced PI3K and MEK/ERK pathway activation and downstream protein/phospho-isoform expression/association after knockdown and pharmacologic inhibition of pathway members. EMT was assessed using epithelial/mesenchymal markers and migration assays. Results In primary colon cancers, loss of nuclear p21 correlated with upstream activation of Activin/PI3K while nuclear p21 expression was associated with TGFβ/MEK/ERK pathway activation. Activin, but not TGFβ, led to PI3K activation via interaction of ACVR1B and p85 independent of SMAD4, resulting in p21 downregulation. In contrast, TGFβ increased p21 via MEK/ERK pathway through a SMAD4-dependent mechanism. While Activin induced EMT via PI3K, TGFβ induced EMT via MEK/ERK activation. In vivo, loss of ACVR2A resulted in loss of pAkt, consistent with Activin-dependent PI3K signaling. Conclusion Although Activin and TGFβ share growth suppressive SMAD signaling in colon cancer, they diverge in their SMAD4-independent pro-migratory signaling utilizing distinct mitogenic signaling pathways that affect EMT. p21 localization in colon cancer may determine a dominant Activin versus TGFβ ligand signaling phenotype warranting further validation as a therapeutic biomarker prior to targeting TGFβ family Receptors.
