- Email: [email protected]
Positive feedback activation of notch signal by obesity enhances colorectal tumorigenicity
abstracts
Annals of Oncology
2015P
Characterization of atypical dMMR (deficient MisMatch Repair) tumors: A study from a large cohort of 4948 cases
M. Jaffrelot1, J. Selves2, N. Fares3, A. Staub4, A.P. Laurenty5, M. Danjoux2, J. Meilleroux2, E. Chipoulet6, C. Toulas7, R. Guimbaud8 1 Oncology, Institut Universitaire du Cancer -Toulouse- Oncopole, Toulouse, France, 2 Department of Pathology, Institut Claudius Regaud - Institut Universitaire du Cancer de Toulouse, Toulouse, France, 3Haute Garonne, Hospital Rangueil, Toulouse, France, 4 Haute Garonne, CH de Montauban, Montauban, France, 5Oncology, Clinique Croix du sud, Quint Fonsegrives, France, 6Oncology, Institut Claudius Regaud - Institut Universitaire du Cancer de Toulouse, Toulouse, France, 7Genetics, Institut Claudius Regaud - Institut Universitaire du Cancer de Toulouse, Toulouse, France, 8Oncology, Institut Universitaire du Cancer -Toulouse- Oncopole, Toulouse, France Background: MMR testing is performed to screen Lynch Syndrome, evaluate the prognosis of colorectal cancer (CRC) and predict the efficacy of PD1/PDL1 blockade in all tumor types. Two methods are available: immunohistochemistry (IHC) using antibodies against MMR proteins and molecular biology (MB) for assessing microsatellite instability (MSI). Classically, dMMR tumor corresponds to loss of expression of two proteins (MLH1 and PMS2 or MSH2 and MSH6) associated with MSI. Atypical profiles of dMMR tumors have sporadically been described. The aim of our study was to describe the frequency and characteristics of these atypical cases. Methods: All MMR testing performed in our center between 2007 and 2017 were checked to select cases with both available IHC and MB. Then, all dMMR cases were reviewed to identify atypical cases which were defined by: isolated loss of expression of one protein, loss of expression of two proteins without MSI, normal expression of the four proteins with MSI, aberrant loss of proteins, or MSI-low. Biological data of atypical cases were controlled and clinical data were collected for each case. Results: 4948 MMR tests were performed, 3800 had both available IHC and MB data, and 585 were dMMR (15 %). Among them, 97 cases were atypical and after biological control, 8 cases were re-classified typical; allowing to finally identify 89 atypical cases: 60 CRC, 10 endometrial carcinoma, 8 digestive non CRC and 11 others types of cancers. A strong correlation with genetic syndromes was observed for those atypical profiles. Conclusions: Even using controlled IHC and MB, 15% of dMMR tumors have an atypical profile. These atypical cases mainly involve non CRC cancer with a strong prediction for Lynch syndrome. Their therapeutic impact particularly for immunotherapy should be now evaluated. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
Volume 30 | Supplement 5 | October 2019
2016P
Modulation of TLR9-dependent autophagy response via inhibition of c-Met signaling influences the survival of HT29 cancer cells
Simon1, T. Dank o2, A. Sebestye´n2, A.L. Kiss3, G. M} uzes1 F.J. Sipos1, L. Nagy1, B. Barta1, A. Division of Immunology, Semmelweis University, Second Dept. of Internal Medicine, Budapest, Hungary, 2Biology, First Department of Pathology and Experimental Cancer Research, Budapest, Hungary, 3Department of Human Morphology and Developmental Biology, Semmelweis University, Budapest, Hungary 1
Background: In HT29 cells, an interplay between self-DNA-induced TLR9- and autophagy responses was found with remarkable effects on survival and differentiation of tumor cells. c-Met activation is known to drive the progression of colorectal cancer by promoting signaling cascades that mainly result in alterations of cell motility, survival and proliferation. c-Met inhibition was shown to inhibit autophagy. In cancer cells the interrelated role of c-Met inhibition and TLR9/autophagy signaling has not yet been clarified, so we aimed to assess this complex interplay. Methods: HT29 cells were incubated for 72 h with genomic (g), hypermethylated (m), and fragmented (f) tumor self-DNAs, and with/without inhibitors of c-Met (diisothiocyanatostilbene), autophagy (chloroquine) and TLR9 (ODN2088), respectively. Cell viability was measured by MTT assay. Transcriptional changes of TLR9-signaling, PI3K, CD95, c-Met, Bcl2, cytochrome-c, and the autophagy process were assayed by Human v3 miRNA Assay (NanoString). Autophagy proteins were detected by immunocytochemistry, while morphology of apoptosis and autophagy by transmission electron microscopy (TEM). Results: Self-DNAs g and f resulted in significant upregulation of Beclin1, Atg16L1, LC3 mRNAs, and downregulation of PI3K, Bcl2, CD95, and cytochrome-c, verified by immunocytochemistry, as well. c-Met inhibition alone altered inversely the autophagyassociated gene- and protein-expressions. In each group of tumor cells using combined inhibition of autophagy, TLR9 and/or c-Met-signaling varying degree of autophagy was observed according to NanoString and TEM. Following combined incubation with c-Met inhibitor and m-DNAs no expected suppression of tumor cell survival and induction of apoptosis and mitophagy were detected. Further, c-Met inhibition changed the cell-protective effect f-DNA on macroautophagy. Conclusions: Our study provided evidence for an intense crosstalk between the inhibited c-Met canonical and non-canonical signaling pathways, and the TLR9/autophagy response with profound impacts on survival, proliferation and death of HT29 cells subjected to intact/modified self-DNAs. Legal entity responsible for the study: Ferenc Sipos. Funding: StartUp. Disclosure: All authors have declared no conflicts of interest.
2017P
Positive feedback activation of notch signal by obesity enhances colorectal tumorigenicity
D. Chu1, Z. Zhang1, J. Zhang1, Y. Wang1, Y. Li1, X. Bu1, E. Li2, J. Zhang1 Gastroenterology, First Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an, China, 2Medical Oncology, First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China 1
Background: Colorectal cancer (CRC) is one of the most common malignancies worldwide. Investigations revealed that the steep rise of CRC incidence was significantly linked to increased obesity rates. However, the molecular interactions of obesity with CRC are still not well understood. The Notch signaling pathway is critical for many cellular processes. It is also a key player in regulating body energy metabolism. Therefore, we aimed to investigate the potential interaction and mechanism of Notch signal between obesity and CRC tumorigenesis.
doi:10.1093/annonc/mdz269 | v807
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz269.035/5577608 by guest on 26 October 2019
autophagy-associated gene- and protein-expressions. Upon combined inhibition of autophagy, TLR9 and/or IGF1R-signaling varying degree of autophagy was detected in all groups of tumor cells according to NanoString and TEM. Incubation with IGF1R inhibitor and with m-DNA no expected suppression of tumor cell survival, induction of apoptotic death, and activation of mitophagy were found. Moreover, IGF1R inhibition affected negatively the cell-protective effect f-DNA on macroautophagy and lipophagy. Conclusions: Our study provided evidence for an interaction between the inhibited IGF1R and TLR9/autophagy signaling with major influences on survival, proliferation and death of HT29 cells subjected to intact/modified self-DNAs. (Funded by StartUp.). Legal entity responsible for the study: Ferenc Sipos. Funding: StartUp. Disclosure: All authors have declared no conflicts of interest.
abstracts
2018P
The pathological and functional roles of BRPF1 in hepatocellular carcinoma
of PTP in PC has never been elucidated yet. In the present study, to develop effective therapeutic strategy for refractory PC, the biological significance of PTP non-receptor type 3 (PTPN3) in PC is investigated. Methods: Three pancreatic ductal adenocarcinoma cell (PDAC) lines were used as target cells. Inhibition or overexpression of PTPN3 was performed using PTPN3 siRNA/ shRNA and plasmid, respectively. Protein expression was analyzed by western blot and immunostaining. Proliferation assay was performed by MTT assay. Migration and invasion were estimated by time-lapse imaging and matrigel invasion assay. Mice xenograft experiments were performed using BALB/c nude mice. Twenty-three surgically resected human PC specimens were used for immunostaining. Results: 1) PTPN3 is highly expressed in PC cells compared to that in normal pancreatic duct cells by immunostaining. 2) Inhibition of PTPN3 significantly decreased migration and invasion in PDAC through inhibition of epithelial mesenchymal transition (EMT) and matrix metalloproteinase (MMP)-2 expression. 3) PTPN3 inhibition significantly decreased proliferation in vitro in PDAC. 4) PTPN3 overexpression led to increased proliferation, migration and invasion in PDAC. 5) Tumor volume in mice injected with PTPN3-inhibited PDAC was significantly lower than that in control mice. The expressions of Ki-67 and VEGF in PTPN3-inhibited PDAC were lower than those in control. 6) Signaling from PTPN3 was through PI3K and MAPK signaling pathways. 7) When intensity of PTPN3 expression was divided into 2 groups; weak and strong, intensity of PTPN3 expression was positively correlated with tumor size (T factor) and clinical staging. Conclusions: These results suggest that PTPN3 contributes to the induction of malignant phenotypes of pancreatic cancer and that PTPN3 could be a new effective and specific therapeutic target for PC. Legal entity responsible for the study: Kyushu University Hospital, Cancer Therapy and Research. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
L.H.C. Cheng, F.H.C. Tsang, L. Wei, C.T. Law, I.O.L. Ng, C.M. Wong Pathology, The University of Hong Kong, Hong Kong, China Background: Bromodomain-containing proteins are important epigenetic regulators that specifically recognize acetylated histones and implicated in regulating gene expression. Bromodomain inhibitors showed a prominent therapeutic effect on metabolic diseases and various types of cancers in clinical trials. In this study, we employed RNASeq to interrogate the expression profile of bromodomain-containing proteins in human hepatocellular carcinoma (HCC). We found that Bromodomain and PHD Finger Containing 1 (BRPF1) was among the most significantly overexpressed bromodomain-containing proteins in HCC. Methods: The expression profile of bromodomain-containing proteins in our HCC patients was analyzed by RNA-Seq. BRPF1 knockout in MHCC97L was accomplished by using lentiviral-based CRISPR gene editing system. Cell proliferation, colony formation, and cell migration assays were performed to assess in vitro function of BRPF1. A nude mice orthotopic liver xenograft model was used to study the role of BRPF1 in HCC tumorigenicity and lung metastasis in vivo. Sphere formation assay and qPCR were performed to study the stemness properties of BRPF1 knockdown cells. Apoptosis and cell cycle arrest assay was performed by flow cytometry. Senescence was detected by B-galactosidase staining, mRNA and protein expression of senescence-associated makers. Results: Clinically, high BRPF1 expression was associated with poorer survival rates in HCC patients. The upregulation of BRPF1 mRNA in HCC was significantly associated with gene copy gain and gene amplification. ROC analysis indicated that BRPF1 was a potential biomarker for HCC detection. shRNA-mediated knockdown and CRISPRmediated knockout of BRPF1 suppressed cell proliferation and colony formation in MHCC97L. Knockout of BRPF1 also reduced tumorigenicity in liver orthotopic injection model in nude mice. We found that BRPF1 expression was elevated in CD133þ liver cancer stem cells. Inactivation of BRPF1 also reduced the expression of genes related to cancer stemness and cancer renewal ability in sphere formation. BRPF1 inhibition induced apoptosis, cell cycle arrest as well as cellular senescence. Conclusions: Our findings suggest that BRPF1 may contribute to HCC development and cancer stemness. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
2019P
Protein tyrosine phosphatase non-receptor type 3 (PTPN3) could be a new therapeutic target for pancreatic cancer
2020P
A Novel bispecific BCMAxCD3 T cell-engaging antibody that treat multiple myeloma (MM) with minimal cytokine secretion
Z. Li1, Q. Li2, G. Zhang1, X. Ma2, Z. Li1, X. Hu1, K. Ouyang2, B. Li1, Z. Liu1 National Engineering Laboratory of High Level Expression in Mammalian Cells, Lunan Pharmaceutical Group, Linyi, China, 2Molecular Discovery, Ampsource Biopharma (Shanghai) Inc, Shanghai, China
1
Background: B-cell maturation antigen (BCMA) is a potential therapeutic target in treatment of patients with MM. BCMA is widely expressed at the surface of myeloma cells and induces proliferative signals. BCMA-directed BCMA antibody conjugated with toxin has recently demonstrated outstanding anti-tumor responses and acceptable tolerability in MM treatment, allowing patients to achieve deeper responses with minimally added toxicity. Methods: We used ELISA and FACS to verify AP163 can be combined with CD3/ BCMA protein on the cell surface. We used the reporter gene method to verify that it induced T cell activation and target cell killing. In vivo, efficacy was studied in NCIH929 and Raji xenograft models in female NPG mice. NPG mice were injected subcutaneously into the right dorsal flank with NCI-H929/Raji cells and human PBMC. Then the mice were treated with AP163 and tumor volume was measured twice/week. Cynomolgus monkeys were mainly used for toxicity testing. Intravenous administration is consistent with clinical administration. Its main toxicological target is the increase of cytokines and a series of adverse reactions. Results: we constructed AP163 which binds to BCMA-expressing cell lines. AP163 induced both types of cell crosslinking, followed by T cell activation, cytokine production, proliferation and redirected target cell killing, with EC50 values in the picomolar range. The affinities of AP163 with human and cynomolgus CD3 are comparable. In vitro, AP163 only induced minimal cytokine release in the presence of BCMA target. When administered to mice bearing human MM xenografts and human T cells, AP163 eradicated subcutaneous tumors in two xenograft models tested and significantly delayed tumor growth at doses as low as 0.04mg/kg. AP163 displayed a 9-h half-life in cynomolgus and was well tolerated in non-human primates, even at high doses up to 5 mg/kg. Conclusions: AP163 clears tumor cells in a BCMAþ and T-cell dependent manner in vitro and in vivo. Our preclinical data suggest that AP163 induces less cytokine secretion than a conventional bispecific in vitro, with reduction of tumor cell in vivo. AP163 is highly efficacious, safe and convenient for the treatment of patients with MM. Legal entity responsible for the study: National Engineering Laboratory of High Level Expression In Mammalian Cells. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
A. Yamasaki, S. Koga, S. Ichimiya, K. Nakayama, Y. Oyama, Y. Fujioka, H. Onishi Cancer Therapy and Research, Kyushu University Hospital, Fukuoka, Japan Background: Pancreatic cancer (PC) is a refractory disease and we have not an effective therapeutic strategy yet. Therefore, the development of a new therapeutic procedure is urgently required. Recently, protein tyrosine phosphatase (PTP) has been attracted as a new therapeutic target for some types of cancers. However, the biological significance
v808 | Tumour Biology and Pathology
Volume 30 | Supplement 5 | October 2019
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz269.035/5577608 by guest on 26 October 2019
Methods: In the present study, we randomly recruited 968 cases of CRC specimens, 262 cases of colorectal intraepithelial neoplasia specimens and 185 cases of normal epithelium specimens. Target protein expression was investigated by immunohistochemistry. C57BL/6J mice were randomly allocated intogroups fed with standard rodent chow, high-fat diet (HFD), and HFD treated with DBZ or DAPT. Potential differentially expressed proteins were screened by iTRAQ, and these identified proteins were then analyzed. Results: Notch1 intracellular domain and DLL4 was up-regulated in overweight participants compared with normal-weight ones. In overweight participants, Notch1 was increased from normal epithelium, intraepithelial neoplasia to CRC. Obesity was identified at week 5 in mice fed with HFD, which began to lead to upregulation of DLL4 and consequent increased Notch1 activity in colorectal tissues. While mice treated with DBZ and DAPT were almost resistant to HFD-induced obesity then. After 10 weeks, Notch1 activity in mice fed with HFD was significantly up-regulated compared with those fed with standard rodent chow. In addition, glucose tolerance and insulin sensitivity were also ameliorated by DBZ and DAPT treatment, through a PP2A-SHIP2 dependent manner. Conclusions: Notch signaling activation is linked to obesity in both nonmalignant participants and CRC patients. Obesity induced by HFD can increase Notch activity by DLL4-Notch1 pathway. While inhibition of Notch signaling can attenuate high fat diet–induced obesity by improving insulin resistance.These results indicate that activation of Notch signaling by its positive feedback with obesity could be a molecular bridge that connecting obesity and CRC. Legal entity responsible for the study: Xian Jiaotong University. Funding: National Nature Science Foundation of China. Disclosure: All authors have declared no conflicts of interest.
Annals of Oncology
Annals of Oncology
2015P
Characterization of atypical dMMR (deficient MisMatch Repair) tumors: A study from a large cohort of 4948 cases
M. Jaffrelot1, J. Selves2, N. Fares3, A. Staub4, A.P. Laurenty5, M. Danjoux2, J. Meilleroux2, E. Chipoulet6, C. Toulas7, R. Guimbaud8 1 Oncology, Institut Universitaire du Cancer -Toulouse- Oncopole, Toulouse, France, 2 Department of Pathology, Institut Claudius Regaud - Institut Universitaire du Cancer de Toulouse, Toulouse, France, 3Haute Garonne, Hospital Rangueil, Toulouse, France, 4 Haute Garonne, CH de Montauban, Montauban, France, 5Oncology, Clinique Croix du sud, Quint Fonsegrives, France, 6Oncology, Institut Claudius Regaud - Institut Universitaire du Cancer de Toulouse, Toulouse, France, 7Genetics, Institut Claudius Regaud - Institut Universitaire du Cancer de Toulouse, Toulouse, France, 8Oncology, Institut Universitaire du Cancer -Toulouse- Oncopole, Toulouse, France Background: MMR testing is performed to screen Lynch Syndrome, evaluate the prognosis of colorectal cancer (CRC) and predict the efficacy of PD1/PDL1 blockade in all tumor types. Two methods are available: immunohistochemistry (IHC) using antibodies against MMR proteins and molecular biology (MB) for assessing microsatellite instability (MSI). Classically, dMMR tumor corresponds to loss of expression of two proteins (MLH1 and PMS2 or MSH2 and MSH6) associated with MSI. Atypical profiles of dMMR tumors have sporadically been described. The aim of our study was to describe the frequency and characteristics of these atypical cases. Methods: All MMR testing performed in our center between 2007 and 2017 were checked to select cases with both available IHC and MB. Then, all dMMR cases were reviewed to identify atypical cases which were defined by: isolated loss of expression of one protein, loss of expression of two proteins without MSI, normal expression of the four proteins with MSI, aberrant loss of proteins, or MSI-low. Biological data of atypical cases were controlled and clinical data were collected for each case. Results: 4948 MMR tests were performed, 3800 had both available IHC and MB data, and 585 were dMMR (15 %). Among them, 97 cases were atypical and after biological control, 8 cases were re-classified typical; allowing to finally identify 89 atypical cases: 60 CRC, 10 endometrial carcinoma, 8 digestive non CRC and 11 others types of cancers. A strong correlation with genetic syndromes was observed for those atypical profiles. Conclusions: Even using controlled IHC and MB, 15% of dMMR tumors have an atypical profile. These atypical cases mainly involve non CRC cancer with a strong prediction for Lynch syndrome. Their therapeutic impact particularly for immunotherapy should be now evaluated. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
Volume 30 | Supplement 5 | October 2019
2016P
Modulation of TLR9-dependent autophagy response via inhibition of c-Met signaling influences the survival of HT29 cancer cells
Simon1, T. Dank o2, A. Sebestye´n2, A.L. Kiss3, G. M} uzes1 F.J. Sipos1, L. Nagy1, B. Barta1, A. Division of Immunology, Semmelweis University, Second Dept. of Internal Medicine, Budapest, Hungary, 2Biology, First Department of Pathology and Experimental Cancer Research, Budapest, Hungary, 3Department of Human Morphology and Developmental Biology, Semmelweis University, Budapest, Hungary 1
Background: In HT29 cells, an interplay between self-DNA-induced TLR9- and autophagy responses was found with remarkable effects on survival and differentiation of tumor cells. c-Met activation is known to drive the progression of colorectal cancer by promoting signaling cascades that mainly result in alterations of cell motility, survival and proliferation. c-Met inhibition was shown to inhibit autophagy. In cancer cells the interrelated role of c-Met inhibition and TLR9/autophagy signaling has not yet been clarified, so we aimed to assess this complex interplay. Methods: HT29 cells were incubated for 72 h with genomic (g), hypermethylated (m), and fragmented (f) tumor self-DNAs, and with/without inhibitors of c-Met (diisothiocyanatostilbene), autophagy (chloroquine) and TLR9 (ODN2088), respectively. Cell viability was measured by MTT assay. Transcriptional changes of TLR9-signaling, PI3K, CD95, c-Met, Bcl2, cytochrome-c, and the autophagy process were assayed by Human v3 miRNA Assay (NanoString). Autophagy proteins were detected by immunocytochemistry, while morphology of apoptosis and autophagy by transmission electron microscopy (TEM). Results: Self-DNAs g and f resulted in significant upregulation of Beclin1, Atg16L1, LC3 mRNAs, and downregulation of PI3K, Bcl2, CD95, and cytochrome-c, verified by immunocytochemistry, as well. c-Met inhibition alone altered inversely the autophagyassociated gene- and protein-expressions. In each group of tumor cells using combined inhibition of autophagy, TLR9 and/or c-Met-signaling varying degree of autophagy was observed according to NanoString and TEM. Following combined incubation with c-Met inhibitor and m-DNAs no expected suppression of tumor cell survival and induction of apoptosis and mitophagy were detected. Further, c-Met inhibition changed the cell-protective effect f-DNA on macroautophagy. Conclusions: Our study provided evidence for an intense crosstalk between the inhibited c-Met canonical and non-canonical signaling pathways, and the TLR9/autophagy response with profound impacts on survival, proliferation and death of HT29 cells subjected to intact/modified self-DNAs. Legal entity responsible for the study: Ferenc Sipos. Funding: StartUp. Disclosure: All authors have declared no conflicts of interest.
2017P
Positive feedback activation of notch signal by obesity enhances colorectal tumorigenicity
D. Chu1, Z. Zhang1, J. Zhang1, Y. Wang1, Y. Li1, X. Bu1, E. Li2, J. Zhang1 Gastroenterology, First Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an, China, 2Medical Oncology, First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China 1
Background: Colorectal cancer (CRC) is one of the most common malignancies worldwide. Investigations revealed that the steep rise of CRC incidence was significantly linked to increased obesity rates. However, the molecular interactions of obesity with CRC are still not well understood. The Notch signaling pathway is critical for many cellular processes. It is also a key player in regulating body energy metabolism. Therefore, we aimed to investigate the potential interaction and mechanism of Notch signal between obesity and CRC tumorigenesis.
doi:10.1093/annonc/mdz269 | v807
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz269.035/5577608 by guest on 26 October 2019
autophagy-associated gene- and protein-expressions. Upon combined inhibition of autophagy, TLR9 and/or IGF1R-signaling varying degree of autophagy was detected in all groups of tumor cells according to NanoString and TEM. Incubation with IGF1R inhibitor and with m-DNA no expected suppression of tumor cell survival, induction of apoptotic death, and activation of mitophagy were found. Moreover, IGF1R inhibition affected negatively the cell-protective effect f-DNA on macroautophagy and lipophagy. Conclusions: Our study provided evidence for an interaction between the inhibited IGF1R and TLR9/autophagy signaling with major influences on survival, proliferation and death of HT29 cells subjected to intact/modified self-DNAs. (Funded by StartUp.). Legal entity responsible for the study: Ferenc Sipos. Funding: StartUp. Disclosure: All authors have declared no conflicts of interest.
abstracts
2018P
The pathological and functional roles of BRPF1 in hepatocellular carcinoma
of PTP in PC has never been elucidated yet. In the present study, to develop effective therapeutic strategy for refractory PC, the biological significance of PTP non-receptor type 3 (PTPN3) in PC is investigated. Methods: Three pancreatic ductal adenocarcinoma cell (PDAC) lines were used as target cells. Inhibition or overexpression of PTPN3 was performed using PTPN3 siRNA/ shRNA and plasmid, respectively. Protein expression was analyzed by western blot and immunostaining. Proliferation assay was performed by MTT assay. Migration and invasion were estimated by time-lapse imaging and matrigel invasion assay. Mice xenograft experiments were performed using BALB/c nude mice. Twenty-three surgically resected human PC specimens were used for immunostaining. Results: 1) PTPN3 is highly expressed in PC cells compared to that in normal pancreatic duct cells by immunostaining. 2) Inhibition of PTPN3 significantly decreased migration and invasion in PDAC through inhibition of epithelial mesenchymal transition (EMT) and matrix metalloproteinase (MMP)-2 expression. 3) PTPN3 inhibition significantly decreased proliferation in vitro in PDAC. 4) PTPN3 overexpression led to increased proliferation, migration and invasion in PDAC. 5) Tumor volume in mice injected with PTPN3-inhibited PDAC was significantly lower than that in control mice. The expressions of Ki-67 and VEGF in PTPN3-inhibited PDAC were lower than those in control. 6) Signaling from PTPN3 was through PI3K and MAPK signaling pathways. 7) When intensity of PTPN3 expression was divided into 2 groups; weak and strong, intensity of PTPN3 expression was positively correlated with tumor size (T factor) and clinical staging. Conclusions: These results suggest that PTPN3 contributes to the induction of malignant phenotypes of pancreatic cancer and that PTPN3 could be a new effective and specific therapeutic target for PC. Legal entity responsible for the study: Kyushu University Hospital, Cancer Therapy and Research. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
L.H.C. Cheng, F.H.C. Tsang, L. Wei, C.T. Law, I.O.L. Ng, C.M. Wong Pathology, The University of Hong Kong, Hong Kong, China Background: Bromodomain-containing proteins are important epigenetic regulators that specifically recognize acetylated histones and implicated in regulating gene expression. Bromodomain inhibitors showed a prominent therapeutic effect on metabolic diseases and various types of cancers in clinical trials. In this study, we employed RNASeq to interrogate the expression profile of bromodomain-containing proteins in human hepatocellular carcinoma (HCC). We found that Bromodomain and PHD Finger Containing 1 (BRPF1) was among the most significantly overexpressed bromodomain-containing proteins in HCC. Methods: The expression profile of bromodomain-containing proteins in our HCC patients was analyzed by RNA-Seq. BRPF1 knockout in MHCC97L was accomplished by using lentiviral-based CRISPR gene editing system. Cell proliferation, colony formation, and cell migration assays were performed to assess in vitro function of BRPF1. A nude mice orthotopic liver xenograft model was used to study the role of BRPF1 in HCC tumorigenicity and lung metastasis in vivo. Sphere formation assay and qPCR were performed to study the stemness properties of BRPF1 knockdown cells. Apoptosis and cell cycle arrest assay was performed by flow cytometry. Senescence was detected by B-galactosidase staining, mRNA and protein expression of senescence-associated makers. Results: Clinically, high BRPF1 expression was associated with poorer survival rates in HCC patients. The upregulation of BRPF1 mRNA in HCC was significantly associated with gene copy gain and gene amplification. ROC analysis indicated that BRPF1 was a potential biomarker for HCC detection. shRNA-mediated knockdown and CRISPRmediated knockout of BRPF1 suppressed cell proliferation and colony formation in MHCC97L. Knockout of BRPF1 also reduced tumorigenicity in liver orthotopic injection model in nude mice. We found that BRPF1 expression was elevated in CD133þ liver cancer stem cells. Inactivation of BRPF1 also reduced the expression of genes related to cancer stemness and cancer renewal ability in sphere formation. BRPF1 inhibition induced apoptosis, cell cycle arrest as well as cellular senescence. Conclusions: Our findings suggest that BRPF1 may contribute to HCC development and cancer stemness. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
2019P
Protein tyrosine phosphatase non-receptor type 3 (PTPN3) could be a new therapeutic target for pancreatic cancer
2020P
A Novel bispecific BCMAxCD3 T cell-engaging antibody that treat multiple myeloma (MM) with minimal cytokine secretion
Z. Li1, Q. Li2, G. Zhang1, X. Ma2, Z. Li1, X. Hu1, K. Ouyang2, B. Li1, Z. Liu1 National Engineering Laboratory of High Level Expression in Mammalian Cells, Lunan Pharmaceutical Group, Linyi, China, 2Molecular Discovery, Ampsource Biopharma (Shanghai) Inc, Shanghai, China
1
Background: B-cell maturation antigen (BCMA) is a potential therapeutic target in treatment of patients with MM. BCMA is widely expressed at the surface of myeloma cells and induces proliferative signals. BCMA-directed BCMA antibody conjugated with toxin has recently demonstrated outstanding anti-tumor responses and acceptable tolerability in MM treatment, allowing patients to achieve deeper responses with minimally added toxicity. Methods: We used ELISA and FACS to verify AP163 can be combined with CD3/ BCMA protein on the cell surface. We used the reporter gene method to verify that it induced T cell activation and target cell killing. In vivo, efficacy was studied in NCIH929 and Raji xenograft models in female NPG mice. NPG mice were injected subcutaneously into the right dorsal flank with NCI-H929/Raji cells and human PBMC. Then the mice were treated with AP163 and tumor volume was measured twice/week. Cynomolgus monkeys were mainly used for toxicity testing. Intravenous administration is consistent with clinical administration. Its main toxicological target is the increase of cytokines and a series of adverse reactions. Results: we constructed AP163 which binds to BCMA-expressing cell lines. AP163 induced both types of cell crosslinking, followed by T cell activation, cytokine production, proliferation and redirected target cell killing, with EC50 values in the picomolar range. The affinities of AP163 with human and cynomolgus CD3 are comparable. In vitro, AP163 only induced minimal cytokine release in the presence of BCMA target. When administered to mice bearing human MM xenografts and human T cells, AP163 eradicated subcutaneous tumors in two xenograft models tested and significantly delayed tumor growth at doses as low as 0.04mg/kg. AP163 displayed a 9-h half-life in cynomolgus and was well tolerated in non-human primates, even at high doses up to 5 mg/kg. Conclusions: AP163 clears tumor cells in a BCMAþ and T-cell dependent manner in vitro and in vivo. Our preclinical data suggest that AP163 induces less cytokine secretion than a conventional bispecific in vitro, with reduction of tumor cell in vivo. AP163 is highly efficacious, safe and convenient for the treatment of patients with MM. Legal entity responsible for the study: National Engineering Laboratory of High Level Expression In Mammalian Cells. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
A. Yamasaki, S. Koga, S. Ichimiya, K. Nakayama, Y. Oyama, Y. Fujioka, H. Onishi Cancer Therapy and Research, Kyushu University Hospital, Fukuoka, Japan Background: Pancreatic cancer (PC) is a refractory disease and we have not an effective therapeutic strategy yet. Therefore, the development of a new therapeutic procedure is urgently required. Recently, protein tyrosine phosphatase (PTP) has been attracted as a new therapeutic target for some types of cancers. However, the biological significance
v808 | Tumour Biology and Pathology
Volume 30 | Supplement 5 | October 2019
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz269.035/5577608 by guest on 26 October 2019
Methods: In the present study, we randomly recruited 968 cases of CRC specimens, 262 cases of colorectal intraepithelial neoplasia specimens and 185 cases of normal epithelium specimens. Target protein expression was investigated by immunohistochemistry. C57BL/6J mice were randomly allocated intogroups fed with standard rodent chow, high-fat diet (HFD), and HFD treated with DBZ or DAPT. Potential differentially expressed proteins were screened by iTRAQ, and these identified proteins were then analyzed. Results: Notch1 intracellular domain and DLL4 was up-regulated in overweight participants compared with normal-weight ones. In overweight participants, Notch1 was increased from normal epithelium, intraepithelial neoplasia to CRC. Obesity was identified at week 5 in mice fed with HFD, which began to lead to upregulation of DLL4 and consequent increased Notch1 activity in colorectal tissues. While mice treated with DBZ and DAPT were almost resistant to HFD-induced obesity then. After 10 weeks, Notch1 activity in mice fed with HFD was significantly up-regulated compared with those fed with standard rodent chow. In addition, glucose tolerance and insulin sensitivity were also ameliorated by DBZ and DAPT treatment, through a PP2A-SHIP2 dependent manner. Conclusions: Notch signaling activation is linked to obesity in both nonmalignant participants and CRC patients. Obesity induced by HFD can increase Notch activity by DLL4-Notch1 pathway. While inhibition of Notch signaling can attenuate high fat diet–induced obesity by improving insulin resistance.These results indicate that activation of Notch signaling by its positive feedback with obesity could be a molecular bridge that connecting obesity and CRC. Legal entity responsible for the study: Xian Jiaotong University. Funding: National Nature Science Foundation of China. Disclosure: All authors have declared no conflicts of interest.
Annals of Oncology