Shou Liu

Nonlinear Growth Kinetics of Breast Cancer Stem Cells: Implications for Cancer Stem Cell Targeted Therapy

Cancer stem cells (CSCs) have been identified in primary breast cancer tissues and cell lines. The CSC population varies widely among cancerous tissues and cell lines, and is often associated with aggressive breast cancers. Despite of intensive research, how the CSC population is regulated within a tumor is still not well understood so far. In this paper, we present a mathematical model to explore the growth kinetics of CSC population both in vitro and in vivo. Our mathematical models and supporting experiments suggest that there exist non-linear growth kinetics of CSCs and negative feedback mechanisms to control the balance between the population of CSCs and that of non-stem cancer cells. The model predictions can help us explain a few long-standing questions in the field of cancer stem cell research, and can be potentially used to predict the efficicacy of anti-cancer therapy.

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.

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.

Overexpression of miR-489 derails mammary hierarchy structure and inhibits HER2/neu-induced tumorigenesis

Although it has been demonstrated that transformed progenitor cell population can contribute to tumor initiation, factors contributing to this malignant transformation are poorly known. Using in vitro and xenograft-based models, previous studies demonstrated that miR-489 acts as a tumor suppressor miRNA by targeting various oncogenic pathways. It has been demonstrated that miR-489 directly targets HER2 and inhibits the HER2 signaling pathway; however, its role in mammary gland development and HER2-induced tumor initiation hasn’t been studied. To dissect the role of miR-489, we sorted different populations of mammary epithelial cells and determined that miR-489 was highly expressed in mammary stem cells. MMTV-miR-489 mice that overexpressed miR-489 in mammary epithelial cells were developed and these mice exhibited an inhibition of mammary gland development in early ages with a specific impact on highly proliferative cells. Double transgenic MMTV-Her2-miR489 mice were then generated to observe how miR-489 overexpression affects HER2-induced tumorigenesis. miR-489 overexpression delayed HER2-induced tumor initiation significantly. Moreover, miR-489 overexpression inhibited tumor growth and lung metastasis. miR-489 overexpression reduced mammary progenitor cell population significantly in preneoplastic mammary glands of MMTV-Her2 mice which showed a putative transformed population in HER2-induced tumorigenesis. The miR-489 overexpression reduced CD49fhiCD61hi populations in tumors that have stem-like properties, and miR-489 overexpression altered the HER2 signaling pathway in mammary tumors. Altogether, these data indicate that the inhibition of HER2-induced tumorigenesis by miR-489 overexpression was due to altering progenitor cell populations while decreasing tumor growth and metastasis via influencing tumor promoting genes DEK and SHP2.

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 3424: Role of miR-489 in mammary gland development and Her2 mediated tumorigenesis

HER2 overexpression is linked with poor prognosis and outcome in breast cancer. In our previous study, we have found miR-489 was specifically down regulated by HER2 overexpression. Restoration of miR-489 in multiple breast cancer cell lines significantly inhibited cell growth in vitro and decreased tumor growth in xenograft mice. To study role of miR-489 in Her2 mediated tumorigenesis, for the first time we generated MMTV-miR-489 transgenic mice, which overexpress miR-489 specifically in mammary gland. Our qRT-PCR data has confirmed transgenic mice have significantly more miR-489 expression than FVB mice. We used western blot to further validate our model system and found significant downregulation of miR-489 targets DEK and PTPN11. To find out whether miR-489 has any role in mammary gland development, mammary gland whole mount was performed from wild type FVB mice and MMTV-miR-489 mice at different age. Mammary gland from MMTV-miR-489 mice demonstrated reduction in growth at early age. Our immunohistochemistry staining demonstrated significantly reduction in Ki-67 positive cells in MMTV-miR-489 mammary gland at early age, further confirming reduced growth in MMTV-miR-489 mice. However, we found no significant effect on weight of litters of MMTV-miR-489 female since after 8-week mammary gland was able to recover growth. To find out effect of miR-489 overexpression on Her2 mediated tumorigenesis, we generated double transgenic mice MMTV-Her2/miR-489 by crossing MMTV-miR-489 mice with MMTV-Her2 mice. We have observed significant delay in tumor onset and reduced tumor growth in MMTV-Her2/ miR-489 mice compare to MMTV-Her2 mice. We have also observed less number of metastatic site in lung by performing H and E staining of lung. Our IHC data showed reduction in PTPN11 and DEK in miR-489 overexpress mammary tumor. Surprisingly, we found significant reduction of miR-489 expression in mammary tumors of MMTV-Her2-miR-489 when compared with normal mammary gland of same mice. Overall, our results indicated miR-489 overexpression suppresses mammary gland development at early age, reduced mammary tumorigenesis and decrease lung metastasis by targeting PTPN11 and DEK. Citation Format: Yogin Patel, Mithil Soni, Shou Liu, Hexin Chen. Role of miR-489 in mammary gland development and Her2 mediated tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3424. doi:10.1158/1538-7445.AM2017-3424

Abstract LB-54: HOXB7 regulates breast cancer stem cells via induction of TGFβ2

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA HOX genes encode a family of homeodomain-containing transcription factors involved in the determination of cell fate and identity during embryonic development. HOXB7 is overexpressed in more than 70% of breast cancers. Overexpression of HOXB7 in breast epithelial cells and induces epithelial-mesenchymal transition. In this study, we found that overexpression of the HOXB7 gene in breast cell lines increases cancer stem cell populations and promotes cell invasion, in contrast, knockdown of HOXB7 expression decreases the proportions of cancer stem cells. Colonogenic assay and xenograft transplantation experiments further confirmed that overexpression of HOXB7 in breast cancer cells increases their tumorigenecity both in vitro and in vivo. Mechanistically we found that HOXB7 strongly induces TGFβ2 expression and activates the canonical TGFβ/SMAD signaling pathways, which may be responsible for induction of EMT leading to expansion of cancer stem cell population. Overall, our results suggest that HOXB7 may be a critical factor which regulates the self-renewal and differentiation of breast cancer stem cells and therefore a potential therapeutic target in breast cancer. Citation Format: Shou Liu, Sarah Johnson, Hexin Chen. HOXB7 regulates breast cancer stem cells via induction of TGFβ2. [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 LB-54. doi:10.1158/1538-7445.AM2014-LB-54

Abstract 3104: HOXB7 promotes tumor progression via activation of the transformation growth factor-β signaling pathway.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Our previous studies have shown that overexpression of Hoxb7 delays tumors onset, but promotes tumor progression and metastasis in oncogene Her2/neu-induced mammary tumorigenesis, which is reminiscent of the dual role of TGF-β in breast cancer. To investigate whether HOXB7 directly or indirectly regulates the TGF-β signaling, we examined the phosphorylation levels of SMAD3, an important mediator of TGF-beta signaling, in primary mammary tumors derived from the Hoxb7 and Her2/neu double-transgenic mice and the Her2/neu mono-transgenic mice. Phosphorylation of SMAD3 is detected in a higher percentage of primary tumor tissues from double transgenic mice than that from Her2/neu mono-transgenic mice (69.2% versus 15.4%, p < 0.05), suggesting that Hoxb7 may activate TGF-β/Smad signaling in breast tumor tissues. In line with this finding, overexpression of HOXB7 induces expression of TGF-β ligands in both mouse and human breast cancer cell lines, leading to increased cell motility and invasiveness. The acquainted phenotypes in HOXB7-overepressing cells can be reversed by pharmacologic inhibition of TGF-β signaling. Promoter deletion analysis and Chromotin-immunprocipiration assay further demonstrated that HOXB7 can directly bind to and activate the promoter of TGFβ2. More importantly, expression of HOXB7 and TGFβ2 is strongly correlated in primary breast cancer tissues and associated with advanced stages of tumor progression and reduced survival rate. Taken together, our results suggest that HOXB7 may promote tumor migration, invasion and possibly metastasis through activation of the TGF-β signaling pathway. Citation Format: Shou Liu, Hongmei Zhang, Hexin Chen. HOXB7 promotes tumor progression via activation of the transformation growth factor-β signaling pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3104. doi:10.1158/1538-7445.AM2013-3104 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.