Background: Hurthle cell carcinoma of the thyroid is a rare form of thyroid cancer. It may present as a low grade tumour or can present as a more aggressive metastatic carcinoma. Hurthle cell carcinoma has the highest incidence of metastasis among all differentiated thyroid cancers. Most commonly haematogenous spread to lungs, bones and brain, however spread to regional lymph nodes is not uncommon. The breast is a rare site for metastasis from extramammary sources. We present the first case of breast metastasis from Hurthle cell carcinoma of the thyroid.

Metastatic potential in basal-like breast cancers typically correspond with increased enrichment of EpCAM-/CD49f- cancer stem cells (CSC). With this premise in mind, it is important to better understand the mechanistic driver of these cell populations and their distinctive potential to interact with the tumor microenvironment (TME) for cancer promotion. Previous work from our lab has compared the 450K DNA methylation profile of EpCAM-/CD49f- poor breast cancer cell lines to that of EpCAM-/CD49f- enriched breast cancer cell lines and found the IL32 promoter to be hypomethylated in EpCAM-/CD49f- enriched cell lines, a result which corresponded basal-like patient samples in TCGA. By identifying IL32 being differentially regulated in CSC-enriched cell lines, we further sought to characterize IL32’s role in breast cancer aggressiveness. We first were able to identify several overarching mechanisms altered in siIL32 treated SUM15PT cells by RNAseq differential expression analysis (FDR p-value <0.01). Most notable from our RNAseq results was the significant enrichment of upregulated pathways involved in extracellular matrix (ECM) organization as well as significant enrichment of downregulated pathways involved in cellular and replicative stress responses. Particular examples of transcripts differentially expressed between control and siIL32-treated SUM159PT cells included COL6A1, ITGB3, and CD24 that were upregulated as well as NQO1, HMOX1, and CXCL2/CXCL3 that were downregulated. Furthermore, IL32 suppression decreased SUM159PT invasion in both an ECM-matrix cell invasion assay and a chick CAM xenograft/angiogenesis model. From our RNAseq results, we then performed a multi-pathway protein phosphorylation array to determine whether any key signaling events were affected by siIL32 knockdown in SUM159PT cells. Based on this approach, we were able observe a significant decrease in phosphorylated JNK and phosphorylated NFKB in siL32-treated cells when compared to control, both of which are well-established events that can coordinate both cell stress responses and cellular invasion. Collectively, our results reflect the notion that differential IL32 expression by promoter hypomethylation in breast CSCs plays a role to mitigating intracellular stress and subsequently allowing for breast cancer cell invasion and metastasis. Citation Format: Megan A. Wilson, Elayne M. Benson, Emma Gray, Paige Cairns, Maria Ouzounova, Hasan Korkaya, Austin Y. Shull. Epigenetic driven IL32 expression contributes to a JNK related cell stress response in breast cancer stem cells to promote cellular invasion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3595.

Metastatic basal-like breast cancers are believed to correspond with EpCAM-/Cd49f- cancer stem cell (CSC) enrichment. As well, basal-like breast cancers typically correspond with tumor inflammation and immunoediting phenotypes. However, the exact interplay between CSCs and the inflammatory signature of basal-like breast cancers is not well understood. To provide insight regarding the clinical overlap between breast cancer stem cells and tumor inflammation, we compared the 450K DNA methylation profile of EpCAM-/CD49f- CSCs from the isogenic MCF10A p53-/PTEN- breast cell line against the corresponding EpCAM+/CD49f+ and EpCAM-/CD49f+ subpopulations to determine whether differential DNA methylation occurred within the promoters of immune-related genes in CSCs. In addition, we also overlapped the 450K DNA methylation profile from 16 established breast cancer cell lines of varying EpCAM-/CD49f- concentrations to compare against the isolated CSCs. Based on our results, we identified 1432 differentially methylated promoter regions overall (ANOVA FDR p-value <0.001) and found IL32 to be differentially hypomethylated in the EpCAM-/CD49f- enriched cell lines. This hypomethylation of IL32 corresponded with increased expression of the beta isoform of IL32. Results from the cell lines were mirrored in The Cancer Genome Atlas (TCGA) breast cancer datasets, which revealed decreased promoter DNA methylation and increased gene expression of IL32 in basal-like patients. Further analysis of TCGA data using Gene Set Enrichment Analysis (GSEA) revealed that transcripts that tightly correlate with IL32 expression were preferentially involved in NF-kappaB mediated inflammation, with specific examples including REL, CCL5, PIK3CD, and IDO1. Furthermore, publicly available H3K27Ac and BRD4 ChIPseq data revealed that the IL32 promoter in the basal-like breast cancer cell line SUM159PT contains a high presence of H3K27 acetylation and BRD4 recruitment, with the latter event being disrupted by JQ1 treatment. These results complemented qRT-PCR results showing the IL32-beta isoform being quickly suppressed by 1uM JQ1 in SUM159PT as well as chick chorioallanotoic membrane (CAM) xenograft assays demonstrating suppressed metastasis and neovascularization of SUM159PT treated with JQ1. Collectively, these findings highlight the potential impact of IL32 promoter hypomethylation in basal-like breast cancer stem cells and how the overall epigenetic signature may predispose CSCs towards an immunomodulatory phenotype. Citation Format: Emma V. Gray, Caroline E. Dyar, Maria Ouzounova, Max S. Wicha, Hasan Korkaya, Austin Y. Shull. IL32 expression is epigenetically regulated in EpCAM-/Cd49f- basal-like breast cancers and can be suppressed by the bromodomain inhibitor JQ1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3683.

Basal‐like breast cancers typically correspond with increased enrichment of EpCAM‐/CD49f‐ cancer stem cells (CSC) and a propensity toward metastasis. However, the molecular mechanisms underlying these general characteristics are not well understood. To provide further insight concerning CSCs and their intrinsic metastatic mechanisms, we compared the 450K DNA methylation profile of EpCAM‐/CD49f‐ poor breast cancer cell lines to that of EpCAM‐/CD49f‐ enriched breast cancer cell lines. From our results, we were able to determine and highlight IL32 as a gene whose promoter is hypomethylated in EpCAM‐/CD49f‐ enriched cell lines. The hypomethylated IL32 promoter corresponded with increased IL32 expression in both cell lines and basal‐like breast cancer patients from The Cancer Genome Atlas (TCGA) database. Interestingly, increased IL32 expression preferentially occurred for the IL32‐beta transcript and corresponds with previous reports demonstrating that IL32‐beta is not secreted from the cell like other canonical interleukins and preferentially localizes to the mitochondria in breast cancer cells. Additionally, expression of the beta‐transcript could be suppressed when CSC‐enriched cells were treated with the BET‐bromodomain inhibitor JQ1. Because of this phenomenon, we sought to determine the effects of suppressing IL32 in the EpCAM‐/CD49f‐ enriched cell line SUM159PT via siRNA‐mediated knockdown and subsequent RNAseq differential expression analysis. From our results, we determined that transcripts involved in extracellular matrix (ECM) organization and collagen/integrin interaction were preferentially affected by IL32 silencing. Additionally, IL32 suppression decreased the invasiveness of SUM159PT based on an ECM‐matrix cell invasion assay. Collectively, our results reflect the notion that differential IL32 expression by promoter hypomethylation in breast CSCs plays a role in ECM remodeling for purposes of breast cancer cell invasion and metastasis.

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