Chunfa Jie

Proteomic profiling of cancer stem cells derived from primary tumors of HER2/Neu transgenic mice

Human epidermal growth factor receptor 2 (HER2) overexpression leads to mammary tumorigenesis and its elevated levels lead to increase in cancer stem cells (CSCs), invasion, and metastasis. CSCs are resistant to radiation/chemotherapeutic drugs and are believed to be responsible for recurrence/relapse of cancer. CSCs are isolated using flow cytometry based sorting, although reliable, this technology hinders the convenient identification of molecular targets of CSCs. Therefore to understand the molecular players of increased CSC through HER2 overexpression and to develop meaningful targets for combination therapy, we isolated and characterized breast CSCs through convenient tumorsphere culture. We identified the altered protein expression in CSC as compared to non‐CSC using LC‐MS/MS and confirmed those results using qRT‐PCR and Western blotting. Ferritin heavy chain 1 (FTH1) was identified as a candidate gene, which is involved in iron metabolism and iron depletion significantly decreased the self‐renewal of CSCs. We further performed in silico analysis of altered genes in tumorsphere and identified a set of genes (PTMA, S100A4, S100A6, TNXRD1, COX‐1, COX‐2, KRT14, and FTH1), representing possible molecular targets, which in combination showed a promise to be used as prognostic markers for breast cancer.

HOXB7 promotes malignant progression by activating the TGFβ signaling pathway.

Overexpression of HOXB7 in breast cancer cells induces an epithelial-mesenchymal transition and promotes tumor progression and lung metastasis. However, the underlying mechanisms for HOXB7-induced aggressive phenotypes in breast cancer remain largely unknown. Here, we report that phosphorylation of SMAD3 was detected in a higher percentage in primary mammary tumor tissues from double-transgenic MMTV-Hoxb7/Her2 mice than tumors from single-transgenic Her2/neu mice, suggesting activation of TGFβ/SMAD3 signaling by HOXB7 in breast tumor tissues. As predicted, TGFβ2 was high in four MMTV-Hoxb7/Her2 transgenic mouse tumor cell lines and two breast cancer cell lines transfected with HOXB7, whereas TGFβ2 was low in HOXB7-depleted cells. HOXB7 directly bound to and activated the TGFβ2 promoter in luciferase and chromatin immunoprecipitation assays. Increased migration and invasion as a result of HOXB7 overexpression in breast cancer cells were reversed by knockdown of TGFβ2 or pharmacologic inhibition of TGFβ signaling. Furthermore, knockdown of TGFβ2 in HOXB7-overexpressing MDA-MB-231 breast cancer cells dramatically inhibited metastasis to the lung. Interestingly, HOXB7 overexpression also induced tumor-associated macrophage (TAM) recruitment and acquisition of an M2 tumor-promoting phenotype. TGFβ2 mediated HOXB7-induced activation of macrophages, suggesting that TAMs may contribute to HOXB7-promoted tumor metastasis. Providing clinical relevance to these findings, by real-time PCR analysis, there was a strong correlation between HOXB7 and TGFβ2 expression in primary breast carcinomas. Taken together, our results suggest that HOXB7 promotes tumor progression in a cell-autonomous and non-cell-autonomous manner through activation of the TGFβ signaling pathway.

A novel double-negative feedback loop between miR-489 and the HER2-SHP2-MAPK signaling axis regulates breast cancer cell proliferation and tumor growth

Human epidermal growth factor receptor 2 (HER2 or ErBb2) is a receptor tyrosine kinase overexpressed in 20-30% of breast cancers and associated with poor prognosis and outcome. Dysregulation of several microRNAs (miRNAs) plays a key role in breast cancer progression and metastasis. In this study, we screened and identified miRNAs dysregualted in HER2-positive breast cancer cells. Our molecular study demonstrated that miR-489 was specifically downregulated by the HER2-downstream signaling, especially through the MAPK pathway. Restoration or overexpression of miR-489 in HER2-positive breast cancer cells significantly inhibited cell growth in vitro and decreased the tumorigenecity and tumor growth in xenograft mice. Mechanistically, we found that overexpression of miR-489 led to the decreased levels of HER2 and SHP2 and thus attenuated HER2-downstream signaling. Furthermore, we for the first time demonstrated that HER2 is a direct target of miR-489 and therefore HER2-SHP2-MAPK and miR-489 signaling pathways form a mutually inhibitory loop. Using quantitative real-time PCR analysis and Fluorescent in situ hybridization technique (FISH), we found that miR-489 was expressed at significantly lower level in tumor tissues compared to the adjacent normal tissues. Downregulation of miR-489 in breast cancers was associated with aggressive tumor phenotypes. Overall, our results define a double-negative feedback loop involving miR-489 and the HER2-SHP2-MAPK signaling axis that can regulate breast cancer cell proliferation and tumor progression and might have therapeutic relevance for HER2-positive breast cancer.

Differential effect of long-term drug selection with doxorubicin and vorinostat on neuroblastoma cells with cancer stem cell characteristics

Numerous studies have confirmed that cancer stem cells (CSCs) are more resistant to chemotherapy; however, there is a paucity of data exploring the effect of long-term drug treatment on the CSC sub-population. The purpose of this study was to investigate whether long-term doxorubicin treatment could expand the neuroblastoma cells with CSC characteristics and histone acetylation could affect stemness gene expression during the development of drug resistance. Using n-myc amplified SK-N-Be(2)C and non-n-myc amplified SK-N-SH human neuroblastoma cells, our laboratory generated doxorubicin-resistant cell lines in parallel over 1 year; one cell line intermittently treated with the histone deacetylase inhibitor (HDACi) vorinostat and the other without exposure to HDACi. Cells’ sensitivity to chemotherapeutic drugs, the ability to form tumorspheres, and capacity for in vitro invasion were examined. Cell-surface markers and side populations (SPs) were analyzed using flow cytometry. Differentially expressed stemness genes were identified through whole genome analysis and confirmed with real-time PCR. Our results indicated that vorinostat increased the sensitivity of only SK-N-Be(2)C-resistant cells to chemotherapy, made cells lose the ability to form tumorspheres, and reduced in vitro invasion and the SP percentage. CD133 was not enriched in doxorubicin-resistant or vorinostat-treated doxorubicin-resistant cells. Nine stemness-linked genes (ABCB1, ABCC4, LMO2, SOX2, ERCC5, S100A10, IGFBP3, TCF3, and VIM) were downregulated in vorinostat-treated doxorubicin-resistant SK-N-Be(2)C cells relative to doxorubicin-resistant cells. A sub-population of cells with CSC characteristics is enriched during prolonged drug selection of n-myc amplified SK-N-Be(2)C neuroblastoma cells. Vorinostat treatment affects the reversal of drug resistance in SK-N-Be(2)C cells and may be associated with downregulation of stemness gene expression. This work may be valuable for clinicians to design treatment protocols specific for different neuroblastoma patients.

The Effect of Vorinostat on the Development of Resistance to Doxorubicin in Neuroblastoma

Histone deacetylase (HDAC) inhibitors, especially vorinostat, are currently under investigation as potential adjuncts in the treatment of neuroblastoma. The effect of vorinostat co-treatment on the development of resistance to other chemotherapeutic agents is unknown. In the present study, we treated two human neuroblastoma cell lines [SK-N-SH and SK-N-Be(2)C] with progressively increasing doses of doxorubicin under two conditions: with and without vorinsotat co-therapy. The resultant doxorubicin-resistant (DoxR) and vorinostat-treated doxorubicin resistant (DoxR-v) cells were equally resistant to doxorubicin despite significantly lower P-glycoprotein expression in the DoxR-v cells. Whole genome analysis was performed using the Ilumina Human HT-12 v4 Expression Beadchip to identify genes with differential expression unique to the DoxR-v cells. We uncovered a number of genes whose differential expression in the DoxR-v cells might contribute to their resistant phenotype, including hypoxia inducible factor-2. Finally, we used Gene Ontology to categorize the biological functions of the differentially expressed genes unique to the DoxR-v cells and found that genes involved in cellular metabolism were especially affected.

HER2 overexpression triggers an IL-1α pro-inflammatory circuit to drive tumorigenesis and promote chemotherapy resistance

Systemic inflammation in breast cancer correlates with poor prognosis, but the molecular underpinnings of this connection are not well understood. In this study, we explored the relationship between HER2 overexpression, inflammation, and expansion of the mammary stem/progenitor and cancer stem-like cell (CSC) population in breast cancer. HER2-positive epithelial cells initiated and sustained an inflammatory milieu needed to promote tumorigenesis. HER2 induced a feedforward activation loop of IL1α and IL6 that stimulated NFκB and STAT3 pathways for generation and maintenance of breast CSC. In mice, Il1a genetic deficiency delayed MMTV-Her2-induced tumorigenesis and reduced inflammatory cytokine expression as well as CSC in primary tumors. In clinical specimens of human breast tumor tissues, tissue microarray analysis revealed a strong positive correlation between IL1α/IL6 expression and CSC-positive phenotype. Pharmacologic blockade of IL1α signaling reduced the CSC population and improved chemotherapeutic efficacy. Our findings suggest new therapeutic or prevention strategies for HER2-positive breast cancers.Significance: IL1α signaling driven by HER2 promotes chronic inflammation needed to support cancer stem-like cell maintenance in HER2-positive breast cancers. Cancer Res; 78(8); 2040-51. ©2018 AACR.

Autophagy, Cell Viability, and Chemoresistance Are Regulated By miR-489 in Breast Cancer

It is postulated that the complexity and heterogeneity in cancer may hinder most efforts that target a single pathway. Thus, discovery of novel therapeutic agents targeting multiple pathways, such as miRNAs, holds promise for future cancer therapy. One such miRNA, miR-489, is downregulated in a majority of breast cancer cells and several drug-resistant breast cancer cell lines, but its role and underlying mechanism for tumor suppression and drug resistance needs further investigation. The current study identifies autophagy as a novel pathway targeted by miR-489 and reports Unc-51 like autophagy activating kinase 1 (ULK1) and lysosomal protein transmembrane 4 beta (LAPTM4B) to be direct targets of miR-489. Furthermore, the data demonstrate autophagy inhibition and LAPTM4B downregulation as a major mechanism responsible for miR-489–mediated doxorubicin sensitization. Finally, miR-489 and LAPTM4B levels were inversely correlated in human tumor clinical specimens, and more importantly, miR-489 expression levels predict overall survival in patients with 8q22 amplification (the region in which LAPTM4B resides). Implications: These findings expand the understanding of miR-489–mediated tumor suppression and chemosensitization in and suggest a strategy for using miR-489 as a therapeutic sensitizer in a defined subgroup of resistant breast cancer patients. Mol Cancer Res; 16(9); 1348–60. ©2018 AACR.

Abstract 781: The effect of vorinostat on the development of resistance to doxorubicin in neuroblastoma

Histone deacetylase (HDAC) inhibitors, especially vorinostat, are currently under investigation as potential adjuncts in the treatment of neuroblastoma. The effect of vorinostat co-treatment on the development of resistance to other chemotherapeutic agents is unknown. In the present study, we treated two human neuroblastoma cell lines [SK-N-SH and SK-N-Be(2)C] with progressively increasing doses of doxorubicin under two conditions: with and without vorinsotat co-therapy. The resultant doxorubicin-resistant (DoxR) and vorinostat-treated doxorubicin resistant (DoxR-v) cells were equally resistant to doxorubicin despite significantly lower P-glycoprotein expression in the DoxR-v cells. Whole genome analysis was performed using the Ilumina Human HT-12 v4 Expression Beadchip to identify genes with differential expression unique to the DoxR-v cells. We uncovered a number of genes whose differential expression in the DoxR-v cells might contribute to their resistant phenotype, including hypoxia inducible factor-2. Finally, we used Gene Ontology to categorize the biological functions of the differentially expressed genes unique to the DoxR-v cells and found that genes involved in cellular metabolism were especially affected. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 781. doi:1538-7445.AM2012-781

Abstract 335: Human neuroblastoma cells undergo TGF-beta mediated mesenchymal change in the transition to drug resistance

Background: Hypoxia inducible factor 1-alpha (HIF1-alpha) is overexpressed in doxorubicin-resistant neuroblastoma cell lines relative to drug sensitive parental cells lines, and has been implicated in inducing epithelial to mesenchymal transition (EMT). We hypothesized that the transition of human neuroblastoma cells from a drug-sensitive to drug-resistant phenotype involves mesenchymal change. Methods: A doxorubicin resistant (DoxR) human neuroblastoma cell line was generated by incubating parental wild type (WT) SK-N-Be(2)C cells in stepwise concentrations of doxorubicin. Cells were deemed resistant after surviving in a concentration of drug approximately 2 Log above the wild type IC50, confirmed by MTT colorimetric survival assay. Cell migration and capacity for invasion were assayed using the scratch test and Matrigel invasion culture system. EMT pathway related genes differentially expressed in the DoxR cells relative to the parental (WT) lines were analyzed using the Ilumina Human HT-12 v4 Expression Beadchip. Genes of interest with a fold change in expression >1.5 and an adjusted p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 335. doi:1538-7445.AM2012-335