Poster T6 - Transcript Characterization of lncRNAs in Triple Negative Breast Cancer

Introduction: Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer and has the highest rate of metastasis for all breast cancer subtypes. TNBC is often diagnosed in late stages due to its high rate of proliferation and the lack of efficient and effective methods of diagnosis. There are currently a limited number of tools available for monitoring TNBC. Certain long non-coding RNA (lncRNA), such as H19, show potential for use as biomarkers for TNBC prognosis and monitoring. Typical expression of the H19 gene produces a lncRNA active in fetal development for regulation of cell proliferation. H19 expressed in cancer tissue is significantly higher than in non-cancerous tissue and has been shown to heavily influence tumorigenesis and metastasis within cancer cells. Our objective is to characterize the presence of H19, other lncRNAs, and their isoforms in TNBC for use as future biomarkers.

Materials and

Methods: We performed direct RNA sequencing using Oxford Nanopore Technologies on TNBC patient tissue and plasma, along with MDA-MB-231 (TNBC) and MCF-12A (human breast epithelial) cells. Exons of selected non-coding RNAs known to be associated with breast cancer were used to search against the sequencing results. We are also transfecting cells with different transcripts of H19, to interrogate which splice variants are related to cancer progression, cell proliferation, and EMT. 48 hours after transfection, the cells are fixed and immunostained and assessed for stemness via flow cytometry (results visualized in Figure 1).

Results, Conclusions, and Discussions: TNBC patient tissue, MCF-12A, and MDA-MB-231 cells showed various exon hits. TNBC patient plasma did not result in any hits from the selected lncRNA exon search. TNBC-specific exon hits were found for H19, DANCR, MALAT1, NEAT1, SAMMSON, SNHG-12, HISLA, and TINCR. No hits were found for H19 in cell lines, and only a few hits were found in TNBC tissue samples. LINC02620 (BCRT-1) had no TNBC-specific exon hits. In the future, we will continue to sequence patient plasma and tissue samples to determine which lncRNAs are selectively expressed at specific stages of tumor development. Additionally, we investigated the function of the H19 T3 transcript isoform through direct transfection of the T3 transcript to the cells. The results of the transfection and immunostaining procedure are visualized in Figure 1. There is a tendency that T3 transcript may reduce the ALDH activity, pending additional data points. ALDH is used as one proxy measurement for stemness and possible chemotherapeutic resistance.
This study provides insight into future advancements in TNBC diagnosis and prognosis management through the characterization of multiple lncRNAs as potential biomarkers. Identifying associated degrees of stemness with ectopic lncRNA expression can aid in TNBC stage diagnosis and progression monitoring. This is improved by the isoform-level resolution we are aiming for. From the improved analytical understanding of this study on TNBC lncRNA expression, the development of better biomarker screens can be achieved for TNBC detection and treatment guidance.
lncRNA ectopic expression is a rapidly expanding research topic because it was previously thought that DNA outside of genes was “junk” DNA. Now it is understood that lncRNAs have highly critical roles in regulatory processes that have yet to be fully elucidated. The function of lncRNAs in cancer and tumorigenesis is even more of a hot topic because lncRNAs can better explain possible routes of tumorigenesis than analysis of protein-coding genes alone. Research on lncRNAs provides an opportunity to understand their role in cells and in cancer, as well as possible monitoring biomarkers for progression.

Acknowledgements (Optional): National Institute Of General Medical Sciences of the NIH P20GM139769.
CP is partially supported by NIH NIGMS 3P20GM121342-03S1.
COBRE Grant P20GM109094
Prisma Health Transformative Seed Grant
This work was supported in part by the Clemson University Creative Inquiry Program.