The expression and significance of insulin-like growth factor-1 receptor and its pathway on breast cancer stem/progenitors

Introduction

Dysregulation of the insulin-like growth factor-1 receptor (IGF-1R)/phosphatidylinositol-3-kinase (PI3K)/Akt pathway was shown to correlate with breast cancer disease progression. Cancer stem cells are a subpopulation within cancer cells that participate in tumor initiation, radio/chemoresistance and metastasis. In breast cancer, breast cancer stem cells (BCSCs) were identified as CD24^-CD44⁺ cells or cells with high intracellular aldehyde dehydrogenase activity (ALDH⁺). Elucidation of the role of IGF-1R in BCSCs is crucial to the design of breast cancer therapies targeting BCSCs.

Methods

IGF-1R expression in BCSCs and noncancer stem cells sorted from xenografts of human primary breast cancers was examined by fluorescence-activated cell sorting (FACS), western blot analysis and immunoprecipitation. The role of IGF-1R in BCSCs was assessed by IGF-1R blockade with chemical inhibitor and gene silencing. Involvement of PI3K/Akt/mammalian target of rapamycin (mTOR) as the downstream pathway was studied by their phosphorylation status upon IGF-1R inhibition and the effects of chemical inhibitors of these signaling molecules on BCSCs. We also studied 16 clinical specimens of breast cancer for the expression of phosphor-Akt in the BCSCs by FACS.

Results

Expression of phosphorylated IGF-1R was greater in BCSCs than in non-BCSCs from xenografts of human breast cancer, which were supported by western blot and immunoprecipitation experiments. The sorted IGF-1R-expressing cells displayed features of cancer stem/progenitors such as mammosphere formation in vitro and tumorigenicity in vivo, both of which were suppressed by knockdown of IGF-1R. A specific inhibitor of the IGF-1R, picropodophyllin suppressed phospho-Akt^Ser473 and preferentially decreased ALDH⁺ BCSC populations of human breast cancer cells. Furthermore, picropodophyllin inhibited the capacity of CD24^-CD44⁺ BCSCs to undergo the epithelial-mesenchymal transition process with downregulation of mesenchymal markers. Inhibitors of signal molecules downstream of IGF-1R including PI3K/Akt/mTOR also reduced the ALDH⁺ population of breast cancer cells. Furthermore, the mTOR inhibitor, rapamycin, suppressed BCSCs in vitro and in vivo.

Conclusion

Our data support the notion that IGF-1R is a marker of stemness, and IGF-1R and its downstream PI3K/Akt/mTOR pathway are attractive targets for therapy directed against breast cancer stem/progenitors.     

Hypoxia promotes the phenotypic change of aldehyde dehydrogenase activity of breast cancer stem cells

Stable breast cancer cell (BCC) lines are valuable tools for the identification of breast cancer stem cell (BCSC) phenotypes that develop in response to several stimuli as well as for studying the basic mechanisms associated with the initiation and maintenance of BCSCs. However, the characteristics of individual, BCC‐derived BCSCs varies and these cells show distinct phenotypes depending on the different BCSC markers used for their isolation. Aldehyde dehydrogenase (ALDH) activity is just such a recognized biomarker of BCSCs with a CD44⁺/CD24^? phenotype. We isolated BCSCs with high ALDH activity (CD44⁺/CD24^?/Aldefluor^pos) from a primary culture of human breast cancer tissue and observed that the cells had stem cell properties compared to BCSCs with no ALDH activity (CD44⁺/CD24^?/Aldefluor^neg). Moreover, we found Aldefluor^pos BCSCs had a greater hypoxic response and subsequent induction of HIF‐1α expression compared to the Aldefluor^neg BCSCs. We also found that knocking down HIF‐1α, but not HIF‐2α, in Aldefluor^pos BCSCs led to a significant reduction of the stem cell properties through a decrease in the mRNA levels of genes associated with the epithelial‐mesenchymal transition. Indeed, HIF‐1α overexpression in Aldefluor^neg BCSCs led to Slug and Snail mRNA increase and the associated repression of E‐cadherin and increase in Vimentin. Of note, prolonged hypoxic stimulation promoted the phenotypic changes of Aldefluor^neg BCSCs including ALDH activity, tumorigenesis and metastasis, suggesting that hypoxia in the tumor environment may influence BCSC fate and breast cancer clinical outcomes.     

miR-155 increases stemness and decitabine resistance in triple-negative breast cancer cells by inhibiting TSPAN5

Effective therapeutic targets for triple-negative breast cancer (TNBC), a special type of breast cancer (BC) with rapid metastasis and poor prognosis, are lacking, especially for patients with chemotherapy resistance. Decitabine (DCA) is a Food and Drug Administration-approved DNA methyltransferase inhibitor that has been proven effective for the treatment of tumors. However, its antitumor effect in cancer cells is limited by multidrug resistance. Cancer stem cells (CSCs), which are thought to act as seeds during tumor formation, regulate tumorigenesis, metastasis, and drug resistance through complex signaling. Our previous study found that miR-155 is upregulated in BC, but whether and how miR-155 regulates DCA resistance is unclear. In this study, we demonstrated that miR-155 was upregulated in CD24⁻CD44⁺ BC stem cells (BCSCs). In addition, the overexpression of miR-155 increased the number of CD24⁻CD44⁺ CSCs, DCA resistance and tumor clone formation in MDA-231 and BT-549 BC cells, and knockdown of miR-155 inhibited DCA resistance and stemness in BCSCs in vitro. Moreover, miR-155 induced stemness and DCA resistance by inhibiting the direct target gene tetraspanin-5 (TSPAN5). We further confirmed that overexpression of TSPAN5 abrogated the effect of miR-155 in promoting stemness and DCA resistance in BC cells. Our data show that miR-155 increases stemness and DCA resistance in BC cells by targeting TSPAN5. These data provide a therapeutic strategy and mechanistic basis for future possible clinical applications targeting the miR-155/TSPAN5 signaling axis in the treatment of TNBC.     

POMC maintains tumor‐initiating properties of tumor tissue‐derived long‐term‐cultured breast cancer stem cells

The identification and understanding of the molecular network of cancer stem cells (CSCs) have had a profound impact on our view of carcinogenesis and treatment strategy. Unfortunately, a major problem is that serial passages of CSCs from clinical solid tumor specimens currently are not available in any lab, and thus, reported data are difficult to confirm and intensively interrogated. Here, we have generated two tumor tissue‐derived breast CSC (BCSC) lines that showed prolonged maintenance over 20 serial passages in vitro , while retaining their tumor‐initiating biological properties. We then deciphered the intrinsic mechanism using analyses of mRNA expression array profiles. It has been determined that pro‐opiomelanocortin (POMC) is closely related with protein phosphorylation mediated by G‐protein‐coupled estrogen receptor (GPER) in BCSC. Following, knockdown of POMC inhibits properties of mammosphere formation, CD44⁺CD24^? population, CD44 expression, and clonogenicity ability in BCSC. We found that inhibition of POMC attenuates phosphorylation of AKT2 and GSK3β in BCSC. Further in vivo investigations demonstrated that POMC interference regulates proliferation of BCSC‐bearing tumors. Combination of the clinical results that POMC positive expression is frequently upregulated in human breast cancer and POMC positivity correlated with a poor prognosis, POMC is a potential therapeutic target for BCSC. In conclusion, we have successfully established two long‐term‐cultured BCSC from clinical specimens. We further indicated that POMC acts as a potential therapeutic target and prognostic marker for future treatment of BCSC.     

G-Protein-Coupled Estrogen Receptor Enhances the Stemness of Triple-Negative Breast Cancer Cells and Promotes Malignant Characteristics

G-protein coupled estrogen receptor (GPER) is a transmembrane receptor that mediates non-genomic effects of estrogen. This study aimed to investigate the role of GPER in the stemness formation and malignancies in triple negative breast cancer (TNBC) cells. Spheroids of MDA-MB-468 cells were induced by mammosphere culture, and the proportion of the CD44⁺ /CD24^−/low stem cell subpopulation was detected. Malignant characteristics, expression of GPER and stemness-related markers, and tumorigenesis in a xenograft assay were compared between the mammospheres and adherent cultured cells. The impacts of 17β-estradiol (E2) and the GPER-specific antagonist G15 were studied in in vitro assays. The proportion of the CD44⁺ /CD24^−/low subpopulation was increased in the mammospheres of MDA-MB-468 cells, which also showed higher expression of GPER and stemness-related markers than adherent cultured cells. The abilities of spherical colonies to proliferate, invade, and form colonies in soft agar were enhanced. Spherical cells exhibited stronger tumorigenesis ability than adherent cells in the xenograft assay. E2 treatment enhanced tumorigenicity of both adherent and spherical cells. Spherical cells treated with E2 had stronger proliferation, invasion, and colony formation abilities than other groups. Pretreatment with G15 effectively blocked the stimulation by E2. In conclusion, the expression of GPER in TNBC cells is positively related to stemness and malignant features.     

Mortalin maintains breast cancer stem cells stemness via activation of Wnt/GSK3β/β-catenin signaling pathway

Previous research indicated that mortalin overexpressed in breast cancer and contributed to carcinogenesis. Mortalin was also demonstrated to promote Epithelial-mesenchymal transition (EMT) and was considered as a factor for maintaining the stemness of the cancer stem cells. However, the underlying mechanisms about mortalin maintaining the stemness of breast cancer stem cells (BCSCs) remain unclear. Here, we identified that increased expression of mortalin in breast cancer was associated with poorer overall survival rate. Mortalin was elevated in breast cancer cell lines and BCSC-enriched populations. Additionally, knockdown of mortalin significantly inhibited the cell proliferation, migration and EMT, as well as sphere forming capacity and stemness genes expression. Further study revealed that mortalin promoted EMT and maintained BCSCs stemness via activating the Wnt/GSK3β/β-catenin signaling pathway in vivo and in vitro. Taken together, these findings unveiled the mechanism of mortalin in maintaining and regulating the stemness of BCSCs, and may offer novel therapeutic strategies for breast cancer treatment.     

Microarray-based analysis of microRNA expression in breast cancer stem cells

Background

This study aimed to determine the miRNA profile in breast cancer stem cells (BCSCs) and to explore the functions of characteristic BCSC miRNAs.

Methods

We isolated ESA⁺CD44⁺CD24^-/low BCSCs from MCF-7 cells using fluorescence-activated cell sorting (FACS). A human breast cancer xenograft assay was performed to validate the stem cell properties of the isolated cells, and microarray analysis was performed to screen for BCSC-related miRNAs. These BCSC-related miRNAs were selected for bioinformatic analysis and target prediction using online software programs.

Results

The ESA⁺CD44⁺CD24^-/low cells had up to 100- to 1000-fold greater tumor-initiating capability than the MCF-7 cells. Tumors initiated from the ESA⁺CD44⁺CD24^-/low cells were included of luminal epithelial and myoepithelial cells, indicating stem cell properties. We also obtained miRNA profiles of ESA⁺CD44⁺CD24^-/low BCSCs. Most of the possible targets of potential tumorigenesis-related miRNAs were oncogenes, anti-oncogenes or regulatory genes.

Conclusions

We identified a subset of miRNAs that were differentially expressed in BCSCs, providing a starting point to explore the functions of these miRNAs. Evaluating characteristic BCSC miRNAs represents a new method for studying breast cancer-initiating cells and developing therapeutic strategies aimed at eradicating the tumorigenic subpopulation of cells in breast cancer.     

乳腺癌治疗新靶点GPER1研究进展

目的：总结国内外对乳腺癌治疗新靶点G蛋白偶联雌激素受体（Gprotein-coupledestrogenreceptor1,GPER1／GPR30）的研究进展。方法：应用Medline、PubMed及CNKI期刊全文数据库检索系统,分别以“G蛋白偶联雌激素受体（GPER1／GPR30）”和“乳腺癌”、“G蛋白偶联雌激素受体（GPER1／GPR30）”和“乳腺癌内分泌治疗”为关键词,检索1997-2013年国内外的相关文献。纳入标准：1）GPER1参与的信号转导通路与乳腺癌;2）GPER1与乳腺癌抗雌激素治疗耐药性;3）GPER1与乳腺癌病理过程。依据纳入标准,纳入分析的参考文献为52篇。结果：GPER1是一种新型雌激素受体,它不但构建了慢速基因效应和快速非基因效应的交互通话,还通过表皮生长因子受体（epidermalgrowthfactorreceptor,EGFR）活化后参与蛋白激酶途径及通过第二信使cAMP、Ca2＋途径介导快速非基因效应,发挥间接转录调控作用。GPER1介导了乳腺癌的发生发展,其激活可能促使乳腺癌细胞产生他莫昔芬（tamoxifen,TAM）耐药。结论：GPER1是新型的乳腺肿瘤标志。以GPER1及GPER1介导的信号通路为靶点的治疗将有望成为乳腺癌预防和治疗的新方案。     

Targeting highly expressed extracellular HSP90 in breast cancer stem cells inhibits tumor growth in vitro and in vivo

Breast cancer stem cells (BCSC) have been identified in breast carcinoma as CD44⁺/CD24^?/low cells, which display tumorigenic activity and have the ability to self-renew, differentiate and metastasize. Previous studies showed that extracellular HSP90 (eHSP90) participates in the invasion and metastatic processes of various cancers including breast cancer. Here, we show for the first time that eHSP90 is over-expressed in mammosphere cultures that are derived from the MDA-MB-231, MDA-MB-453 and MCF-7 breast cancer cell lines. These mammospheres are highly enriched in cells of the CD44⁺/CD24^?/low BCSC phenotype and additionally show high expression of the BCSC markers CD49f and Sox2. Thus our results indicate that eHSP90 represents a potential novel BCSC marker. Moreover, we present evidence that eHSP90 is functionally involved in BCSC activity in vitro and in vivo. Selective neutralization of eHSP90, using the monoclonal antibody mAb 4C5, has the capacity to inhibit stem cell activity in vitro because the formation of mammosphere-derived colonies is dramatically reduced in its presence. In vivo, the treatment of mice with mAb4C5 using a prophylactic protocol, significantly inhibited the primary growth of MDA-MB-231 and mammosphere-derived tumors. More importantly, administration of this antibody in a therapeutic protocol caused a statistically significant regression of established tumors derived from MDA-MB-231 originating mammospheres. Tumor regression was even greater when mAb 4C5 was administered in combination with paclitaxel. Overall, our findings implicate eHSP90 as a potential novel BCSC biomarker. Moreover they show that eHSP90 participates in BCSC-derived primary tumor growth. Finally, we provide additional support for the possible therapeutic value of mAb4C5 in the treatment of breast cancer.     

雌激素受体GPER在乳腺癌中的研究现状

G蛋白偶联雌激素受体（G protein-coupled estrogen receptor, GPER, 也称GPR30）在多种类型细胞中均可介导雌激素信号,其在乳腺癌、子宫内膜癌等激素敏感性肿瘤细胞中的作用尤为明显。雌激素及抗雌激素药物通过激活GPER促进下游信号通路的活化及靶基因的表达进而参与乳腺癌细胞的增殖、迁移、侵袭及他莫昔芬耐药等恶性生物学行为。目前发现表皮生长因子受体（epidermal growth factor receptor, EGFR）的转活是GPER引发下游生物学效应的关键靶点。此外,GPER还被认为是预测三阴乳腺癌患者预后的潜在生物学标志物之一,对GPER的深入研究可能为乳腺癌患者的综合性评估和临床治疗打开更广阔的前景。     

Blocking the formation of radiation–induced breast cancer stem cells

The goal of adjuvant (post-surgery) radiation therapy (RT) for breast cancer (BC) is to eliminate residual cancer cells, leading to better local tumor control and thus improving patient survival. However, radioresistance increases the risk of tumor recurrence and negatively affects survival. Recent evidence shows that breast cancer stem cells (BCSCs) are radiation-resistant and that relatively differentiated BC cells can be reprogrammed into induced BCSCs (iBCSCs) via radiation-induced re-expression of the stemness genes. Here we show that in irradiation (IR)-treated mice bearing syngeneic mammary tumors, IR-induced stemness correlated with increased spontaneous lung metastasis (51.7%). However, IR-induced stemness was blocked by targeting the NF-κB- stemness gene pathway with disulfiram (DSF)and Copper (Cu²⁺). DSF is an inhibitor of aldehyde dehydrogenase (ALDH) and an FDA-approved drug for treating alcoholism. DSF binds to Cu²⁺ to form DSF-Cu complexes (DSF/Cu), which act as a potent apoptosis inducer and an effective proteasome inhibitor, which, in turn, inhibits NF-κB activation. Treatment of mice with RT and DSF significantly inhibited mammary primary tumor growth (79.4%) and spontaneous lung metastasis (89.6%) compared to vehicle treated mice. This anti-tumor efficacy was associated with decreased stem cell properties (or stemness) in tumors. We expect that these results will spark clinical investigation of RT and DSF as a novel combinatorial treatment for breast cancer.     

The influence of miR-34a expression on stemness and cytotoxic susceptibility of breast cancer stem cells

In this study, we investigate the effect of miR-34a expression and biological characteristics of breast cancer stem cells (BCSCs). The mammospheres were formed from murine breast cancer cell line 4T1 and regarded as murine BCSCs. Identification of stemness molecules and cloning experiments validate the biological characteristics of BCSCs we have established. We showed that miR-34a, as a tumor suppressor, could separately reduce the stemness of BCSCs and activate the cytotoxic susceptibility of BCSCs to natural killer (NK) cells in vitro via down regulating the expression of Notch1 signaling molecules. Moreover, miR-34a could completely restrain established mice breast tumor xenografts in vivo in the NOD/SCID mice that have functional NK cells at a normal level, whereas it was less effective in NOD/SCID/ CD122/IL-2Rβ mice that do not have functional NK cells. We conclude that miR-34a is a crucial, dual tumor suppressor and BCSCs-targeting immunotherapeutic agent and has shown efficacy in the treatment of murine breast cancer. The results also suggest that impaired NK cells could contribute to the resistance to therapies.     

Flubendazole elicits anti‐metastatic effects in triple‐negative breast cancer via STAT3 inhibition

Tumor metastasis remains the cause of 90% of cancer‐related deaths. Cancer stem cells (CSC) are thought to be responsible for the aggressive and metastatic nature of triple‐negative breast cancers (TNBC), and new therapeutic strategies are being devised to target them. Flubendazole (FLU) is a widely used anthelmintic agent that also exhibits anticancer activity in several cancer types. The aim of this study was to characterize the mechanism of action of FLU on breast cancer stem cell (BCSC)‐like properties and metastasis in TNBC. FLU treatment caused a significant induction of apoptosis, accompanied by G2/M phase accumulation, caspase‐3/‐7 activation and the dysregulation of STAT3 activation in TNBC cells. The latter phenomenon was associated with impairment of cancer stem‐like traits, concomitant with a reduction in the CD24^low/CD44^high, CD24^high/CD49f^high subpopulation, ALDH1 activity and mammosphere formation. The BCSC‐enriched populations exhibited enhanced metastasis with higher STAT3 activation, while FLU administration inhibited tumor growth, angiogenesis and lung and liver metastasis, coinciding with decreased MMP‐2 and MMP‐9 levels in circulating blood. FLU kills not only rapid proliferating tumor cells but also effectively eradicates BCSC‐like cells in vitro and in vivo. Our findings warrant further investigation of FLU as a treatment for metastatic TNBC.     

GPER Mediates Estrogen-Induced Signaling and Proliferation in Human Breast Epithelial Cells and Normal and Malignant Breast

17β-Estradiol (estrogen), through receptor binding and activation, is required for mammary gland development. Estrogen stimulates epithelial proliferation in the mammary gland, promoting ductal elongation and morphogenesis. In addition to a developmental role, estrogen promotes proliferation in tumorigenic settings, particularly breast cancer. The proliferative effects of estrogen in the normal breast and breast tumors are attributed to estrogen receptor α. Although in vitro studies have demonstrated that the G protein-coupled estrogen receptor (GPER, previously called GPR30) can modulate proliferation in breast cancer cells both positively and negatively depending on cellular context, its role in proliferation in the intact normal or malignant breast remains unclear. Estrogen-induced GPER-dependent proliferation was assessed in the immortalized nontumorigenic human breast epithelial cell line, MCF10A, and an ex vivo organ culture model employing human breast tissue from reduction mammoplasty or tumor resections. Stimulation by estrogen and the GPER-selective agonist G-1 increased the mitotic index in MCF10A cells and proportion of cells in the cell cycle in human breast and breast cancer explants, suggesting increased proliferation. Inhibition of candidate signaling pathways that may link GPER activation to proliferation revealed a dependence on Src, epidermal growth factor receptor transactivation by heparin-bound EGF and subsequent ERK phosphorylation. Proliferation was not dependent on matrix metalloproteinase cleavage of membrane-bound pro-HB-EGF. The contribution of GPER to estrogen-induced proliferation in MCF10A cells and breast tissue was confirmed by the ability of GPER-selective antagonist G36 to abrogate estrogen- and G-1-induced proliferation, and the ability of siRNA knockdown of GPER to reduce estrogen- and G-1-induced proliferation in MCF10A cells. This is the first study to demonstrate GPER-dependent proliferation in primary normal and malignant human tissue, revealing a role for GPER in estrogen-induced breast physiology and pathology.     

Lack of G protein-coupled estrogen receptor (GPER) in the plasma membrane is associated with excellent long-term prognosis in breast cancer

G protein-coupled estrogen receptor (GPER), or GPR30, is a membrane receptor reported to mediate non-genomic estrogen responses. Tamoxifen is a partial agonist at GPER in vitro. Here, we investigated if GPER expression is prognostic in primary breast cancer, if the receptor is treatment-predictive for adjuvant tamoxifen, and if receptor subcellular localization has any impact on the prognostic value. Total and plasma membrane (PM) GPER expression was analyzed by immunohistochemistry in breast tumors from 742 postmenopausal lymph node-negative patients subsequently randomized for tamoxifen treatment for 2–5 years versus no systemic treatment, regardless of estrogen receptor (ER) status, and with a median follow-up of 17 years for patients free of event. PM GPER expression was a strong independent prognostic factor for poor prognosis in breast cancer without treatment-predictive information for tamoxifen. In the tamoxifen-treated ER-positive and progesterone receptor (PgR)-positive patient subgroup, the absence of PM GPER (53 % of all ER-positive tumors) predicted 91 % 20-year distant disease-free survival, compared to 73 % in the presence of GPER (p = 0.001). Total GPER expression showed positive correlations with ER and PgR and negative correlation with histological grade, but the correlations were biphasic. On the other hand, PM GPER expression showed strong negative correlations with ER and PgR, and strong positive correlation with HER2 overexpression and high histological grade. GPER overexpression and PM localization are critical events in breast cancer progression, and lack of GPER in the PM is associated with excellent long-term prognosis in ER-positive and PgR-positive tamoxifen-treated primary breast cancer.