Lili Tang

IL-32 promotes breast cancer cell growth and invasiveness.

Interleukin (IL)-32 is a newly identified cytokine in humans and primates. It has been established that IL-32 may antagonize cancer growth. However, to the best of our knowledge, the direct effect of IL-32 on breast cancer cell growth has not yet been investigated. In addition, rodents lack the expression of IL-32; hence, the effects of IL-32 on breast cancer xenografts in nude mice have not been studied. The present study aimed to examine the potential regulatory effects of IL-32 on breast cancer cells in nude mice. The effects of IL-32 on tumor cell growth in cell cuture and a tumor xenograft model were investigated, as well as the effects of IL-32 on apoptosis. The effects of IL-32 on cell proliferation and apoptosis were investigated by MTT assay and TUNEL staining, respectively. The results revealed that IL-32 increases the proliferation rate of cancer cells and decreases the rate of apoptosis, In addition, IL-32 was found to enhance the growth of tumor xenografts in vivo. In summary, IL-32 may represent a useful therapeutic target for human breast cancer.

Reversal of Multidrug Resistance by Gefitinib Via RAF1/ERK Pathway in Pancreatic Cancer Cell Line

Pancreatic cancer is a devastating malignancy, characterized by intrinsic or acquired resistance to conventional chemotherapies. Recent evidences suggest an involvement of tyrosine kinase pathway in the regulation of multidrug resistance (MDR) protein gene expression. The aim of this study was to test whether gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor could regulate the MDR protein gene expression and sensitize the resistant cancer cells to chemotherapy. The gene expression of MDR proteins (MRP1, MRP2, MRP3, and PGP) were evaluated by quantitative RT‐PCR, and expression levels of various tyrosine kinases were investigated by quantitative RT‐PCR and Western Blot in pancreatic cancer cell line. MTT assay was used for evaluating the effect of chemotherapeutic agents. Chemotherapeutics induced drug resistance by regulating the gene expression of MDR proteins (MRP1, MRP2, and MRP3), and increased the gene expression of RAF1/ERK and the phosphorylation of ERK in pancreatic cancer Bxpc‐3 cells. Gefitinib caused an inhibition of p‐ERK tyrosine kinase activation in a dose‐dependent manner, and reversed gemcitabine‐induced RAF1/ERK gene expression and p‐ERK activation. In addition, a reversal of MDR proteins gene expression was achieved by gefitinib, which sensitized resistant cells to gemcitabine. This study demonstrated that MDR of Bxpc‐3 cell is involved in the RAF1/ERK tyrosine kinase pathway. Gefitinib reverses the MDR protein gene expression and restores sensitivity of resistant cells to gemcitabine via RAF1/ERK signaling pathway. Combination of gefitinib with conventional chemotherapeutic agents may offer a new approach for the treatment of patients with pancreatic cancer. Anat Rec, 2012.

Associations between body mass index and molecular subtypes as well as other clinical characteristics of breast cancer in Chinese women.

Background: Several studies have shown a positive association between body mass index (BMI) and the development of hormone receptor-positive breast cancer in postmenopausal women; however, the associations between BMI groups and molecular subtypes have yet to be well defined in premenopausal breast cancer patients. Methods: A total of 2465 female breast cancer patients diagnosed at our institution were recruited for this study. Clinicopathologic information (including age, body height and weight, as well as tumor subtypes and stages) was collected; analyses of these characteristics and the associations between them were performed. Results: A total of 1951 cases were included in the study. The mean age was 47.3 years, the majority of patients were of normal weight, premenopausal, had stage 2 cancer, and did not present with positive nodes. The prevalence of the luminal A, luminal B, human epidermal growth factor receptor 2+, and triple-negative subtypes were 57.8%, 11.6%, 6.1%, and 24.5%, respectively. There were significant differences in the clinicopathologic features among BMI groups in premenopausal patients. The case-only odds ratio (OR) analysis revealed that normal weight patients tended to have luminal B cancer (OR = 1.4, P = 0.206), and overweight and obese patients tended to have triple-negative cancer in premenopausal patients (OR = 2.8, OR = 3.7, respectively; P < 0.001). Conclusion: In Chinese women, breast cancer came with these characteristics: young mean age (premenopause), luminal A subtype, and the majority of them were within a normal weight range. In premenopausal patients, underweight patients tended to have luminal A, lower human epidermal growth factor receptor 2+ expression, stage 1 and no positive node cancer. However, overweight and obese patients tended to have a triple-negative, stage 3, and lymph node metastatic cancer.

Haplotype analysis of eight genes of the monoubiquitinated FANCD2–DNA damage–repair pathway in breast cancer patients

BACKGROUND Ten genes are associated with increased susceptibility to inherited breast cancer have also been associated with population breast cancer risk, and all are involved directly or indirectly in the monoubiquitinated FANCD2-DNA damage repair pathway. We analyzed 13 haplotype blocks in eight of these genes to estimate the breast cancer risk conferred by individual haplotypes. METHODS Haplotype blocks were constructed with 48 tag single-nucleotide polymorphisms (tSNPs) identified in eight breast cancer susceptibility genes, TP53, PTEN, CHEK2, ATM, NBS1, RAD50, BRIP1, and PALB2. Genotyping was performed by SNPscan on 734 female patients and 672 female age-matched controls. RESULTS Forty-five tSNPs were successfully genotyped by SNPscan, and call rates for each tSNP were above 98.9%. Thirteen haplotype blocks of eight genes were constructed with 41 successfully genotyped tSNPs. We found that seven haplotypes from four haplotype blocks located within three genes (NBS1, PTEN, and BRIP1) were significantly associated with breast cancer risk. Among these, four haplotypes (ATC in block 1 of NBS1, GCCCC and GCCCT in block 2 of NBS1, and GCT in block 2 of BRIP1) were correlated with breast cancer risk in sporadic cases (OR (95% CI) 1.350(1.124-1.623), 0.752(0.584-0.969), 0.803(0.649-0.993), and 0.776(0.604-0.997), respectively), and only one haplotype (GGCCT in block 2 of NBS1) was significantly associated with breast cancer risk in familial and early-onset cases (OR(95% CI) 1.902(1.134-3.191)). CONCLUSIONS Four haplotypes within two genes (NBS1 and BRIP1) involved in the monoubiquitinated FANCD2-DNA damage-repair pathway are significantly associated with increased sporadic breast cancer risk, while one haplotype within NBS1 is correlated with an increased risk of familial or early-onset breast cancer, indicating that specific haplotypes may be distinct predictors of breast cancer.

E3 Ubiquitin Ligase UBR5 Drives the Growth and Metastasis of Triple-Negative Breast Cancer

Patients with triple-negative breast cancers (TNBC) are at high risk for recurrence and metastasis at an early time despite standard treatment, underscoring the need for novel therapeutic modalities. Here, we report for the first time a distinctive and profound role of the E3 ubiquitin ligase UBR5 in the growth and metastasis of TNBC. An analysis of primary TNBC specimen by whole-exon sequencing revealed strong gene amplifications of UBR5 associated with the disease. UBR5 overexpression in TNBC tissues was confirmed at mRNA and protein levels. CRISPR/Cas9-mediated deletion of ubr5 in an experimental murine mammary carcinoma model of TNBC dramatically abrogated tumor growth and metastasis in vivo, which could be reversed completely via reconstitution with wild-type UBR5 but not a catalytically inactive mutant. Loss of UBR5 caused an impairment in angiogenesis within the tumor, associated with increased apoptosis, necrosis, and growth arrest. Absence of UBR5 in the tumor triggered aberrant epithelial-to-mesenchymal transition, principally via abrogated expression of E-cadherin, which resulted in severely reduced tumor metastasis to secondary organs. Use of NOD/SCID mice revealed that tumor-derived UBR5 facilitated tumor growth in a manner completely dependent upon immune cells in the microenvironment, whereas it promoted metastasis in a tumor cell-autonomous fashion. Our findings unveil UBR5 as a novel and critical regulator of tumor growth, metastasis, and immune response and highlight the potential for UBR5 as an effective therapeutic target for the treatment of highly aggressive breast and ovarian cancers that fail conventional therapy. Cancer Res; 77(8); 2090-101. ©2017 AACR.

Mutation screening of 10 cancer susceptibility genes in unselected breast cancer patients.

Variants of cancer susceptibility genes other than BRCA1/2 have been proved to be associated with increased risks of breast cancer. This study was performed to investigate the spectrum and prevalence of mutations in 10 cancer susceptibility genes in paired tumor/normal tissues of 292 unselected Chinese breast cancer patients. We performed an analysis of germline and somatic variants in ATM, CDH1, CHEK2, ESR1, GATA3, MAP3K1, MSH2, PALB2, RB1 and STK11 genes by integrating microfluidic PCR‐based target enrichment and next‐generation sequencing technologies. In total, 3 germline and 25 somatic deleterious mutations were found among 27 patients (9.25%), and 17 of them were novel mutations. Most deleterious mutations were prevalent in luminal A invasive breast cancer (P = .014). We also observed 83 variants of uncertain significance (VUS) in 100 patients (34.25%), 23 of which were predicted to be deleterious by in silico prediction programs (MetaSVM and MetaLR). VUS carriers had higher positive rate of lymph node metastasis than non‐carriers (P = .008) and were predominantly present in ER+ tumors (P = .018). Our findings would enhance the understanding of the molecular mechanisms of breast cancer in Chinese population.

Integrated analysis of promoter methylation and expression of telomere related genes in breast cancer

Telomeres at the ends of eukaryotic chromosomes play a critical role in tumorgenesis. Using microfluidic PCR and next-generation bisulfite sequencing technology, we investigated the promoter methylation of 29 telomere related genes in paired tumor and normal tissues from 184 breast cancer patients. The expression of significantly differentially methylated genes was quantified using qPCR method. We observed that the average methylation level of the 29 telomere related genes was significant higher in tumor than that in normal tissues (P = 4.30E-21). A total of 4 genes (RAD50, RTEL, TERC and TRF1) showed significant hyper-methylation in breast tumor tissues. RAD51D showed significant methylation difference among the four breast cancer subtypes. The methylation of TERC showed significant association with ER status of breast cancer. The expression profiles of the 4 hyper-methylated genes showed significantly reduced expression in tumor tissues. The integration analysis of methylation and expression of these 4 genes showed a good performance in breast cancer prediction (AUC = 0.947). Our results revealed the methylation pattern of telomere related genes in breast cancer and suggested a novel 4-gene panel might be a valuable biomarker for breast cancer diagnosis.

Abstract 4893: High-throughput mutation sequencing of the full exon regions in BRCA1 and BRCA2 genes using nanofluidic-PCR prepared libraries

Breast cancer is one of the most common cancers and also the principal cause of cancer-related death among women in the world. BRCA1 and BRCA2 genes contribute to DNA repair and transcriptional regulation in response to DNA damage. Mutations of BRCA1/2 genes greatly increase lifetime risk to develop breast cancer and these mutations are frequently observed in hereditary breast cancer. In addition, recent studies suggested that breast cancer patients who carry a BRCA1 or BRCA2 germline mutation benefit from poly (ADP-ribose) polymerase (PARP) inhibitors. Therefore, it would be of great interest to develop a high-throughput approach for mutation screening of the full exon regions in BRCA1 and BRCA2 gene. We have developed a targeted gene exon-sequencing approach using a nanofluidic PCR platform, the Access Array™ system, which enables simultaneous amplification of 48 genetic regions (amplicons) from 48 samples in parallel. We have applied this system to generate the libraries of BRCA1 and BRCA2 coding regions. Total 120 target-specific primer pairs were designed covering all 22 exons of BRCA1 and 26 exons of BRCA2. A primer design strategy was applied to incorporate sample-specific barcodes and Illumina sequencing adaptors into each amplicon, allowing direct sequencing without further library preparation. The exon regions of these 2 genes have been amplified on Access Arrays using 48 multiplex mixes of the 120 primer sets in paired tumor/normal tissues from 144 breast cancer patients. We will present the next-generation sequencing data of BRCA1 and BRCA2 mutation screening collected from amplicon libraries generated using the Access Array system. Our results indicate the excellent utilization of this approach in the mutation screening of BRCA1 and BRCA2 in clinical samples. Note: This abstract was not presented at the meeting. Citation Format: Jun Wang, Guoli Li, Ming Chen, Jianfu Heng, Xinwu Guo, Limin Peng, Fan Zhang, Zibo Li, Shouman Wang, Zhi Xiao, Lizhong Dai, Wenjun Yi, Lili Tang. High-throughput mutation sequencing of the full exon regions in BRCA1 and BRCA2 genes using nanofluidic-PCR prepared libraries. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4893. doi:10.1158/1538-7445.AM2015-4893

Cluster of differentiation 147 mediates chemoresistance in breast cancer by affecting vacuolar H+-ATPase expression and activity

Vacuolar H+-ATPase (V-ATPase) serves a key role in adjusting and maintaining the intracellular pH, as well as in regulating the drug resistance of tumor cells. In recent years, the expression level of V-ATPase has been considered to be able to predict the sensitivity of breast cancer cells to chemotherapy drugs. Cluster of differentiation 147 (CD147) is known to serve a key role in the development and progression of breast cancer. The present study aimed to identify the role CD147 and V-ATPase in chemoresistance in breast cancer, and to characterize the regulation of CD147 on V-ATPase. Firstly, the expression levels of CD147 and V-ATPase were detected in chemotherapy-resistance breast cancer samples. It was demonstrated that V-ATPase was highly expressed in chemotherapy-resistance breast cancer samples, and that its expression was correlated with CD147 expression. Subsequently, MCF-7 and MDA-MB-231 cells were used to study the regulatory effect of CD147 on the expression and function of V-ATPase. Gene transfection or small interfering RNA transfection were used to control the expression of CD147 in the two cell lines. The results revealed that the overexpression of CD147 increased the expression of V-ATPase in MCF-7 cells, whereas CD147 knockdown decreased V-ATPase expression in MDA-MB-231 cells. It was also observed that CD147 affected the V-ATPase activity, regulating the transmembrane pH gradient of cancer cells. These results demonstrated that CD147 was associated with the sensitivity of chemotherapeutic drugs of epirubicin and docetaxel, while pantoprazole was able to partially reverse the CD147-mediated chemoresistance in breast cancer. Therefore, the current study provided a possible mechanism for further examination of drug resistance in breast cancer.

Abstract P1-08-03: Association analysis of single-nucleotide polymorphisms in FANCD2-DNA damage repair pathway genes with breast cancer risk

Purpose: The aim of the study was to estimate breast cancer risk conferred by individual single-nucleotide polymorphisms (SNPs) of breast cancer susceptibility genes in the monoubiquitinated FANCD2–DNA damage repair pathway. Methods: We selected 48 tagging SNPs (tSNPs) from eight breast cancer susceptibility genes (TP53,PTEN,NBS1,BRIP1,PALB2,ATM,CHEK2 and RAD50) involved in the monoubiquitinated FANCD2–DNA damage repair pathway.The 48 tSNPs were genetyped by SNPscan in 734 women with breast cancer(534 sporadic cases and 200 early-onset and familial cases) and 672 sex- and age-matched healthy controls from Hunan and Sichuan Province of China.The odds ratio were calculated by logistic regression analysis under co-dominant model ,dominant model and recessive model respectively. Results: Forty-five tSNPs were successfully genotyped by SNPscan, and the call rates for each tSNP were above 98.9%. We found that 13 tSNPs of five genes (PALB2, TP53, NBS1, PTEN, and BRIP1) were significantly associated with breast cancer risk. A total of five tSNPs (rs2299941 of PTEN, rs2735385, rs6999227, rs1805812, and rs1061302 of NBS1) were tightly associated with breast cancer risk in sporadic cases, and five other tSNPs (rs1042522 of TP53, rs2735343 of PTEN, rs7220719, rs16945628, and rs11871753 of BRIP1) were tightly associated with breast cancer risk in early-onset and familial cases. We have not found significant tSNPs in the other three genes (ATM, RAD50, and CHEK2).Three tSNPs of TP53(rs12951053, rs1042522 and rs8064946),three tSNPs of BRIP1(rs16945628, rs7220719 and rs11871753) and rs2735343 of PTEN can significantly increase the risk of breast cancer,and most of these were under the analysis of early-onset and familial cases.Four tSNPs of NBS1(rs2735385, rs6999227, rs1805812 and rs1061302),rs2299941 of PTEN and rs513313 of PALB2 can significantly decrease the risk of breast cancer,and most of these were under the analysis of sporadic cases. Conclusions: Some of the tSNPs of five breast cancer susceptibility genes (PALB2, TP53, NBS1, PTEN, and BRIP1) involved in the monoubiquitinated FANCD2–DNA damage repair pathway were significantly associated with breast cancer risk in women from Hunan and Sichuan Province of China.Some locuses can increase breast cancer risk,and others can decrease breast cancer risk.But the majority of the tSNPs are located in the intron domain and their functions are unknown, so larger studies are needed to research the functions of these genes further. Citation Format: Chen F, Tang L, Huang J. Association analysis of single-nucleotide polymorphisms in FANCD2-DNA damage repair pathway genes with breast cancer risk. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-08-03.