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Breast cancer (BrCa) is the most common form of cancer in women worldwide, impacting nearly 2.1 million women each year. Importantly, current treatment options for metastatic breast cancer fail to elicit an anti-tumor response, leading to a 5-year survival rate below 30%. While most classes of therapy focus on inhibiting or targeting specific proteins, long non-coding RNAs (lncRNAs) offer a unique, relatively untapped, source of potential therapeutic targets. Ongoing experiments are evaluating the regulatory role of the lncRNA YTHDF3-AS1 (ENSG00000270673), which has the potential to develop into a novel therapeutic intervention of BrCa. Through genome-wide analysis, we found that YTHDF3-AS1 expression correlates with essential developmental regulatory genes such as CA3, VPS28, EXOSC4, and TM2D2 and according to pathway analysis is likely linked with the Wnt pathway. Additionally, TCGA data reveals that over 5% of patients with BrCa have an increased copy number of YTHDF3-AS1. Although the role of YTHDF3-AS1 has yet to be explored, YTHDF3 expression was recently shown to be a critical driver of breast cancer metastasis. Of note, YTHDF3-AS1 is upstream of the protein-coding region, but within the promoter region of the YTHDF3 gene. Mechanistically, this allows the lncRNA YTHDF3-AS1 to potentially upregulate the expression of the protein-coding YTHDF3 gene, as two known functions of antisense lncRNAs describe the ability to 1) induce nearby gene expression through binding to the promoter region and 2) stabilize the RNA product of the nearby gene through binding to the 5’UTR. Interestingly, this manner of relationship between antisense (or "divergent") lncRNAs and their protein-coding partner was shown to be highly prevalent in pluripotent stem cells, with the downregulation of both the antisense and protein-coding transcripts following differentiation. Together, our preliminary analysis and previous literature suggest that the lncRNA YTHDF3-AS1 may contribute to the upregulation of YTHDF3, which is a known metastatic promoting protein. Our current study aims to determine the functional role of YTHDF3-AS1 in BrCa oncogenesis, with an emphasis on the mechanistic actions of this lncRNA for the development of future therapeutic approaches. Citation Format: Alakesh Bera, Surya Radhakrishnan, Eric Russ, Diya Biswas, Madhan Subramanian, Harvey B. Pollard, Hai Hu, Craig D. Shriver, Roopa Biswas, Meera Srivastava. Functional role of long non-coding RNA YTHDF3-AS1 in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5828.

The chromosome 8q24 gene contains functional risk loci for multiple cancers, including prostate, colon, and breast. Breast cancer (BrCa) is one of the deadliest diseases among women. Our previous studies using TCGA clinical survival data (Cell. 2018; 173(2):400-416.e11) indicated that several genes are contributed significantly towards the survival of patients with BrCa. Over 75 such most significant genes were screened for further studies in terms of their contribution towards BrCa patient's survival. We found two genes (EXOSC4 and VPS28) with over 10% genetic copy number alterations located at chromosome 8q24 gene loci. Exosome Component 4 (EXOSC4) is located at 8q24.3 and associated with a non-catalytic component of the RNA exosome complex, which has 3' to 5' exoribonuclease activity. It also participates in cellular RNA processing and degradation events. However, the functional role of EXOSC4 towards BrCa progression or survival remains unknown. To understand the molecular basis of an elevated level of EXOSC4 in BrCa propagation, we screened the expression of this protein in different BrCa cell-lines. We also compared the expression of VPS28 within same cell lines. Data indicated that EXOSC4 was co-expressed with VPS28 and Myc, and was also secreted into extracellular space. Therefore, we next evaluated the level of EXOSC4 in patient's serum samples by reverse phase protein array (RPPA). We have well defined and categorized BrCa patients serum samples (n=240) from the Clinical Breast Care Project. The data indicated that the elevated level of EXOSC4 is associated with worse overall survival. In addition, results also showed that the high level of EXOSC4 is linked with breast cancer recurrence.In summary, through multiple modality studies we have confirmed the copy number alteration of EXOSC4 is associated with BrCa patient survival. Once validated, serum level of EXOSC4 could be a predictive biomarker for BrCa progression and survival. Additional experiments are ongoing to evaluate the regulatory role of the EXOSC4 gene which has the potential to develop into a novel therapeutic intervention of BrCa. Funding Sources: This work was supported by the grant to Dr. Meera Srivastava (DoD, DAMD17-03-1-0107). The work was also supported by the Collaborative Health Initiative Research Program (CHIRP) funding (CHIRP ID# IAA-A-HL-14-007). Disclaimers: The contents of this publication are the sole responsibility of the author(s) and do not necessarily reflect the views, opinions or policies of The Uniformed Services University of the Health Sciences (USUHS), The Henry M. Jackson Foundation (HJF), the Department of Defense (DoD), the Departments of the Army, Navy, or Air Force. Correspondence: Email: meera.srivastava@usuhs.edu Citation Format: Alakesh Bera, John Karaian, Madhan Subramanian, Michael Eklund, Eric Russ, Harvey B. Pollard, Hai Hu, Craig D. Shriver, Meera Srivastava. EXOSC4, a novel gene at chromosome 8q24 loci is linked with breast cancer progression and is a prognostic marker for breast cancer survival [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4322.

Background: Quantitative estimation of circulating cell-free DNA (cfDNA) isolated from serum by noninvasive procedures can serve as a potential biomarker for the early detection of many cancers. However, a simple, straightforward technique is unavailable to estimate the cfDNA in clinical labs. Moreover, the prognostic value of cfDNA in patients with breast cancer (BrCa) is currently under debate. The aim of this study was to develop a simple yet effective quantitative method for measuring the cfDNA in serum and to eventually investigate the relationship between cfDNA and the occurrence of recurrence in BrCa patients. Methods: A total of 240 patient cases (n=240) were selected and are comprised of different subtypes of breast cancer patients and control individuals. We selected 21 serum samples from patients which showed recurrence after 4-7 years of disease-free survival. For the compare studies, each of the recurrent and non-recurrent serum samples was incubated with the SYBR Green I (2 μM). A standard graph was also made with known DNA concentration to calculate the amount of cfDNA in these recurrent and non-recurrent serum samples. Additionally, a comparative study was also performed with the serum of patients with non-recurrent BrCa versus healthy patients. Results: We develop a simple fluorescent based measuring technique which can easily estimate the cfDNA in one step. SYBR Green binds to DNA, and as a result, the fluorescence of SYBR Green increases substantially. Global Wilcoxon analyses were performed to compare the cfDNA amount between non-recurrent and recurrent patients. There is a significant difference in fluorescent intensities between recurrent patients' samples versus non-recurrent patients which are directly proportional to the cfDNA levels. The amount of cfDNA is higher in recurrent patient (ratio is 1.3 up; p= 0.03; AUC=0.76) compared to similar non-recurrent patients. While we compared the fluorescence data between normal/healthy patients versus non-recurrent is turned out as non-significant (healthy to non-recurrent ratio = 1.03; p= 0.20, AUC=0.61). Conclusion: In this current study, we developed a straightforward one-step technique to measure the amount of cfDNA in serum, which can easily translate into a clinical diagnostic tool. To the best of our knowledge, this is the first report which demonstrates serum cfDNA as an early detection marker for recurrent breast cancer patients. The relatively high level of cfDNA in the serum of recurrent breast cancer patients compared to non-recurrent breast cancer patients indicates an uncovered circulating genetic information which triggers the cancer recurrence pathway to relapse cancer in the near future. Citation Format: Bera A, Eidelman O, Russ E, Landa A, Karaian J, Eklund M, Hu H, Pollard HB, Shriver CD, Srivastava M. Circulating cell-free DNA in serum as a marker for the early detection of tumor recurrence in breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-26.

Analysis of somatic protein-coding mutations, particularly via tumor exome sequencing, has driven discovery of novel cancer genes in recent years. However, ~97% of the human genome is non-coding and not addressable by exome-targeting methods. Tumor whole genome sequencing, by contrast, allows for genome-wide discovery of somatic events and may identify novel non-coding driver mutations. In conjunction, flexible tools employing principled statistical methods are needed for discovery of recurrent somatic driver mutations from tumor whole genomes. Here, we developed a computational toolset that addresses these needs and describe its application to a novel cohort of lung adenocarcinoma tumors generated by the APOLLO (Applied Proteogenomics OrganizationaL Learning and Outcomes) consortium. Our toolset flexibly computes feature-wise and hotspot somatic mutation enrichment statistics in both protein-coding and non-coding (e.g. promoters, enhancers) genomic regions. Additionally, we incorporated an affinity propagation-based clustering procedure that groups test regions by user-specified genomic covariates that can influence local mutation properties. We applied our toolset to several TCGA datasets of tumor exome sequencing. We found strong agreement between our results and those of other tools: among the top 15 genes called at FDR < 0.01 by MutSigCV, we observed a median overlap of 75.7% with genes ranked in the top 15 by our methods. We next applied our toolset to tumor whole genome sequencing data from our APOLLO lung adenocarcinoma cohort. During analysis, we simultaneously controlled for several genomic covariates, including GC/CpG content, replication timing, and proximal mRNA expression. Gene-centric analysis identified several significantly mutated genes, including KRAS, STK11, and TP53 (FDR < 0.005). We examined matched mRNA-seq data from this cohort and found significant over-expression of KRAS mRNA in samples possessing a non-silent coding mutation in this gene (p < 0.02). We additionally assessed somatic mutation enrichment in promoters, 3' UTRs, non-coding genes, and lung-specific enhancers. From these results, we found significant enrichment of somatic mutations in the body of the long non-coding RNA NEAT1. Among the tumors with somatic NEAT1 mutations, a single sample was hypermutated at this locus and corresponded with the lowest observed RNA expression for this lncRNA in our cohort (TPM of 12.2 vs. overall mean TPM of 84.2). In conclusion, we developed a flexible toolset for interrogating both coding and non-coding landscapes from tumor whole genomes. We applied our methods to a novel cohort of lung adenocarcinoma tumors and identified recurrently mutated genes and non-coding regions, including the NEAT1 lncRNA. Citation Format: Anthony R. Soltis, Coralie Viollet, Harvey B. Pollard, Christopher A. Moskaluk, Robert F. Browning, Clifton L. Dalgard, Craig D. Shriver, Matthew D. Wilkerson. Flexible discovery of recurrent coding and non-coding mutations in tumor whole genomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2281.

Tumor suppressor function for Annexin A7 (ANXA7; 10q21) is based on cancer-prone phenotype in Anxa7(+/−) mouse and ANXA7 prognostic role in human cancers. Because ANXA7-caused liposome aggregation can be promoted by arachidonic acid (AA), we hypothesized that the phospholipid-binding tumor suppressor ANXA7 is associated with AA cascade. In a comparative study of ANXA7 versus canonical tumor suppressor p53 effects on AA lipoxygenation pathway in the p53-mutant and androgen-insensitive DU145 prostate cancer cells, both tumor suppressors altered gene expression of major 5-lipoxygenase (LOX) and 15-LOXs, including response to T helper 2 (Th2)-cytokine [interleukin-4 (IL-4)] and endogenous steroids (mimicked by dexamethasone). Wild-type and mutant ANXA7 distinctly affected expression of the dexamethasone-induced 15-LOX-2 (a prostate-specific endogenous tumor suppressor) as well as the IL-4-induced 15-LOX-1. On the other hand, wild-type p53 restored 5-LOX expression in DU145 to levels comparable to benign prostate epithelial cells. Using mass spectrometry of DNA affinity–enriched nuclear proteins, we detected different proteins that were bound to adjacent p53 and estrogen response elements in the 5-LOX promoter in DU145 cells introduced with ANXA7 versus p53. Sex hormone regulator 17-β hydroxysteroid dehydrogenase 4 was identified under p53 introduction, which induced the 5-LOX expression. Meantime, nuclear proteins bound to the same 5-LOX promoter site under introduction of ANXA7 (that was associated with the repressed 5-LOX) were identified as zinc finger proteins ZNF433 and Aiolos, pyrin domain–containing NALP10, and the p53-regulating DNA repair enzyme APEX1. Thus, ANXA7 and p53 can distinctly regulate LOX transcription that is potentially relevant to the AA-mediated cell growth control in tumor suppression. (Cancer Res 2006; 66(19): 9609-16)

Background: Accurate detection of somatic mutations is critical for informing targeted therapy options. Prevalent non-cancer cell admixture complicates this detection in breast cancer. Conventional mutation detection relies on DNA sequencing; however in prior work, we demonstrated that combining RNA and DNA sequencing increases mutation signal strength, or mutant allele fraction (MAF). The ratio of RNA MAF versus DNA MAF (RNA:DNA MAF) was greatest in low purity breast tumors. We hypothesized that this elevation is biologically driven and would be conserved in a second, distinct tissue specimen of the same tumors. Here, we compare mutation characteristics between two tissue blocks in a cohort of breast tumors (n = 8) to evaluate possible preservation of RNA versus DNA mutation signal throughout the tumor. Methods: We selected four high purity and four low purity breast tumors (“Block1”) from The Cancer Genome Atlas (TCGA) cohort and associated ABSOLUTE purity analysis. For these tumors, we acquired a second tissue block (“Block2”) not analyzed by TCGA, cut sections, analyzed sections by H&E stains, and extracted nucleic acids. Whole genome DNA sequencing and mRNA sequencing was performed for Block2 specimens using Illumina X and NextSeq 500 sequencers, respectively. Somatic mutations in Block2 were detected using UNCeqR and compared to published UNCeqR somatic mutations from TCGA. We then evaluate MAF characteristics in the entire TCGA breast tumor cohort (n = 695). Results: Tumor purity estimates, determined by histology and by sequencing, were reduced in Block2 of the low purity tumor set versus the high purity tumor set, consistent with Block1 analysis. Molecular properties of genome-wide gene expression and somatic DNA copy number were highly similar between block-mated specimens (p < 0.01). We then identified expressed mutations present in Block1 and Block2 of the same tumor and compared the MAFs on these common mutations. DNA MAF and RNA MAF were each significantly correlated between Block1 and Block2 (p < 1e-12 in both cases). The average RNA:DNA MAF was 2.5 for the cohort, indicating that RNA mutation signal is greater than DNA in general. In Block2 specimens, the RNA:DNA MAFs were significantly greater in the low purity tumor set than the high purity tumor set (mean 2.7 versus 2.1, p < 6e-5), reflecting the same trend observed in Block1 specimens. Analyzing the entire TCGA cohort, RNA:DNA MAF was positively correlated with proliferation pathway gene expression (p < 3e-16 ) and was greatest in the Basal subtype versus other subtypes (p < 2e-9). Conclusion: Mutant allele fraction both of DNA and of RNA was conserved across breast tumor subsections. Low purity and basal subtype breast tumors had elevated RNA:DNA MAF supporting a relationship to underlying biology and identifying classes of tumors with pronounced benefit for DNA and RNA integrated mutation analysis. Citation Format: Jerez Te, Coralie Viollet, Xijun Zhang, Jatinder Singh, Jeffrey A. Hooke, Harvey B. Pollard, Hai Hu, Craig D. Shriver, Clifton L. Dalgard, Matthew D. Wilkerson. Reproducible elevation of RNA versus DNA mutation signal in low purity breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5403. doi:10.1158/1538-7445.AM2017-5403