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Breast cancer is the most commonly diagnosed and the second leading cause of cancer-related mortality among women worldwide. miR-518f-5p has been shown to modulate the expression of the metastasis suppressor CD9 in prostate cancer. However, the role of miR-518f-5p and CD9 in breast cancer is unknown. Therefore, this study aimed to elucidate the role of miR-518f-5p and the mechanisms responsible for decreased CD9 expression in breast cancer, as well as the role of CD9 in de novo tumor formation and metastasis. miR-518f-5p function was assessed using migration, adhesion, and proliferation assays. miR-518f-5p was overexpressed in breast cancer cell lines that displayed significantly lower CD9 expression as well as less endogenous CD9 3′UTR activity, as assessed using qPCR and dual luciferase assays. Transfection of miR-518f-5p significantly decreased CD9 protein expression and increased breast cell migration in vitro. Cd9 deletion in the MMTV/PyMT mouse model impaired tumor growth, but had no effect on tumor initiation or metastasis. Therefore, inhibition of miR-518f-5p may restore CD9 expression and aid in the treatment of breast cancer metastasis.

Triple negative breast cancer (TNBC) is a highly aggressive subtype with a high rate of metastasis, early distant recurrence and resistance to therapy leading to worse survival than other breast cancer subtypes. There are no well-established biomarkers that can determine women who will do better and those who are likely to have poorer outcomes with TNBC, nor are there targeted therapies. Thus, the identification of prognostic and/or predictive biomarkers will enable tailored therapies based on their likelihood of disease outcomes and may prevent over- and under-diagnosis. Previous studies from our laboratory have identified four genes (ANP32E, DSC2, ANKRD30A and IL6ST/gp130) that are specific to TNBC and were associated with lymph node metastasis (LNmets), the earliest indicator of tumor progression via distal spread. This study aimed to validate these findings using absolute quantitation by digital droplet PCR (ddPCR) and to determine relationships with clinicopathological features and survival. Our analysis confirmed all four genes displayed significant expression differences between TNBC cases and non-TNBC cases. Moreover, low IL6ST expression was significantly associated with grade 3 disease, hormone receptor negativity and earlier age at diagnosis; low ANKRD30A expression was associated with tumor size; and high ANP32E expression was significantly associated with grade and the number of positive lymph nodes. Individually, three of the four genes were associated with relapse-free survival in TNBC and in combination, all four genes were significantly associated with TNBC survival, but not in hormone receptor-positive cases. Collectively our results suggest that the four genes may have utility in TNBC prognostication.

Background: Triple negative breast cancer (TNBC) is a highly metastatic and aggressive subtype of breast cancer which lacks receptors for-estrogen, progesterone and human epidermal growth factor 2. It accounts for approximately 10-20% of all breast cancer cases. It is commonly diagnosed in younger African-American women less than 40 years. There are no targeted therapies for this breast cancer subtype, therefore, chemotherapy and surgery remain the only options for treatment. However, there is a high chance of recurrence within three years of diagnosis and the majority of deaths occurs within the five years of diagnosis. High genomic aberration is a common event in this cancer where the differentially expressed genes in TNBC could be due to the variations in copy number of those genes. The dissemination of primary cancer cells to the lymphatic system represents one of the first signs of meta-static spread. In TNBC, any LN involvement is associated with worse disease-free and overall survival. Therefore, identification of copy number variations (CNVs) within LN metastases in this highly aggressive breast cancer subtype may serve as an indicator of prognosis. Objectives: The aim of the current study was to define regions of copy number gain or loss that are associated with metastasis to the lymph node in TNBC and their association with gene expression. Methods: Partek Genomic Suite was used to analyse the copy number variations in the study cohort containing 23 invasive ductal carcinoma (IDC), 12 lymph node metastasis (LNmets) and 3 normal adjacent tissues (NAT); as well as in validation cohort containing 70 IDC samples (Avery-Kiejda KA, Genom Data.2017;14:1-4).All the breast cancer samples were TNBC. The genomic segmentation algorithm was used for copy number detection. Moreover, their association with gene expression was also analysed using previous gene expression datasets (Accession No.GSE78758). Additionally, GO-enrichment and pathway analysis was performed to identify functional groups and pathways affected by copy number changes. Results: More CNV regions were amplified compared to loss in both the study and the validation cohort. The whole genome copy number profile was similar between IDC and lymph node metastases with 81% (263 in total) of amplified CNVs present in LNmets also present in matched IDCs, implying that many of the alterations in the primary tumour were carried over in metastasis. Very few genes were deleted and of those that were, only 2 were common to IDCs and LNmets. No significant CNVs were observed in NAT. Frequent amplification was mainly observed in chromosome 1q, 8q and 10p whereas the deletion in 4q, 5q and 14q was common in both IDC and metastasis samples. In total, 95 genes (66 gains and 29 losses) were only present in LNmet and not in IDCs from either the study or validation cohorts. According to GO-enrichment analysis, these LNmet associated genes were mainly enriched for DNA-binding transcription activity, cell fate specification and negative regulation of EGFR signalling pathway. Conclusion: This study has identified several regions of CNV in a well-defined TNBC cohort that could play major role in metastasis to lymph node. Citation Format: Mamta Pariyar, Andrea Mathe, Rodney Scott, Kelly Avery-Kiejda. Identification of copy number variation associated with lymph node metastasis in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-26.

BackgroundTriple negative breast cancer (TNBC) is a highly metastatic and aggressive subtype of breast cancer and cases presenting with lymph node involvement have worse outcomes. This study aimed to determine the regions of copy number variation (CNV) associated with lymph node metastasis in TNBC patients. MethodsCNV analyses were performed in a study cohort of 23 invasive ductal carcinomas (IDC), 12 lymph node metastases (LNmets) and 7 normal adjacent tissues (NAT); as well as in a second cohort containing 70 TNBC IDC samples and the same 7 NATs. CNVs associated genes were analysed using GO-enrichment and Pathway analysis and also integrated with gene expression analysis. The prognostic role for genes showing CNV based change in mRNA expression was determined using Kaplan-Meier (KM) plotter database. ResultsFor the IDCs, frequent amplification was evident in chromosomal regions 8q, 1q, 2(p and q), 3q24, 10p and 12p and deletion in 3p, 4 (p and q), 5q, 14q and 17p in contrast to LNmets showing frequent amplification of 4q, 2p, 3q24, 1q, 10p, 12p, 8q, 20p, 21q and 6p and deletion in 1p, 4q (4q21.1, 4q26) and 5q. A total of 686 (441 amplified and 245 deleted) genes were associated with lymph node metastasis. The LNmet-associated genes were highly enriched for “regulation of complement activation”, “regulation of protein activation cascade”, “regulation of humoral immune response”, “oxytocin signalling pathway” and “trail binding” pathways. Moreover, 6/686 LNmet-associated genes showed CNV-based changes in their mRNA expression of which, high expression of ASPM and KIF14 was significantly associated with worse relapse free survival.ConclusionThis study has identified several CNV regions in TNBC that could play a major role in metastasis to the lymph node.