Chungyeul Kim

HRG-β1-driven ErbB3 signaling induces epithelial-mesenchymal transition in breast cancer cells

BackgroundHeregulin (HRG; also known as neuregulin) is a ligand for ErbB3. One of its isotypes, HRG-β1, binds to ErbB3 and forms heterodimers with other ErbB family members, thereby enhancing the proliferation and tumorigenesis of breast cancer cells. HRG stimulation may contribute to the progression of epithelial–mesenchymal transition (EMT) and tumor metastasis in breast cancer. Majority of studies regarding EMT has been concentrated on TGF-β signaling. Therefore, we investigated whether the HRG-β1 and ErbB3 activate Smad2 signaling during process of EMT in breast cancer cells.MethodsThe SK-BR-3 and MCF7 breast cancer cell lines were used. The expressions of phospho-Smad2 and EMT markers were observed by western blotting and immunofluorescence assays after treatment with HRG-β1. The cell motility and invasiveness were determined by wound healing and matrigel invasion assays. Smad2 and ErbB3 small interfering RNA (siRNA) transfections were performed to assess the involvement of ErbB3 and Smad2 in HRG-β1-induced EMT.ResultsHRG-β1 induced EMT through activation of Smad2. The expression of E-cadherin was decreased after HRG-β1 treatment, while the expressions of Snail, vimentin, and fibronectin were increased. The HRG-β1-induced expressions of Snail, vimentin, and fibronectin, and nuclear colocalization of phospho-Smad2 and Snail were inhibited by pretreatment with a PI3k inhibitor, LY294002, or two phospho-Smad2 inhibitors, PD169316 or SB203580 and cancer cell migration by HRG-β1 was inhibited. Knockdown of Smad2 by siRNA transfection suppressed the expressions of Snail and fibronectin in response to HRG-β1 stimulation and knockdown of ErbB3 suppressed the expressions of phospho-Smad2, Snail, and fibronectin induced by HRG-β1, whereas E-cadherin was increased compared with control siRNA-transfected cells. Knockdown of ErbB3 and Smad2 also decreased SK-BR-3 and MCF7 cell invasion.ConclusionsOur data suggest that HRG-β1 and ErbB3 induce EMT, cancer cell migration and invasion through the PI3k/Akt-phospho-Smad2-Snail signaling pathway in SK-BR-3 and MCF7 breast cancer cells.

Anticancer effect of metformin on estrogen receptor-positive and tamoxifen-resistant breast cancer cell lines

Acquisition of tamoxifen resistance (TR) during anti-estrogenic therapy using tamoxifen is a major obstacle in the treatment of estrogen receptor (ER)-positive breast cancer. As a biguanide derivative, metformin is commonly used to treat type II diabetes. It has recently emerged as a potential anticancer agent. The objective of the present study was to investigate the anticancer activity of metformin in relation to ERα expression and its signaling pathway in ERα-positive MCF-7 and MDA-MB-361 breast cancer cells as well as TR MCF-7 breast cancer cells. Metformin inhibited both protein and mRNA levels of ERα in the presence or absence of estrogen (E2) in the MCF-7, TR MCF-7 and MDA-MB-361 cells. Metformin repressed E2-inducible estrogen response element (ERE) luciferase activity, protein levels and mRNA levels of E2/ERα-regulated genes [including c-Myc, cyclin D1, progesterone receptor (PR) and pS2] to a greater degree than tamoxifen, resulting in inhibition of cell proliferation of MCF-7, TR MCF-7 and MDA-MB-361 cells. Collectively, our results suggest that one of the anticancer mechanisms of metformin could be attributable to the repression of expression and transcriptional activity of ERα. Metformin may be a good therapeutic agent for treating ERα-positive breast cancer by inhibiting the expression and function of ERα. In addition, metformin may be useful to treat tamoxifen-resistant breast cancer.

Detection of human papillomavirus in Korean breast cancer patients by real-time polymerase chain reaction and meta-analysis of human papillomavirus and breast cancer

Background Human papillomavirus (HPV) is a well-established oncogenic virus of cervical, anogenital, and oropharyngeal cancer. Various subtypes of HPV have been detected in 0% to 60% of breast cancers. The roles of HPV in the carcinogenesis of breast cancer remain controversial. This study was performed to determine the prevalence of HPV-positive breast cancer in Korean patients and to evaluate the possibility of carcinogenic effect of HPV on breast. Methods Meta-analysis was performed in 22 case-control studies for HPV infection in breast cancer. A total of 123 breast cancers, nine intraductal papillomas and 13 nipple tissues of patients with proven cervical HPV infection were tested by real-time polymerase chain reaction to detect 28 subtypes of HPV. Breast cancers were composed of 106 formalin-fixed and paraffin embedded (FFPE) breast cancer samples and 17 touch imprint cytology samples of breast cancers. Results The overall odds ratio between breast cancer and HPV infection was 5.43 (95% confidence interval, 3.24 to 9.12) with I2 = 34.5% in meta-analysis of published studies with case-control setting and it was statistically significant. HPV was detected in 22 cases of breast cancers (17.9%) and two cases of intaductal papillomas (22.2%). However, these cases had weak positivity. Conclusions These results failed to serve as significant evidence to support the relationship between HPV and breast cancer. Further study with larger epidemiologic population is merited to determine the relationship between HPV and breast cancer.

Anti-cancer effect of metformin by suppressing signaling pathway of HER2 and HER3 in tamoxifen-resistant breast cancer cells.

Development of new therapeutic strategies is becoming increasingly important to overcome tamoxifen resistance. Recently, much interest has been focused on anti-tumor effects of metformin commonly used to treat type II diabetes. Increased protein expression and signaling of epidermal growth factor receptor (EGFR) family is a possible mechanism involved in tamoxifen resistance. Since HER2/HER3 heterodimers are able to induce strong downstream signaling and activate various biological responses such as cellular proliferation and growth, we investigated the anti-cancer effect of metformin by inhibition of signaling pathway via downregulation of HER2 and HER3 using tamoxifen-resistant MCF-7 (TR MCF-7) cells. Compared to MCF-7 cells, TR MCF-7 cells showed increased expression of EGFR, HER2, and HER3, and metformin inhibited the expression of these proteins in a dose- and time-dependent manner. Metformin inhibited activation of HER2 (Tyr1248)/HER3 (Tyr1289)/Akt (Ser473) as well as cell proliferation and colony formation by estrogenic promotion in MCF-7 and TR MCF-7 cells. Known as a HER3 ligand, heregulin (HRG)-β1-induced phosphorylation of HER2, HER3 and Akt, and protein interaction of HER2/HER3 and colony formation were inhibited by metformin in both cells. Consistent with the results in the two cell lines, we identified that metformin inhibited HER2/HER3/Akt signaling axis activated by HRG-β1 using the HER2 and HER3-overexpressing breast cancer cell line SK-BR-3. Lastly, lapatinib-induced HER3 upregulation was significantly inhibited by treatment of metformin in HER3 siRNA-transfected TR MCF-7 cells. These data suggest that metformin might overcome tamoxifen resistance through the inhibition of expression and signaling of receptor tyrosine kinase HER2 and HER3.

Genomic Copy Number Signatures Uncovered a Genetically Distinct Group from Adenocarcinoma and Squamous Cell Carcinoma in Non–Small Cell Lung Cancer ☆ ☆☆

Adenocarcinoma (AC) and squamous cell carcinoma (SCC) of non-small cell lung carcinoma (NSCLC) have different clinical presentations, morphologies, treatments, and prognoses. Recent studies suggested that fundamental genetic alterations related to carcinogenesis of each tumor type may be different. In this study, we investigated the genomic alterations of 47 primary NSCLC samples (22 ACs and 25 SCCs) as well as the corresponding normal tissue using array comparative genomic hybridization. Frequent copy number alterations (CNAs), which were identified in more than 68% of all of the cases, were evaluated in each subtype (SCC and AC), and a CNA signature was established. Among these CNAs, 37 genes from the SCCs and 15 genes from the ACs were located in a region of gain, and 4 genes from the SCCs and 13 genes from the ACs were located in a region of loss. The most frequent gain was located on 3q26-29 including the gene TP63 in SCCs and 7q11.23 and 7q36.3 in ACs. Moreover, we identified 3 genetically distinct groups (group I [16 SCC] with CNA signature of SCC; group II [7 SCC + 8 AC], which has a genetically distinctive CNA signature from SCC and AC; and group III [2 SCC + 14 AC] with CNA signature of AC) by gene clustering extracted from CNAs, which are associated with a prognosis. The present study contributed to the molecular characterization of AC and SCC of NSCLC and showed a subtype of tumor that has a unique genetic CNA signature. However, further study about the significance of these 3 distinct groups and their usefulness as a diagnostic marker of identified CNAs is necessary.

Recurrent solitary fibrous tumor of the breast: Magnetic resonance imaging and pathologic findings

A 63‐year‐old woman visited our institution for right mastalgia lasting for two months. On admission, mammography and ultrasonography (US) were performed and revealed relatively well‐ circumscribed, ovoid shaped mass. This patient received excisional mass biopsy and wide excision, and the pathologic diagnosis was confirmed as solitary fibrous tumor (SFT) of the breast. After 6 months, she referred to our hospital again for rapidly growing mass for 1 month. The patient underwent US with a 4‐15 MHz linear array transducer (Aix en Provence, Super‐Sonic imagine, France). Her breast US revealed a heterogeneous echoic, bilobulated mass with microlobulated margin at the lower portion of previously post‐operation site (Figure 1A, B). On dynamic contrast‐enhanced magnetic resonance imaging (MRI) of both breasts (3.0T Skyra, Simens, Erlangen, Germany), a bilobulated oval shape with well‐ circumscribed margin was low signal intensity on T1WI (Figure 2A) and heterogeneously high signal intensity on T2WI (Figure 2B). This mass demonstrated a homogeneously enhancing pattern with internal necrotic portion (Figure 2C) with a type III kinetic curve; rapid enhancement and washout pattern analyzing computer‐aided diagnosis (Figure 2D). Once again, we surgically confirmed judging pathologically identical to the previous tumors; SFT Microscopic examination (Figure 3) demonstrated a proliferation of bland‐looking ovoid to short spindle cells, closely packed and haphazardly arranged within a fibrous stroma (Figure 3A, H&E×200). Immunohistochemically, neoplastic cells showed diffuse staining with CD34 (Figure 3B, ×100) while the cells were completely negative for c‐kit, SMA, S‐100, CD10, and desmin. Solitary fibrous tumor (SFT) is an uncommon group of spindle cell neoplasm, categorized under intermediate fibroblastic tumor having rarely metastasis. In addition, SFT is very rare in the breast, with <20 cases reported in the English literature without any case involving local recurrence or metastases. Our case is very unusual in that SFT of the breast was recurrent and is the first report showing MRI findings. MRI of SFT is relatively nonspecific, representing a well‐ defined lobulated lesion with geographic enhancement. Most

Optimization of RNA Extraction from Formalin-Fixed Paraffin-Embedded Blocks for Targeted Next-Generation Sequencing

Purpose Breast cancer has a high prevalence in Korea. To achieve personalized therapy for breast cancer, long-term follow-up specimens are needed for next-generation sequencing (NGS) and multigene analysis. Formalin-fixed paraffin-embedded (FFPE) samples are easier to store than fresh frozen (FF) samples. The objective of this study was to optimize RNA extraction from FFPE blocks for NGS. Methods RNA quality from FF and FFPE tissues (n=5), expected RNA amount per unit area, the relationship between archiving time and quantity/quality of FFPE-extracted RNA (n=14), differences in quantitative real-time polymerase chain reaction (qRT-PCR) and NGS results, and comparisons of both techniques with tissue processing at different institutions (n=96) were determined in this study. Results The quality of RNA did not show any statistically significant difference between paired FF and FFPE specimens (p=0.49). Analysis of tumor cellularity gave an expected RNA amount of 33.25 ng/mm2. Archiving time affected RNA quality, showing a negative correlation with RNA integrity number and a positive correlation with threshold cycle. However, RNA from samples as old as 10 years showed a 100% success rate in qRT-PCR using short primers, showing that the effect of archiving time can be overcome by proper experiment design. NGS showed a higher success rate than qRT-PCR. Specimens from institution B (n=46), which were often stored in a refrigerator for more than 6 hours and fixed without slicing, showed lower success rates and worse results than specimens from the other institutes. Conclusion Archived FFPE tissues can be used to extract RNA for NGS if they are properly processed before fixation. The expected amount of RNA per unit size calculated in this study will be useful for other researchers.

Adrenal Collision Tumor: Coexistence of Pigmented Adrenal Cortical Oncocytoma and Ganglioneuroma

Background. Adrenal collision tumors (ACTs), in which distinct tumors coexist without intermingling in the same adrenal gland, are rare and their actual prevalence is unknown. ACTs commonly consist of adrenal cortical adenoma, pheochromocytoma, or metastatic malignant tumor. Case Report. A 32-year-old woman who had been experiencing gastric discomfort for one month was referred to our hospital with abnormal imaging findings. The physical examination and the laboratory data including endocrine studies were unremarkable. Abdomen computed tomography (CT) and magnetic resonance imaging (MRI) showed two adjacent masses in the left suprarenal fossa, and a laparoscopic left adrenalectomy was done. Histological and immunohistochemical (IHC) examinations revealed two distinct tumors: a pigmented adrenal cortical oncocytoma (ACO) and a ganglioneuroma, respectively. Conclusion. Both tumors are rare in the adrenal gland and exist as ACTs only exceptionally rarely. This is the first reported case of coexisting oncocytoma and ganglioneuroma in the same adrenal gland to our knowledge.

Abstract 4229: The anti-estrogen effect of metformin in ERα+ breast cancer and tamoxifen resistant cell lines

Anti-estrogen therapy is still the most important systemic treatment to manage estrogen responsive breast cancer. Tamoxifen resistance is a major obstacle in treatment of ER-positive breast cancer. Recently, metformin, which is a biguanide derivative which is commonly used in the treatment of type II diabetes, has been emerged as a potential anti-cancer agent. We investigated if metformin had anti-estrogen effect in ER-positive breast cancer cell lines which are MCF-7, MDA-MB-361 as well as tamoxifen resistant cells (TR-MCF-7). Metformin inhibited the cell proliferation and the expression of ERα in dose-dependent manner in MCF-7 and TR-MCF-7 cells. Under stimulation of 17β-estradiol (E2), metformin inhibited the cell proliferation and expression of mRNA and protein of ERα in MCF-7 and TR-MCF-7 cells, while tamoxifen didn9t affect E2 induced cell proliferation and ERα expression. This result indicates that metformin, as anti-estrogen agent, is superior to tamoxifen for targeting ER in breast cancer. We also identified that metformin inhibited E2 induced cell proliferation and expression of mRNA and protein of ERα in MDA-MB 361 cell line (ER+/Her-2+). We also analyzed estrogen responsive element (ERE) activity using by luciferase assay and identified metformin suppressed E2-inducible luciferase activity and expression of direct ER-regulated genes, such as c-myc, Cyclin D1 and pS2 and FOXA1, which were not inhibited by tamoxifen. In conclusion, metformin has a direct anti proliferative effect and anti-estrogen effect in MCF-7, TR-MCF-7 as well as MDA-MB-361 cell lines. These results suggest that metformin can be used as anti- estrogen in ER positive breast cancer (luminal A & B) and tamoxifen resistant breast cancer. Citation Format: Jinkyoung Kim, Jiyun Lee, Soonyoung Jang, Chungyeul Kim, Aeree Kim. The anti-estrogen effect of metformin in ERα+ breast cancer and tamoxifen resistant cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4229. doi:10.1158/1538-7445.AM2014-4229

Abstract P4-06-02: Changes in intrinsic subtype of breast cancer during tumor progression in same patient

Introduction: Hormone receptor(ER, PR), Epidermal growth factor receptor 2 (HER2) and Ki67 are important prognostic factors and key variables in classification of intrinsic subtype which is essential for selection of adjuvant therapy in breast cancer management. There have been previous reports that instability of hormonal and HER2 status during tumor progression. Recently breast cancer treatment guidelines recommended using intrinsic subtype for treatment option. Especially intrinsic subtypes were determined by using 4 immunohistochemical (IHC) assays that is ER (estrogen receptor), PR (progesterone receptor), HER2 and Ki67. The purpose of this study was to investigate whether the intrinsic subtype changes as tumor progresses from ductal carcinoma in situ (DCIS) to lymph node metastasis Methods : 90 cases of breast cancer were constructed with 2-mm core tissue microarrays (TMA). All cases simultaneously have DCIS, invasive carcinoma (IDC) and lymph node metastasis. IHC and Silver in situ hybridization (SISH) assay was performed on TMA. All of the cases after 2005 had informed consent from the patients for using their medical information and tumor tissues. Evaluation of hormone receptors expression was based on the Allred scoring method. For HER2, membranous staining was evaluated according to the guidelines of the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP). Using the results of the immunohistochemical analyses, tumors were classified into four subtypes. To define luminal A type is an ER positive and/or PR positive, HER2 negative and Ki67 low. As well as luminal B type is an ER positive and/or PR positive, HER2 positive or Ki67 high. HER2 type is HER2 positive, ER negative and PR negative and Ki67 low or high. Basal like type is classified as exhibited ER, PR, and HER2 negative. Results : Of the 90 cases, the lesions with DCIS showed 39 were luminal A, 24 were luminal B, 19 were HER2, and 8 were basal like. The lesions with IDC showed 31 with luminal A, 26 with luminal B, 23 with HER2, and 10 with basal like. Also, 28 were luminal A, 29 were luminal B, 21 were HER2, and 12 were basal like for lymph node metastasis. Overall 25% breast cancer change their intrinsic phenotype during progression. Conclusions : This study demonstrated that a subset of breast cancers can change their intrinsic subtype during cancer progression. These changes have huge impact on patient prognosis and management because each subtype have own different optimized treatment options according to St Gallen and NCCN guideline. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-06-02.