PDF file - 24K, HER2/Neu antibody and TCN plus anti-CTLA-4 mAb triple combination treatment increased serum IFN-gamma level

PDF file - 255K, The observation of more lymphocytes/leukocytes infiltration after HER2/Neu antibody and Akt inhibitor TCN combination treatment

PDF file - 206K, No significant changes in the number of tumor infiltrating T cells (including CD8+ and CD4+ ) upon the addition of anti-CTLA-4 mAb to HER2/Neu antibody and triciribine combination treatment

PDF file - 410K, Histological and immunohistochemical analysis of tumors derived from PTEN-/-/ErbB2KI mice receiving single or combination treatment

PDF file - 85K, Characterization of PTEN-/-/NIC mouse model

PDF file - 36K, Evaluation of markers of other tumor-infiltrating immune cells

PDF file - 137K, Depletion of CD8+ cells, CD4+ cells or both during HER2/Neu antibody and triciribine combination treatment

PDF file - 25K, HER2/Neu antibody 7.16.4 and Akt inhibitor TCN combination treatment reduced tumor multiplicity and lung metastasis in PTEN-/-/NIC mice

PDF file - 37K, Anti-CTLA-4 mAb alone, anti-CTLA-4 mAb combined with HER2/Neu antibody only, or with TCN only showed no significant enhancement of antitumor effect

PDF file - 82K, Primer sequences for quantitative real-time reverse transcription-PCR

Background: Activation of autocrine VEGF-VEGFR2 signalling in tumour cells activates cell proliferation, survival, and angiogenesis, all of which are crucial for tumour progression. Ovarian cancer-associated antigen 66 (OVA66) is now known to be overexpressed in multiple tumours and plays a role in tumour development, but the underlying mechanisms has not been fully investigated. Methods: We employed ovarian and cervical cancer cells and mouse models to detect the role of OVA66 in angiogenesis, growth, and metastasis of cancer cells. Immunofluorescence and western blot were used to determine the function of OVA66 in regulating autocrine VEGF-VEGFR2 signalling. Immunohistochemistry and bioinformatics analysis were used to detect the correlation of OVA66 and VEGF expression. Findings: OVA66 overexpression in the cancer cell lines promoted VEGF secretion, tumour growth and angiogenesis in vitro and in vivo. Conversely, shRNA-mediated OVA66 knockdown had the opposite effects. Mechanistically, OVA66 overexpression was found to boost an autocrine VEGF-VEGFR2 positive-feedback signalling loop in the tumour cells, leading to amplified effect of VEGF on tumour angiogenesis and proliferation and increased migration in vitro and in vivo, respectively. Finally, we identified a significant positive correlation between the expression levels of OVA66 and VEGF in ovarian and cervical cancer specimens, and found that OVA66 was significantly associated with advanced ovarian cancer. Interpretation: We identify a novel function for OVA66 in regulating an autocrine VEGF-VEGFR2 feed-forward signalling loop that promotes tumour progression and angiogenesis. (c) 2019 Published by Elsevier B.V.

机体防御与免疫课程属于基础医学的横向整合课程之一,对课程内容、教学形式和考核方式均进行了改进.文章分析了国内外医学免疫学课程的实施现状,总结了该课程的实践成果.

Ovarian cancer-associated antigen 12 (OVA12) was first identified in an ovarian carcinoma complementary DNA (cDNA) expression library and has been shown to play an important role in tumor growth. Here, we found that overexpression of OVA12 accelerated tumor growth in different tumor cells, whereas OVA12 depletion was associated with the opposite effect. Moreover, knocking down OVA12 led to a significant increase in the protein levels of p53, and the overexpression of OVA12 significantly decreased endogenous p53 levels. In addition, OVA12 stimulated p53 polyubiquitination and degradation by the proteasome and promoted tumor growth at least partially through the p53 pathway. Taken together, these results indicate that OVA12 is a negative regulator of p53 and that inhibition of OVA12 expression might serve as a therapeutic target to restore tumor suppression.

It has been widely suggested that mammosphere-forming cells from tumor cell lines or primary tumors represent the population of cancer stem cells (CSCs), which is supposed to lead to the failure of routine chemotherapy and the recurrence of the disease. However, it is still difficult to obtain CSCs from primary breast cancer for further investigation. We performed a modified culture system to generate mammosphere-forming cells derived from freshly isolated human breast cancer samples and the breast cancer cell line MCF-7. Cancer stem cell-like phenotypes such as CD44 and CD24 were measured by flow cytometry while alkaline phosphatase (AP) and mammaglobin (MGB1) expression was evaluated immunohistochemically. The expression levels of Klf4, Nanog, Oct4, Sox2 and mdr1 genes were analyzed by quantitative real‑time PCR. Resistance to chemotherapeutic drugs was detected through the apoptosis assay upon drug treatments together with the detection of drug-resistant gene mdr1. The results revealed that we successfully obtained mammosphere‑forming cells from the primary breast cancer in conditioned medium after 14 days of culture. Mammosphere-forming cells from primary breast cancer displayed a CD44hiCD24lo phenotype as well as positive AP and MGB1 reactivity. Stem cell-related genes such as Klf4, Nanog and Oct4 were detectably expressed in these cells. These cells formed tumor-like structures in the lymph nodes of nude mice, which were morphologically and histologically similar to breast cancer. Compared to the breast cancer cell line MCF-7 or mammosphere-forming cells from MCF-7 cells, the mammosphere-forming cells from the primary breast cancer exhibited resistance to three of four first-line chemotherapeutic drugs investigated through the induction of apoptosis, which was largely associated with the increased expression of drug-resistant gene mdr1 upon drug treatment. In conclusion, mammosphere-forming cells generated from the primary breast cancer exhibit CSC-like properties together with multiple drug resistance. Determination of the sensitivity of these primary cancer-derived mammosphere-forming cells to chemotherapeutic drugs may thus provide useful instructions for individualized therapy against the recurrence of breast cancer in the future.

Originally identified as an E3 ligase regulating toll-like receptor (TLR) signaling, ring finger protein 216 (RNF216) also plays an essential role in autophagy, which is fundamental to cellular homeostasis.Autophagy dysfunction leads to an array of pathological events, including tumor formation.In this study, we found that RNF216 was upregulated in human colorectal cancer (CRC) tissues and cell lines, and was associated with progression of CRC.RNF216 promoted CRC cell proliferation and migration in vitro and in vivo, largely by enhancing proteasomal degradation of BECN1, a key autophagy regulator and tumor suppressor.RNF216 restricted CRC cell autophagy through BECN1 inhibition under nutritional starvation conditions.RNF216 knockdown increased the autophagy, limiting CRC cell proliferation and migration.Moreover, BECN1 knockdown or autophagy inhibition restored proliferation and migration of RNF216-knockdown CRC cells.Collectively, our results suggested that RNF216 promoted CRC cell proliferation and migration by negatively regulating BECN1-dependent autophagy.This makes RNF216 as a potential biomarker and novel therapeutic target for inhibiting CRC development and progression.

To achieve a better understanding of mechanisms that underlie carcinogenesis and to identify novel target molecules for diagnosis and therapy of carcinoma, we previously identified 24 distinct gene clones by immunoscreening of a cDNA library derived from an ovarian cancer patient through SEREX analysis. Among these genes we focused on a novel gene termed OVA12 and which putatively encodes a 114-amino-acid protein. In the present study, we found that OVA12 was ubiquitously overexpressed in diverse human tumor cell lines. Interestingly, we noticed that overexpression of OVA12 promoted proliferation of cancer cells in vitro and accelerated tumor growth in nude mice as compared to controls. Conversely, specific downregulation of OVA12 inhibited tumor cell proliferation and tumor growth both in vitro and in vivo. Furthermore, OVA12 inhibited 5-FU-induced apoptosis through specific upregulation of Mcl-1 and survivin. These results demonstrate that OVA12 is able to promote tumor growth, suggesting that this antigen might be a new potential target for development of cancer therapy.

Human DCTPP1 (dCTP pyrophosphatase 1), also known as XTP3-transactivated protein A, belongs to MazG-like nucleoside triphosphate pyrophosphatase (NTP-PPase) superfamily. Being a newly identified pyrophosphatase, its relevance to tumorigenesis and the mechanisms are not well investigated. In the present study, we have confirmed our previous study that DCTPP1 was significantly hyperexpressed in breast cancer and further demonstrated its strong association with tumor progression and poor prognosis in breast cancer. Knockdown of DCTPP1 in breast cancer cell line MCF-7 cells remarkably retarded proliferation and colony formation in vitro. The capacity of mammosphere formation of MCF-7 was suppressed with the silence of DCTPP1, which was consistent with the enhanced mammosphere-forming ability in DCTPP1-overexpressed MDA-MB-231 cells. To further dissect the mechanisms of DCTPP1 in promoting tumor cell growth and stemness maintenance, its biochemical properties and biological functions were investigated. DCTPP1 displayed bioactive form with tetrameric structure similar to other MazG domain-containing pyrophosphatases based on structure simulation. A substrate preference for dCTP and its methylated or halogen-modified derivatives over the other canonical (deoxy-) NTPs was demonstrated from enzymatic assay. This substrate preference was also proved in breast cancer cells that the intracellular 5-methyl-dCTP level increased in DCTPP1-deficient MCF-7 cells but decreased in DCTPP1-overexpressed MDA-MB-231 cells. Moreover, global methylation level was elevated in DCTPP1-knockdown MCF-7 cells or mammosphere-forming MCF-7 cells but decreased significantly in DCTPP1-overexpressed MDA-MB-231 cells and its mammospheres. Our results thus indicated that human DCTPP1 was capable of modulating the concentration of intracellular 5-methyl-dCTP. This in turn affected global methylation, contributing to a known phenomenon of hypomethylation related to the cancer cell growth and stemness maintenance. Our current investigations point to the pathological functions of DCTPP1 overexpression in breast cancer cells with aberrant dCTP metabolism and epigenetic modification.

The tumor associated antigen OVA66 has been demonstrated to be highly expressed in malignant tumors and implicated in various cellular processes. To further elucidate its oncogenic character, we established an OVA66 stably overexpressed NIH3T3 cell line and a vector transfected control, named NIH3T3-flagOVA66 and NIH3T3-mock, respectively. NIH3T3-flagOVA66 cells showed faster cell cycling, proliferation, cell migration and more resistance to 5-fluorouracil-induced apoptosis. When NIH3T3-flagOVA66 and NIH3T3-mock cells were injected into nude mice for xenograft tumorigenicity assays, the NIH3T3-flagOVA66 cells formed tumors whereas no tumors were observed in mice inoculated with NIH3T3-mock cells. Analysis of PI3K/AKT and ERK1/2 MAPK signaling pathways by serum stimulation indicated hyperactivation of AKT and ERK1/2 in NIH3T3-flagOVA66 cells compared with NIH3T3-mock cells, while a decreased level of p-AKT and p-ERK1/2 were observed in OVA66 knocked down HeLa cells. To further validate if the p-AKT or p-ERK1/2 is essential for OVA66 induced oncogenic transformation, we treated the cells with the PI3K/AKT specific inhibitor LY294002 and the ERK1/2 MAPK specific inhibitor PD98059 and found either inhibitor can attenuate the cell colony forming ability in soft agar and the cell viability of NIH3T3-flagOVA66 cells, suggesting aberrantly activated AKT and ERK1/2 signaling be indispensible of the tumorigenic role of OVA66. Our results indicate that OVA66 is important in oncogenic transformation, promoting proliferation, cell migration and reducing apoptosis via hyperactivating PI3K/AKT and ERK1/2 MAPK signaling pathway. Thus, OVA66 might be a novel target for early detection, prevention and treatment of tumors in the future.

制备OVA12蛋白和OVA12单抗,探讨其在临床肿瘤辅助诊断中的应用前景.将重组质粒pET32a OVA12转化至BL21(DE3)中,经诱导表达、亲和层析纯化和超滤获得纯化了OVA12蛋白;应用杂交瘤技术制备并通过亲和层析技术纯化得到OVA12单抗;合成OVA12抗原多肽片段,分析单抗识别的抗原表位;建立ELISA法,用纯化的OVA 12蛋白及其抗原多肽分析肿瘤患者血清中抗OVA12抗体的含量;用单抗经免疫组化法检测临床不同类型肿瘤组织中OVA12蛋白的表达差异.结果显示,获得了纯化的OVA12蛋白和两株OVA12单抗;检测得知,肿瘤患者血清中的抗OVA12抗体水平显著高于健康人血清;OVA12蛋白在肿瘤组织中的表达也高于癌旁组织及正常组织,提示OVA 12蛋白和抗OVA12抗体可作为一个新的肿瘤标志物应用于临床肿瘤的辅助诊断.