Transition In Triple-negative Breast Cancer Research Articles

Abstract P6-11-10: Preclinical efficacy of the novel PIM2 kinase inhibitor, JP11646 in triple negative breast cancer models

Abstract Background: Triple negative breast cancer (TNBC) patients have poorer prognosis and there remains a lack of novel targeted therapies for their treatment. PIM2 (Proviral Integrations of Moloney virus 2) belongs to a family of three kinases that have been implicated in the survival and progression of hematologic malignancies and solid tumors. PIM2 has been linked to epithelial to mesenchymal transition in TNBC, which can lead to metastasis and chemotherapeutic resistance. We hypothesized that PIM2 may present as a therapeutic target in TNBC. Materials and Methods: The study involved both in vitro and in vivo studies involving a novel PIM2 inhibitor JP11646 (obtained from Jasco Pharmaceuticals). TNBC cell lines MDA-MB-231 and BT-549 were obtained for our in vitro studies. Cell viability was evaluated using MTT assay. Western Blot assay was used to evaluate relative protein expression. For in vivo studies, female SCID mice were inoculated in the mammary fat pads with 1 × 106 MDA-MB-231 cells. When tumor volumes reached 100 mm3, the mice were treated with JP11646 at the dosage 15mg/kg intraperitoneally for 2 consecutive days weekly for total of 4 weeks as determined from previous experiments. Control animals received vehicle only. The mice were euthanized once tumors reached ∼1,700 mm3. Results: BT-549 cells treated in vitro with 3 different available PIM kinase inhibitors AZD 1208, LGB321 and JP12641 showed only modest reduction in cell viability. However, treatment of both MDA-MB-231 and BT-549 with JP 11646 demonstrated significant reduction in cell viability with IC50 ranging from 40 to 71.6 nM. Treatment with JP11646 demonstrated a novel mechanism of action resulting in downregulation of PIM2 in both cell lines. Treatment with JP11646, but not other PIM kinase inhibitors, resulted in activation of apoptosis as measured by cleaved PARP (cPARP) levels. Anti-PIM2 siRNA treatment but not treatment with non-specific PIM kinase inhibitor AZD1208 resulted in cPARP induction. Inhibition of proteolysis by bortezomib resulted in preservation of PIM2 and inhibition of apoptosis as demonstrated by decreased cPARP levels after treatment with JP11646. PIM2 over-expressing clone of MDA-MB-231 cells showed enhanced proliferation and migration properties both in vitro and in vivo.Treatment of mice with orthotopically implanted MDA-MB-231 tumors with JP 11646 resulted in significant reduction in the tumor growth (p=0.0019) and increased overall survival (p=0.018) as compared to control mice. Conclusions: PIM2 upregulation in TNBC cell line resulted in more aggressive phenotype. JP11646, through novel mechanism of action resulting in degradation of PIM2, showed robust activity in TNBC cell lines both in vitro and in vivo. Further correlative studies in tumors harvested from in vivo experiments are ongoing. These results encourage further exploration of use of JP11646 as a targeted agent in treatment of TNBC. Citation Format: Mehta R, Kothai Guruswamy Sangameswaran D, Bezbatchenko K, Moore J, Gil M, Khoury T, Baldino C, Caserta J, Fetterly, Jr. G, Lee K, Adjei A, Opyrchal M. Preclinical efficacy of the novel PIM2 kinase inhibitor, JP11646 in triple negative breast cancer models [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-11-10.

MiR-212-5p Suppresses the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer by Targeting Prrx2

Background/Aims: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Our study investigated the functional role of miR-212-5p in TNBC. Methods: Realtime PCR was used to quantify miR-212-5p expression levels in 30 paired TNBC samples and adjacent normal tissues. Wound healing and Transwell assays were used to evaluate the effects of miR-212-5p expression on the invasiveness of TNBC cells. Luciferase reporter and Western blot assays were used to verify whether the mRNA encoding Prrx2 is a major target of miR-212-5p. Results: MiR-212-5p was downregulated in TNBC, and its expression levels were related to tumor size, lymph node status and vascular invasion in breast cancer. We also observed that the miR-212-5p expression level was significantly correlated with a better prognosis in TNBC. Ectopic expression of miR-212-5p induced upregulation of E-cadherin expression and downregulation of vimentin expression. The expression of miR212-5p also suppressed the migration and invasion capacity of mesenchymal-like cancer cells accompanied by a morphological shift towards the epithelial phenotype. Moreover, our study observed that miR-212-5p overexpression significantly suppressed Prrx2 by targeting its 3’-untranslated region (3’-UTR) region, and Prrx2 overexpression partially abrogated miR-212-5p-mediated suppression. Conclusions: Our study demonstrated that miR-212-5p inhibits TNBC from acquiring the EMT phenotype by downregulating Prrx2, thereby inhibiting cell migration and invasion during cancer progression.

Molecular markers of epithelial-to-mesenchymal transition in triple-negative breast cancer

Objective To investigate the molecular alterations in breast cancer samples using epithelial-to-mesenchymal transition (EMT) markers such as E-cadherin, vimentin, epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF)-D, and nuclear factor-κB (NF-κB) to reveal their roles in the EMT and breast cancer progression. Methods In 57 cases of invasive ductal breast carcinoma, the expression of E-cadherin, vimentin, EGFR, NF-κB, and PDGF-D was detected using immunohistochemistry. Tumors were categorized into three groups: A [estrogen receptor (ER)+ , and/or progesterone receptor (PR)+ , human epidermal growth factor receptor-2 (Her-2)/neu-], B (ER+ , and/or PR+ , Her-2/neu+ ), and C (triple-negative: ER-, PR-, and Her-2/neu-). Immunostained tissue biopsies were observed under a microscope, evaluated and scored using intensity (0, + , + + , and + + + ) and percentage of positive cells, and data were statistically analyzed. Results Membranous E-cadherin was positive in all 57 cases (100%), whereas cytoplasmic E-cadherin was predominantly positive in groups B and C as compared with group A (21%, 7%, and 0%, respectively). All cases in group A were negative for vimentin and EGFR. There was statistically increased expression of vimentin, EGFR (P<0.01), and NF-κB (P<0.05) in triple-negative cases when compared with groups A and B. Conclusion Vimentin, EGFR, and NF-κB were significantly increased in triple-negative breast cancer, which is consistent with the invasiveness of these tumors. Key words: Triple-negative breast cancer; Epithelial-to-mesenchymal transition; E-cadherin; Vimentin; Epidermal growth factor receptor; Platelet-derived growth factor-D

Silencing of Prrx1b suppresses cellular proliferation, migration, invasion and epithelial-mesenchymal transition in triple-negative breast cancer.

Triple‐negative breast cancer (TNBC) is a highly aggressive tumour subtype associated with poor prognosis. The mechanisms involved in TNBC progression remains largely unknown. To date, there are no effective therapeutic targets for this tumour subtype. Paired‐related homeobox 1b (Prrx1b), one of major isoforms of Prrx1, has been identified as a new epithelial–mesenchymal transition (EMT) inducer. However, the function of Prrx1b in TNBC has not been elucidated. In this study, we found that Prrx1b was significantly up‐regulated in TNBC and associated with tumour size and vascular invasion of breast cancer. Silencing of Prrx1b suppressed the proliferation, migration and invasion of basal‐like cancer cells. Moreover, silencing of Prrx1b prevented Wnt/β‐catenin signaling pathway and induced the mesenchymal‐epithelial transition (MET). Taken together, our data indicated that Prrx1b may be an important regulator of EMT in TNBC cells and a new therapeutic target for interventions against TNBC invasion and metastasis.

SMC1 promotes epithelial-mesenchymal transition in triple-negative breast cancer through upregulating Brachyury.

Triple-negative breast cancer (TNBC) is a special subtype of breast cancer, which is characterized by the negative form of estrogen receptor (ER), progesterone receptor (PR) and human epithelial growth factor receptor 2 (HER2). TNBC accounts for ~15% of all breast cancer forms, and often leads to high mortality and poor prognosis. Structural maintenance of chromosome 1 (SMC1) is a subunit of the cohesion protein complex. Brachyury is a protein that is encoded by the T gene in humans, which is a transcription factor within the T-box complex of genes. Epithelial-mesenchymal transition (EMT) is a ubiquitous process in the body, and in particular, induces metastasis and the proliferation of cancer cells. In the present study, we found that SMC1 expression in TNBC tissues exceeded its expression in adjacent non-tumor tissues. Similarly, the expression of SMC1 in TNBC cell lines (hs578T and HCC1937) was found to be higher than in MCF10a and MCF7 cells. Subsequently, SMC1 was overexpressed and silenced in hs578T and HCC1937 cells through plasmid and siRNA transfection, respectively. The results showed that the high expression of SMC1 often promoted EMT, accompanied by the enhanced expression of Brachyury. Besides, upregulated expression of Brachyury through plasmid transfection also significantly improved the level of EMT, which further indicated that SMC1 increased EMT in TNBC through the induction of Brachyury expression. Taken together, these results contributed to a better understanding of the pathogenesis of TNBC, which also provided an experimental basis for the prevention, diagnosis and treatment of TNBC.

Dual regulation by microRNA-200b-3p and microRNA-200b-5p in the inhibition of epithelial-to-mesenchymal transition in triple-negative breast cancer.

Epithelial to mesenchymal transition (EMT) involves loss of an epithelial phenotype and activation of a mesenchymal one. Enhanced expression of genes associated with a mesenchymal transition includes ZEB1/2, TWIST, and FOXC1. miRNAs are known regulators of gene expression and altered miRNA expression is known to enhance EMT in breast cancer. Here we demonstrate that the tumor suppressive miRNA family, miR-200, is not expressed in triple negative breast cancer (TNBC) cell lines and that miR-200b-3p over-expression represses EMT, which is evident through decreased migration and increased CDH1 expression. Despite the loss of migratory capacity following re-expression of miR-200b-3p, no subsequent loss of the conventional miR-200 family targets and EMT markers ZEB1/2 was observed. Next generation RNA-sequencing analysis showed that enhanced expression of pri-miR-200b lead to ectopic expression of both miR-200b-3p and miR-200b-5p with multiple isomiRs expressed for each of these miRNAs. Furthermore, miR-200b-5p was expressed in the receptor positive, epithelial breast cancer cell lines but not in the TNBC (mesenchymal) cell lines. In addition, a compensatory mechanism for miR-200b-3p/200b-5p targeting, where both miRNAs target the RHOGDI pathway leading to non-canonical repression of EMT, was demonstrated. Collectively, these data are the first to demonstrate dual targeting by miR-200b-3p and miR-200b-5p and a previously undescribed role for microRNA processing and strand expression in EMT and TNBC, the most aggressive breast cancer subtype.

AP-1-mediated chromatin looping regulates ZEB2 transcription: new insights into TNFα-induced epithelial-mesenchymal transition in triple-negative breast cancer.

The molecular determinants of malignant cell behaviour in triple-negative breast cancer (TNBC) are poorly understood. Recent studies have shown that regulators of epithelial-mesenchymal transition (EMT) are potential therapeutic targets for TNBC. In this study, we demonstrate that the inflammatory cytokine TNFα induces EMT in TNBC cells via activation of AP-1 signaling and subsequently induces expression of the EMT regulator ZEB2. We also show that TNFα activates both the PI3K/Akt and MAPK/ERK pathways, which act upstream of AP-1. We further investigated in detail AP-1 regulation of ZEB2 expression. We show that two ZEB2 transcripts derived from distinct promoters are both expressed in breast cancer cell lines and breast tumor samples. Using the chromosome conformation capture assay, we demonstrate that AP-1, when activated by TNFα, binds to a site in promoter 1b of the ZEB2 gene where it regulates the expression of both promoter 1b and 1a, the latter via mediating long range chromatin interactions. Overall, this work provides a plausible mechanism for inflammation-induced metastatic potential in TNBC, involving a novel regulatory mechanism governing ZEB2 isoform expression.

Abstract 3368: MEK inhibitor selumetinib (AZD6244 – ARRY-142886) promotes mesenchymal to epithelial transition in triple-negative breast cancer

Abstract Selumetinib is a highly selective allosteric inhibitor of MEK1/2. Multiple clinical trials of Selumetinib are ongoing in patients with different types of cancer. However, the therapeutic role of Selumetinib in breast cancer has not been well defined. We sought to determine the effect of targeted inhibition of the MEK-ERK pathway by Selumetinib in triple-negative breast cancer (TNBC). We studied the effect of Selumetinib on 2 TNBC cell lines, MDA-MB-231 and SUM-149. In MDA-MB-231 cells, in 2-dimensional (2D) culture, Selumetinib at 0.1μM did not reduce cell viability, but in a 3-dimensional (3D) cell culture model, which mimics the human microenvironment, Selumetinib at 0.1 μM and 1 μM inhibited epithelial to mesenchymal transition (EMT). This result was confirmed by western blotting: expression of the mesenchymal markers fibronectin and vimentin was inhibited, and the epithelial marker beta-catenin was diffusely expressed in both the cytoplasm and the nucleus before treatment but was localized at the plasma membrane after treatment. Selumetinib in 3D cell culture also inhibited projections/filopodia formation, suggesting reversal to a more epithelial phenotype. Results were similar in SUM-149 cells: Selumetinib at 0.1 μM had minimal impact on cell viability in 2D culture, but Selumetinib at 0.1 μM and 1 μM inhibited projections/filopodia in 3D culture. Inhibition of ERK phosphorylation by Selumetinib correlated with a slight increase in the epithelial marker E-cadherin and loss of vimentin. These results suggest that treating TNBC with Selumetinib induces mesenchymal to epithelial transition (MET). In addition, in MDA-MB-231 cells, Selumetinib significantly inhibited anchorage-independent growth, an indicator of in vivo tumorigenicity. Previously, the ERK2 isoform was shown to induce EMT in epithelial cells. We therefore examined whether ERK1 and 2 expression levels correlate with Selumetinib's effect on EMT regulation in TNBC cells. We used shRNA specifically targeting ERK1 and ERK2 (shERK1 and 2). Compared with parental MDA-MB-231 cells, stable clones that constitutively expressed shERK1 or shERK2 showed no difference in growth rate in 2D culture or projection formation in 3D culture. However, in 3D culture, treatment with Selumetinib inhibited spindle-shaped cell morphology and reduced scattering of the parental, vector-transfected, and shERK2 clones but did not inhibit mesenchymal filamentous structures in the shERK1 clones. Our data demonstrate that ERK1 may be necessary for Selumetinib -induced mesenchymal to epithelial transition in TNBC. We are planning in vivo studies to determine if low-dose Selumetinib can inhibit EMT, leading to a reduction of metastasis in a TNBC xenograft model. We will further confirm if ERK1 can serve as a biomarker for MEK inhibitor therapy in TNBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3368. doi:10.1158/1538-7445.AM2011-3368