Hae Hyun Jung

Ets-1 upregulates HER2-induced MMP-1 expression in breast cancer cells.

The human epidermal growth factor receptor-2 (HER2) plays an important role in breast cancer. Enhanced Ets-1 activity has recently been shown to be associated with breast cancer pathogenesis. To test the role of Ets-1 in breast cancer cells in relation to the expression of HER2 and MMP-1, we transiently overexpressed Ets-1 and/or HER2 in MCF-7 breast cancer cells and comprehensively searched for genes related to HER2 and Ets-1 using cDNA microarray analysis. The expression of matrix metalloproteinase (MMP) genes was enhanced by the overexpression of HER2/Ets-1. We analyzed the relationship between HER2-induced MMP-1 expression and the transcription factor Ets-1, which has significant activity in breast cancer pathogenesis. Our results demonstrate that HER2-induced MMP-1 expression is positively regulated by Ets-1 in breast cancer cells. This study confirms that Ets-1 is a downstream effector of oncogenic HER2, associated with MMP-1.

ERK/MAPK pathways play critical roles in EGFR ligands-induced MMP1 expression

Activation of epidermal growth factor receptor (EGFR)-induced signaling pathways has been correlated with tumor progression, invasion and metastasis in a variety of cancers including breast carcinoma, but the underlying mechanism is not well understood. Matrix metalloproteinases (MMPs) have been implicated in cancer invasion and metastasis for their extracellular matrix (ECM)-proteolytic activity. However, the correlation of EGFR pathway with MMP expression in breast cancer has not been established. The aim of this study was to elucidate the interaction between EGFR ligands and their signaling pathway and MMP expression which might be closely related with breast cancer pathogenesis. We investigated the effect of EGF ligands on the MMP1 expression in SK-BR3 cell lines using RT-PCR, Western blot, ELISA and EMSA. Treatments with EGFR ligands, EGF and TGF-α enhanced MMP1 expression at the level of both transcription and translation in SK-BR3 breast cancer cells. EGF and TGF-α treatment resulted in phosphorylation of EGFR, and consequent activation of ERK1/2 pathway. Tyrosine kinase inhibitors of HER family, erlotinib, lapatinib and canertinib suppressed EGF-ligands mediated MMP1 overexpression. The specific MEK inhibitor, U0126, significantly blocks EGF and TGF-α-mediated ERK1/2 activation and subsequent MMP1 induction in SK-BR3 cells. Inhibition of the Akt pathway with LY294002 paradoxically augmented MMP1 expression by reciprocal activation of ERK1/2 pathway. These data suggest that invasive potential of SK-BR3 cell would be affected by these drugs by suppression of EGFR ligands-induced MMP1 expression.

Statin induces inhibition of triple negative breast cancer (TNBC) cells via PI3K pathway.

Primary TNBCs are treated as if they were a single disease entity, yet it is clear they do not behave as a single entity in response to current therapies. Recently, we reported that statins might have a potential benefit for TNBCs associated with ets-1 overexpression. The aim of this study is to investigate the role of PTEN loss in the effects of statin on TNBC cells. In addition, we analyze the relationship between AKT downstream pathways and the effects of statin on TNBC cells. We investigated the effect of a statin on TNBC cells and analyzed the association of PI3K pathways using various TNBC cells in terms of PTEN loss and AKT pathways. Simvastatin treatments resulted in decreased cell viabilities in various TNBC cell lines. Compared with PTEN wild-type TNBC cells, PTEN mutant-type TNBC cells showed a decreased response to simvastatin. Expressions of phosphorylated Akt and total Akt showed an inverse relationship with PTEN expression. The TNBC cell lines, which showed increased expression of p-Akt, appeared to attenuate the expression of p-Akt by PTEN loss in simvastatin-treated TNBC cells. The Akt inhibitor, LY294002, augmented the effect of simvastatin on PTEN wild-type TNBC cells. Simvastatin induces inhibition of TNBC cells via PI3K pathway activation.

Mutational profiling of brain metastasis from breast cancer: matched pair analysis of targeted sequencing between brain metastasis and primary breast cancer

Although breast cancer is the second most common cause of brain metastasis with a notable increase of incidence, genes that mediate breast cancer brain metastasis (BCBM) are not fully understood. To study the molecular nature of brain metastasis, we performed gene expression profiling of brain metastasis and matched primary breast cancer (BC). We used the Ion AmpliSeq Cancer Panel v2 covering 2,855 mutations from 50 cancer genes to analyze 18 primary BC and 42 BCBM including 15 matched pairs. The most common BCBM subtypes were triple-negative (42.9%) and basal-like (36.6%). In a total of 42 BCBM samples, 32 (76.2%) harbored at least one mutation (median 1, range 0–7 mutations). Frequently detected somatic mutations included TP53 (59.5%), MLH1 (14.3%), PIK3CA (14.3%), and KIT (7.1%). We compared BCBM with patient-matched primary BC specimens. There were no significant differences in mutation profiles between the two groups. Notably, gene expression in BCBM such as TP53, PIK3CA, KIT, MLH1, and RB1 also seemed to be present in primary breast cancers. The TP53 mutation frequency was higher in BCBM than in primary BC (59.5% vs 38.9%, respectively). In conclusion, we found actionable gene alterations in BCBM that were maintained in primary BC. Further studies with functional testing and a delineation of the role of these genes in specific steps of the metastatic process should lead to a better understanding of the biology of metastasis and its susceptibility to treatment.

Gene Expression Profiling of Breast Cancer Brain Metastasis

The biology of breast cancer brain metastasis (BCBM) is poorly understood. We aimed to explore genes that are implicated in the process of brain metastasis of primary breast cancer (BC). NanoString nCounter Analysis covering 252 target genes was used for comparison of gene expression levels between 20 primary BCs that relapsed to brain and 41 BCBM samples. PAM50-based intrinsic subtypes such as HER2-enriched and basal-like were clearly over-represented in BCBM. A panel of 22 genes was found to be significantly differentially expressed between primary BC and BCBM. Five of these genes, CXCL12, MMP2, MMP11, VCAM1, and MME, which have previously been associated with tumor progression, angiogenesis, and metastasis, clearly discriminated between primary BC and BCBM. Notably, the five genes were significantly upregulated in primary BC compared to BCBM. Conversely, SOX2 and OLIG2 genes were upregulated in BCBM. These genes may participate in metastatic colonization but not in primary tumor development. Among patient-matched paired samples (n = 17), a PAM50 molecular subtype conversion was observed in eight cases (47.1%), with a trend toward unfavorable subtypes in patients with the distinct gene expression. Our findings, although not conclusive, reveal differentially expressed genes that might mediate the brain metastasis process.

STAT1 and Nmi are downstream targets of Ets‐1 transcription factor in MCF‐7 human breast cancer cell

Ets‐1 is a cellular homologue of the product of the viral ets oncogene of the E26 virus, and it functions as a tissue‐specific transcription factor. It plays an important role in cell proliferation, differentiation, lymphoid cell development, transformation, angiogenesis, and apoptosis. Ets‐1 controls the expression of critical genes involved in these processes by binding to ets binding sites present in the transcriptional regulatory regions. Here, we transiently overexpressed Ets‐1 in MCF‐7 and comprehensively searched for potential downstream targets of Ets‐1 by cDNA microarray analysis. The expressions of several interferon‐related genes including STAT1 and Nmi were augmented by the overexpression of Ets‐1. RT‐PCR and Western blotting confirmed the increase in the levels of STAT1 and Nmi mRNA and protein. In contrast, Ets‐1 siRNA decreased the expression of STAT1 and Nmi proteins. As in our transient transfection experiments, stable overexpression of Ets‐1, also increased the protein expression of STAT1 and Nmi in MCF‐7 cells. Taken together, our results indicate that STAT1 and Nmi are downstream targets of Ets‐1 in MCF‐7 human breast cancer cells.

Role of HER2 mutations in refractory metastatic breast cancers: targeted sequencing results in patients with refractory breast cancer

In women with metastatic breast cancer (MBC), introduction of the anti-HER2 (human epidermal growth factor receptor-2) directed therapies including trastuzumab, pertuzumab, lapatinib, and/or trastuzumab-DM1 has markedly improved overall survival. However, not all cases of HER2-positive breast tumours derive similar benefit from HER2-directed therapy, and a significant number of patients experience disease progression because of primary or acquired resistance to anti-HER2-directed therapies. We integrated genomic and clinicopathological analyses in a cohort of patients with refractory breast cancer to anti-HER2 therapies to identify the molecular basis for clinical heterogeneity. To study the molecular basis underlying refractory MBC, we obtained 36 MBC tumours tissues and used next-generation sequencing to investigate the mutational and transcriptional profiles of 83 genes. We focused on HER2 mutational sites and HER2 pathways to identify the roles of HER2 mutations and the HER2 pathway in the refractoriness to anti-HER2 therapies. Analysis using massively parallel sequencing platform, CancerSCAN™, revealed that HER2 mutations were found in six of 36 patients (16.7%). One patient was ER (estrogen receptor)-positive and HER2-negative and the other five HER2 mutated patients were HER2-positive and HR (hormone receptor)-negative. Most importantly, four of these five patients did not show any durable clinical response to HER2-directed therapies. The HER2 pathway score obtained through transcriptional analyses identified that Growth Receptor Biding protein 2 (GRB2) was the most significantly down regulated gene in the HER2 mutated samples. Detection of HER2 mutations using higher deep DNA sequencing may identify a predictive biomarker of resistance to HER2-directed therapy. Functional validation is warranted.

A seven‐gene signature can predict distant recurrence in patients with triple‐negative breast cancers who receive adjuvant chemotherapy following surgery

The aim of this study was to investigate candidate genes that might function as biomarkers to differentiate triple negative breast cancers (TNBCs) among patients, who received adjuvant chemotherapy after curative surgery. We tested whether the results of a NanoString expression assay that targeted 250 prospectively selected genes and used mRNA extracted from formalin‐fixed, paraffin‐embedded would predict distant recurrence in patients with TNBC. The levels of expression of seven genes were used in a prospectively defined algorithm to allocate each patient to a risk group (low or high). NanoString expression profiles were obtained for 203 tumor tissue blocks. Increased expressions of the five genes (SMAD2, HRAS, KRT6A, TP63 and ETV6) and decreased expression of the two genes (NFKB1 and MDM4) were associated favorable prognosis and were validated with cross‐validation. The Kaplan–Meier estimates of the rates of distant recurrence at 10 years in the low‐ and high‐risk groups according to gene expression signature were 62% [95% confidence interval (CI), 48.6–78.9%] and 85% (95% CI, 79.2–90.7%), respectively. When adjusting for TNM stage, the distant recurrence‐free survival (DRFS)s in the low‐risk group was significantly longer than that in the high‐risk group (p <0.001) for early stage (I and II) and advanced stage (III) tumors. In a multivariate Cox regression model, the gene expression signature provided significant predictive power jointly with the TNM staging system. A seven‐gene signature could be used as a prognostic model to predict DRFS in patients with TNBC who received curative surgery followed by adjuvant chemotherapy.

Clinical implications of genomic profiles in metastatic breast cancer with a focus on TP53 and PIK3CA, the most frequently mutated genes

Breast cancer (BC) has been genetically profiled through large-scale genome analyses. However, the role and clinical implications of genetic alterations in metastatic BC (MBC) have not been evaluated. Therefore, we conducted whole-exome sequencing (WES) and RNA-Seq of 37 MBC samples and targeted deep sequencing of another 29 MBCs. We evaluated somatic mutations from WES and targeted sequencing and assessed gene expression and performed pathway analysis from RNA-Seq. In this analysis, PIK3CA was the most commonly mutated gene in estrogen receptor (ER)-positive BC, while in ER-negative BC, TP53 was the most commonly mutated gene (p = 0.018 and p < 0.001, respectively). TP53 stopgain/loss and frameshift mutation was related to low expression of TP53 in contrast nonsynonymous mutation was related to high expression. The impact of TP53 mutation on clinical outcome varied with regard to ER status. In ER-positive BCs, wild type TP53 had a better prognosis than mutated TP53 (median overall survival (OS) (wild type vs. mutated): 88.5 ± 54.4 vs. 32.6 ± 10.7 (months), p = 0.002). In contrast, mutated TP53 had a protective effect in ER-negative BCs (median OS: 0.10 vs. 32.6 ± 8.2, p = 0.026). However, PIK3CA mutation did not affect patient survival. In gene expression analysis, CALM1, a potential regulator of AKT, was highly expressed in PIK3CA-mutated BCs. In conclusion, mutation of TP53 was associated with expression status and affect clinical outcome according to ER status in MBC. Although mutation of PIK3CA was not related to survival in this study, mutation of PIK3CA altered the expression of other genes and pathways including CALM1 and may be a potential predictive marker of PI3K inhibitor effectiveness.

Statins affect ETS1 -overexpressing triple-negative breast cancer cells by restoring DUSP4 deficiency

We investigated the molecular mechanisms underlying statin-induced growth suppression of triple-negative breast cancer (TNBC) that overexpress the transcription factor ets proto-oncogene 1(ets-1) and downregulate dual specific protein phosphatase 4(dusp4) expression. We examined the gene expression of BC cell lines using the nCounter expression assay, MTT viability assay, cell proliferation assay and Western blot to evaluate the effects of simvastatin. Finally, we performed cell viability testing in TNBC cell line-transfected DUSP4. We demonstrated that ETS1 mRNA and protein were overexpressed in TNBC cells compared with other BC cell lines (P = <0.001) and DUSP4 mRNA was downregulated (P = <0.001). MTT viability assay showed that simvastatin had significant antitumor activity (P = 0.002 in 0.1 μM). In addition, simvastatin could restore dusp4 deficiency and suppress ets-1 expression in TNBC. Lastly, we found that si-DUSP4 RNA transfection overcame the antitumor activity of statins. MAPK pathway inhibitor, U0126 and PI3KCA inhibitor LY294002 also decreased levels of ets-1, phosphor-ERK and phosphor-AKT on Western blot assay. Accordingly, our study indicates that simvastatin potentially affects the activity of transcriptional factors such as ets-1 and dusp4 through the MAPK pathway. In conclusion, statins might be potential candidates for TNBC therapy reducing ets-1 expression via overexpression of dusp4.