Enhanced immortalization, HUWE1 mutations and other biological drivers of breast invasive carcinoma in Black/African American patients

Abstract

Black/African-American (B/AA) breast cancer patients tend to have more aggressive tumor biology compared to White/Caucasians. In this study, a variety of breast tumor molecular expression profiles of patients derived from the two racial groupings were investigated. Breast invasive carcinoma sample data (RNASeq version 2, Reverse Phase Protein Array, mutation, and miRSeq data) from the Cancer Genome Atlas were examined. The results affirm that B/AA patients are more likely than Caucasian patients to harbor the aggressive basal-like or the poor prognosis-associated HER2-enriched molecular subtypes of breast cancer. There is also a higher incidence of the triple-negative breast cancer (TNBC) among B/AA patients than the general population, a fact reflected in the mutation patterns of genes such as PIK3CA and TP53. Furthermore, an immortalization signature gene set, is enriched in samples from B/AA patients. Among stage III patients, TERT, DRAP1, and PQBP1, all members of the immortalization gene signature set, are among master-regulators with increased activity in B/AA patients. Master-regulators driving differences in expression profiles between the two groups include immortalization markers, senescence markers, and immune response and redox gene products. Differences in expression, between B/AA and Caucasian patients, of RB1, hsa-let-7a, E2F1, c-MYC, TERT, and other biomolecules appear to cooperate to enhance entry into the S-phase of the cell cycle in B/AA patients. Higher expression of miR-221, an oncomiR that facilitates entry into the cell cycle S-phase, is regulated by c-MYC, which is expressed more in breast cancer samples from B/AA patients. Furthermore, the cell migration- and invasion-promoting miRNA, miR-135b, has increased relative expression in B/AA patients. Knock down of the immortalization marker TERT inhibited triple-negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) cell viability and decreased expression of TERT, MYC and WNT11. For those patients with available survival data, prognosis of stage II patients 50 years of age or younger at diagnosis, was distinctly poorer in B/AA patients. Also associated with this subset of B/AA patients are missense mutations in HUWE1 and PTEN expression loss. Relative to Caucasian non-responders to endocrine therapy, B/AA non-responders show suppressed expression of a signature gene set on which biological processes including signaling by interleukins, circadian clock, regulation of lipid metabolism by PPARα, FOXO-mediated transcription, and regulation of TP53 degradation are over-represented. Thus, we identify molecular expression patterns suggesting diminished response to oxidative stress, changes in regulation of tumor suppressors/facilitators, and enhanced immortalization in B/AA patients are likely important in defining the more aggressive molecular tumor phenotype reported in B/AA patients.

Keywords: ARACNe, Algorithm for the Reconstruction of Accurate Cellular Networks; African; B/AA, Black/African-American breast cancer patients; B/AA50, Black/African-American stage II breast invasive carcinoma patients diagnosed at age 50 years or younger; BrCA, breast invasive carcinoma; Breast invasive carcinoma; DE, differential expression; DM, differential mutation; EMT, Epithelial-Mesenchymal Transition; GSEA, Gene Set Enrichment Analysis; Immortalization; Molecular subtype; RMA, robust multi-array average; RPPA, Reverse Phase Protein Array; Race; TCGA, the Cancer Genome Atlas; TNBC, triple-negative breast cancer; TRN, Transcriptional Regulatory Network; Triple-negative breast cancer; VIPER, Virtual Inference of Protein activity by Enriched Regulon Analysis; W50, White stage II breast invasive carcinoma patients diagnosed at age 50 years or younger.

Fig. 1

A schematic laying out the steps taken in the studies in this report. RNAseq, Reverse Phase Protein Array (RPPA), miRNA-seq, and mutation data examined were from the Cancer Genome Atlas, July 2016. Specifically, the samples were from subjects diagnosed with breast invasive carcinoma. [DE → Differential Expression; GSEA → Gene Set Enrichment Analysis; TRN → Transcriptional Regulatory Network; DM → Differential Mutation].

Fig. 2

Differential expression of oncogenic and tumor suppressor gene markers in Breast Invasive Carcinoma Samples from White patients and Black or African-American patients (B/AA), TCGA 2016. (A) Immortalization gene, telomerase reverse transcriptase gene (TERT), has increased expression in BRCA samples derived from B/AA (n = 179) relative to White (Caucasian) patients (n = 935). (B) RB1 protein expression is reduced among B/AA patients. The normalized Reverse Phase Protein Array data was derived from 677 white patients and 139 B/AA patients. RB1 was detected using the Rb-M-E antibody. The red boxplot represents normalized expression levels from Caucasian patients; the blue boxplot represents normalized expression levels from B/AA patients. (C) MicroRNA hsa-let-7a expression is decreased in Breast Invasive Carcinoma Samples from B/AA relative to Caucasian patients. The miRNA-seq data was derived from 849 white patients and 188 B/AA patients. Three human let-7-a precursors (hsa-let-7a-1, hsa-let-7a-2, and hsa-let-7a-3) all have suppressed expression in B/AA patients. The red boxplot represents normalized expression levels from white patients; the blue boxplot represents normalized expression levels from black patients. (D) Impact of Differences in MicroRNA and Gene Expression between Caucasian and B/AA patients on Entrance into S Phase of Cell Cycle. RB1 binds to E2F proteins to prevent entry into the S phase of the cell cycle. As there is decreased RB1 expression in B/AA patients, there is less constraint on E2F proteins and entry into the S phase is facilitated. Expression of tumor suppressor, microRNA hsa-let-7a, a negative regulator of c-MYC is suppressed in B/AA patients. Expression of proto-oncogene c-MYC, which positively regulates TERT, is up in B/AA patients as is E2F1 whose targets are needed for the cell cycle S phase. Thus, the differences in expression of these appear to confederate to facilitate entrance into the S phase of the cell cycle in B/AA patients. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

An immortalization signature gene set and differences between breast invasive carcinoma samples from Black or African-American (B/AA) patients and White patients. Using the gene expression data from 925 White patients and 189 Black patients described, a GSEA was performed using MSigDb database gene sets associated with the oxidative stress response regulator, NFE2L2. NFE2L2 has suppressed expression in B/AA patients. (A) An enrichment plot showing a notable cross-section (leading edge) of an immortalization gene set (Dairkee et al., 2007) aggregates at the top (right side of figure has aggregation of bars) of a ranked list of genes in B/AA patients (but not White patients). (B) A heatmap showing the expression of the immortalization gene set. A notable cross-section of this gene set captured in the brown “box” on the heatmap has increased expression in Black patient samples. Blue boxes represent genes with suppressed expression; red boxes represent genes with increased expression. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

The top twenty differentially active transcription regulators along with their corresponding regulons enriched (aggregated at the ends of the list of genes ranked by their levels of expression) in Black/African-American (B/AA) patients and Caucasian patients. HMGA1, TERT, ATF4, TCF3, HESF4, HSF1, PQBP1, FOXE3, DRAP1, TAF6 and IRF3 have increased expression and inferred activity in B/AA patients; ZNF396, CREB1, DENND4A, SP1, BDP1, THRB, ARID4A, RB1, and AR have decreased expression and activity in B/AA patients. In the second column, the genes are rank-sorted (left to right) from the most downregulated in B/AA patients (relative to Caucasians) to the most upregulated, and vertical bars represent targets of the transcription regulators (named in the third column) in the transcriptional regulatory network. Blue bars and boxes represent suppressed expression; red bars and boxes represent increased expression. The fourth and fifth columns represent inferred differential protein activity and gene expression respectively. The numbers in the rightmost column are the respective positions of the master regulators on the ranked gene lists. [Act. → activity; Exp. → expression]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

TERT knockout inhibits cell viability and oncoprotein expression, and induces apoptosis TNBC cells. MDA-MB-231 cells were treated with control and TERT knockout CRISPR/Cas9 plasmids for 72 h and assayed for (A) cell viability and (B) apoptosis induction (red-purple cell nuclei). (C) MDA-MB-231 and (G) MDA-MB-468 cells were treated with control, TERT activation CRISPR/Cas9 plasmids, and TERT knockout CRISPR/Cas9 for 72 h. Cell lysates were immunoblotted for expression of (D, H) TERT, (E, I) c-MYC, and (F, J) Wnt11 proteins. Statistical analysis: Unpaired t-test (*P < 0.05, **P < 0.01; ***P < 0.001; and ****P < 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

The impact of increased c-MYC expression on microRNA expression. Differential (between Black/African-American and White breast cancer patients) microRNA expression data was superposed on a transcriptional regulatory network excerpted from TransmiR, a database for transcription factor-microRNA regulations (Wang et al., 2009). c-MYC targets microRNAs, such as miR-221, which have regulatory influence on the cancer phenotype. White nodes represent microRNAs with increased expression in Blacks, brown nodes represent microRNAs with suppressed expression in blacks. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

(A) Kaplan-Meier (survival) plot capturing the dichotomy in survival between three Black/African-American (B/AA50) and eleven White (W50) patients at stage II breast invasive carcinoma and 50 years old or younger at diagnosis (TCGA, July 2016). The time on the horizontal axis represents the number of days to death. Survival among B/AA patients is significantly short-lived (p < 0.0001). (B) HUWE1 and PIK3CA are differentially mutated between B/AA50 and W50. HUWE1 is frequently mutated in B/AA50; PIK3CA is frequently mutated in W50.

Fig. 8

Gene sets associated with breast cancer resistance to endocrine therapy are expressed differently between Black/African-American (B/AA) and White patients. Pooled gene expression profiles of responders and non-responders to therapy with endocrine drugs (Tamoxifen, Fulvestrant, Anastrozole, Letrozole, Exemestane, Goserelin, or Leuprolide). Responders to these therapies were associated with complete response; non-responders were associated with stable/progressive disease. A) The two gene sets from a study by Creighton et al. (2008) in which two mouse xenograft models of estrogen receptor–positive breast cancer, one with HER2 expansion (“Creighton 3”) and another without HER2 expansion (“Creighton 5”). Leading edge “Creighton 3” (Creighton et al., 2008) genes are suppressed in B/AA non-responders relative to White non-responders. B) Leading edge “Creighton 5” (Creighton et al., 2008) genes are suppressed in B/AA non-responders relative to White non-responders.

Fig. 9

Patterns of frequently mutated genes reflect dominance of triple-negative breast cancer (TNBC) among Black/African-American (B/AA) patients. Most mutated genes among A) stage III B/AA and White Caucasian BrCA patients, and B) TNBC and non-TNBC patients (TCGA, July 2016). PIK3CA mutations (mostly missense mutations), as an example, occur more frequently in White stage III patients (32%) than in B/AA stage III patients (20%); they also occur more frequently in non-TNBC patients (37%), than in TNBC patients (8%). TNBC patients were identified by way of immunohistochemistry (TCGA, July 2016).