HER2 regulates the mammary stem/progenitor cell population driving tumorigenesis and invasion.

The cancer stem cell hypothesis proposes that cancers arise in stem/progenitor cells through disregulation of self-renewal pathways generating tumors, which are driven by a component of 'tumor-initiating cells' retaining stem cell properties. The HER2 gene is amplified in 20-30% of human breast cancers and has been implicated in mammary tumorigenesis as well as in mediating aggressive tumor growth and metastasis. We demonstrate that HER2 overexpression drives mammary carcinogenesis, tumor growth and invasion through its effects on normal and malignant mammary stem cells. HER2 overexpression in normal mammary epithelial cells (NMEC) increases the proportion of stem/progenitor cells as demonstrated by in vitro mammosphere assays and the expression of stem cell marker aldehyde dehydrogenase (ALDH) as well as by generation of hyperplastic lesions in humanized fat pads of NOD (nucleotide-binding oligomerization domain)/SCID (severe combined immunodeficient) mice. Overexpression of HER2 in a series of breast carcinoma cell lines increases the ALDH-expressing 'cancer stem cell' population which displays increased expression of stem cell regulatory genes, increased invasion in vitro and increased tumorigenesis in NOD/SCID mice. The effects of HER2 overexpression on breast cancer stem cells are blocked by trastuzumab in sensitive, but not resistant, cell lines, an effect mediated by the PI3-kinase/Akt pathway. These studies provide support for the cancer stem cell hypothesis by suggesting that the effects of HER2 amplification on carcinogenesis, tumorigenesis and invasion may be due to its effects on normal and malignant mammary stem/progenitor cells. Furthermore, the clinical efficacy of trastuzumab may relate to its ability to target the cancer stem cell population in HER2-amplified tumors

MicroRNA93 regulates proliferation and differentiation of normal and malignant breast stem cells

MicroRNAs (miRNAs) play important roles in normal cellular differentiation and oncogenesis. microRNA93 (mir-93), a member of the mir106b-25 cluster, located in intron 13 of the MCM7 gene, although frequently overexpressed in human malignancies may also function as a tumor suppressor gene. Using a series of breast cancer cell lines representing different stages of differentiation and mouse xenograft models, we demonstrate that mir-93 modulates the fate of breast cancer stem cells (BCSCs) by regulating their proliferation and differentiation states. In "claudin low" SUM159 cells, expression of mir-93 induces Mesenchymal-Epithelial Transition (MET) associated with downregulation of TGFβ signaling and downregulates multiple stem cell regulatory genes, including JAK1, STAT3, AKT3, SOX4, EZH1, and HMGA2, resulting in cancer stem cell (CSC) depletion. Enforced expression of mir-93 completely blocks tumor development in mammary fat pads and development of metastases following intracardiac injection in mouse xenografts. The effect of mir-93 on the CSC population is dependent on the cellular differentiation state, with mir-93 expression increasing the CSC population in MCF7 cells that display a more differentiated "luminal" phenotype. mir-93 also regulates the proliferation and differentiation of normal breast stem cells isolated from reduction mammoplasties. These studies demonstrate that miRNAs can regulate the states and fates of normal and malignant mammary stem cells, findings which have important biological and clinical implications. © 2012 Liu et al

Cancer stem cell heterogeneity in hereditary breast cancer

The cancer stem cell hypothesis proposes that tumors arise in stem or progenitor cells generating in tumors driven by a subcomponent that retains cancer stem cell properties. Recent evidence supports the hypothesis that the BRCA1 gene involved in hereditary breast cancer plays a role in breast stem cell function. Furthermore, studies using mouse BRCA1 knockout models provide evidence for the existence of heterogeneous cancer stem cell populations in tumors generated in these mice. Although these populations may arise from different stem/progenitor cells, they share the expression of a common set of stem cell regulatory genes and show similar characteristics in in vitro mammosphere assays and xenograft models. Furthermore, these 'cancer stem cells' display resistance to chemotherapeutic agents. These studies suggest that breast tumors may display intertumor stem cell heterogeneity. Despite this heterogeneity, cancer stem cells may share common characteristics that can be used for their identification and for therapeutic targeting

Lack of correlation of stem cell markers in breast cancer stem cells

BACKGROUND: Various markers are used to identify the unique sub-population of breast cancer cells with stem cell properties. Whether these markers are expressed in all breast cancers, identify the same population of cells, or equate to therapeutic response is controversial. METHODS: We investigated the expression of multiple cancer stem cell markers in human breast cancer samples and cell lines in vitro and in vivo, comparing across and within samples and relating expression with growth and therapeutic response to doxorubicin, docetaxol and radiotherapy. RESULTS: CD24, CD44, ALDH and SOX2 expression, the ability to form mammospheres and side-population cells are variably present in human cancers and cell lines. Each marker identifies a unique rather than common population of cancer cells. In vivo, cells expressing these markers are not specifically localized to the presumptive stem cell niche at the tumour/stroma interface. Repeated therapy does not consistently enrich cells expressing these markers, although ER-negative cells accumulate. CONCLUSIONS: Commonly employed methods identify different cancer cell sub-populations with no consistent therapeutic implications, rather than a single population of cells. The relationships of breast cancer stem cells to clinical parameters will require identification of specific markers or panels for the individual cancer

The diacylglycerol kinase α/Atypical PKC/β1 integrin pathway in SDF-1α mammary carcinoma invasiveness

Diacylglycerol kinase α (DGKα), by phosphorylating diacylglycerol into phosphatidic acid, provides a key signal driving cell migration and matrix invasion. We previously demonstrated that in epithelial cells activation of DGKα activity promotes cytoskeletal remodeling and matrix invasion by recruiting atypical PKC at ruffling sites and by promoting RCP-mediated recycling of α5β1 integrin to the tip of pseudopods. In here we investigate the signaling pathway by which DGKα mediates SDF-1α-induced matrix invasion of MDA-MB-231 invasive breast carcinoma cells. Indeed we showed that, following SDF-1α stimulation, DGKα is activated and localized at cell protrusion, thus promoting their elongation and mediating SDF-1α induced MMP-9 metalloproteinase secretion and matrix invasion. Phosphatidic acid generated by DGKα promotes localization at cell protrusions of atypical PKCs which play an essential role downstream of DGKα by promoting Rac-mediated protrusion elongation and localized recruitment of β1 integrin and MMP-9. We finally demonstrate that activation of DGKα, atypical PKCs signaling and β1 integrin are all essential for MDA-MB-231 invasiveness. These data indicates the existence of a SDF-1α induced DGKα - atypical PKC - β1 integrin signaling pathway, which is essential for matrix invasion of carcinoma cells

Suppression of apoptosis inhibitor c-FLIP selectively eliminates breast cancer stem cell activity in response to the anti-cancer agent, TRAIL

Introduction It is postulated that breast cancer stem cells (bCSCs) mediate disease recurrence and drive formation of distant metastases - the principal cause of mortality in breast cancer patients. Therapeutic targeting of bCSCs however, is hampered by their heterogeneity and resistance to existing therapeutics. In order to identify strategies to selectively remove bCSCs from breast cancers, irrespective of their clinical subtype, we sought an apoptosis mechanism that would target bCSCs yet would not kill normal cells. Suppression of the apoptosis inhibitor cellular FLICE-Like Inhibitory Protein (c-FLIP) partially sensitizes breast cancer cells to the anti-cancer agent Tumour Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL). Here we demonstrate in breast cancer cell lines that bCSCs are exquisitely sensitive to the de-repression of this pro-apoptotic pathway, resulting in a dramatic reduction in experimental metastases and the loss of bCSC self-renewal. Methods Suppression c-FLIP was performed by siRNA (FLIPi) in four breast cancer cell lines and by conditional gene-knockout in murine mammary glands. Sensitivity of these cells to TRAIL was determined by complementary cell apoptosis assays, including a novel heterotypic cell assay, while tumour-initiating potential of cancer stem cell subpopulations was determined by mammosphere cultures, aldefluor assay and in vivo transplantation. Results Genetic suppression of c-FLIP resulted in the partial sensitization of TRAIL-resistant cancer lines to the pro-apoptotic effects of TRAIL, irrespective of their cellular phenotype, yet normal mammary epithelial cells remained refractory to killing. While 10%-30% of the cancer cell populations remained viable after TRAIL/FLIPi treatment, subsequent mammosphere and aldefluor assays demonstrated that this pro-apoptotic stimulus selectively targeted the functional bCSC pool, eliminating stem cell renewal. This culminated in an 80% reduction in primary tumours and a 98% reduction in metastases following transplantation. The recurrence of residual tumour initiating capacity was consistent with the observation that post-treated adherent cultures re-acquired bCSC-like properties in vitro. Importantly however this recurrent bCSC activity was attenuated following repeated TRAIL/FLIPi treatment. Conclusions We describe an apoptotic mechanism that selectively and repeatedly removes bCSC activity from breast cancer cell lines and suggest that a combined TRAIL/FLIPi therapy could prevent metastatic disease progression in a broad range of breast cancer subtypes. [PROVISIONAL

Markers of Tumor-Initiating Cells Predict Chemoresistance in Breast Cancer

PURPOSE: Evidence is lacking whether the number of breast tumor-initiating cells (BT-ICs) directly correlates with the sensitivity of breast tumors to chemotherapy. Here, we evaluated the association between proportion of BT-ICs and chemoresistance of the tumors. METHODS: Immunohistochemical staining(IHC) was used to examine the expression of aldehyde dehydrogenase 1 (ALDH1) and proliferating cell nuclear antigen, and TUNEL was used to detect the apoptosis index. The significance of various variables in patient survival was analyzed using a Cox proportional hazards model. The percentage of BT-ICs in breast cancer cell lines and primary breast tumors was determined by ALDH1 enzymatic assay, CD44(+)/CD24(-) phenotype and mammosphere formation assay. RESULTS: ALDH1 expression determined by IHC in primary breast cancers was associated with poor clinical response to neoadjuvant chemotherapy and reduced survival in breast cancer patients. Breast tumors that contained higher proportion of BT-ICs with CD44(+)/CD24(-) phenotype, ALDH1 enzymatic activity and sphere forming capacity were more resistant to neoadjuvant chemotherapy. Chemoresistant cell lines AdrR/MCF-7 and SK-3rd, had increased number of cells with sphere forming capacity, CD44(+)/CD24(-) phenotype and side-population. Regardless the proportion of T-ICs, FACS-sorted CD44(+)/CD24(-) cells that derived from primary tumors or breast cancer lines were about 10-60 fold more resistant to chemotherapy relative to the non- CD44(+)/CD24(-) cells and their parental cells. Furthermore, our data demonstrated that MDR1 (multidrug resistance 1) and ABCG2 (ATP-binding cassette sub-family G member 2) were upregulated in CD44(+)/CD24(-) cells. Treatment with lapatinib or salinomycin reduced the proportion of BT-ICs by nearly 50 fold, and thus enhanced the sensitivity of breast cancer cells to chemotherapy by around 30 fold. CONCLUSIONS: These data suggest that the proportion of BT-ICs is associated with chemotherapeutic resistance of breast cancer. It highlights the importance of targeting T-ICs, rather than eliminating the bulk of rapidly dividing and terminally differentiated cells, in novel anti-cancer strategies

Preferential antitumor effect of the Src inhibitor dasatinib associated with a decreased proportion of aldehyde dehydrogenase 1-positive cells in breast cancer cells of the basal B subtype

<p>Abstract</p> <p>Background</p> <p>Recent studies have suggested that the Src inhibitor dasatinib preferentially inhibits the growth of breast cancer cells of the basal-like subtype. To clarify this finding and further investigate combined antitumor effects of dasatinib with cytotoxic agents, a panel of breast cancer cell lines of various subtypes was treated with dasatinib and/or chemotherapeutic agents.</p> <p>Methods</p> <p>Seven human breast cancer cell lines were treated with dasatinib and/or seven chemotherapeutic agents. Effects of the treatments on c-Src activation, cell growth, cell cycle, apoptosis and the proportion of aldehyde dehydrogenase (ALDH) 1-positive cells were examined.</p> <p>Results</p> <p>The 50%-growth inhibitory concentrations (IC₅₀s) of dasatinib were much lower in two basal B cell lines than those in the other cell lines. The IC₅₀s of chemotherapeutic agents were not substantially different among the cell lines. Dasatinib enhanced antitumor activity of etoposide in the basal B cell lines. Dasatinib induced a G1-S blockade with a slight apoptosis, and a combined treatment of dasatinib with etoposide also induced a G1-S blockade in the basal B cell lines. Dasatinib decreased the expression levels of phosphorylated Src in all cell lines. Interestingly, dasatinib significantly decreased the proportion of ALDH1-positive cells in the basal B cell lines but not in the other cell lines.</p> <p>Conclusions</p> <p>The present study indicates that dasatinib preferentially inhibits the growth of breast cancer cells of the basal B subtype associated with a significant loss of putative cancer stem cell population. A combined use of dasatinib with etoposide additively inhibits their growth. Further studies targeting breast cancers of the basal B subtype using dasatinib with cytotoxic agents are warranted.</p

Inverse Relationship between PSA and IL-8 in Prostate Cancer: An Insight into a NF-κB-Mediated Mechanism

Background: Prostate specific antigen (PSA) is traditionally used as an indicator for the presence of prostate cancer (PCa) and radiotherapy is generally used to treat inoperable and locally advanced PCa. However, how cellular PSA level is associated with sensitivity of PCa to radiotherapy is unknown. The previous finding that the RelB-based NF-kB alternative pathway differentially regulates PSA and interleukin-8 (IL-8) in aggressive PCa has directed our attention to the role of RelB in the response of PCa to radiotherapy. Methodology/Principal Findings: RelB and its targets PSA and IL-8 in PCa cells were manipulated by ectopic expression in PCa cells with a low endogenous level of RelB (LNCaP) and by RNAi-based knock-down in PCa cells with a high constitutive level of RelB (PC3). The effects of RelB, PSA and IL-8 on the response of PCa to radiation treatment were examined in vitro and in xenograft tumors. RelB regulates PSA and IL-8 in an inverse manner. When the cellular levels of PSA and IL-8 were directly modulated by genetic manipulations or by the addition of recombinant proteins, the results demonstrate that upregulation of IL-8 enhanced radioresistance of PCa cells and concurrently down-regulated PSA. In contrast, up-regulation of PSA resulted in reduced radioresistance with concurrent down-regulation of IL-8. Conclusion/Significance: RelB plays a critical role in the response of PCa to radiotherapy and the inverse expression of IL-8 and PSA. The results identify a previously unrecognized relationship between IL-8 and PSA in the response of PCa cells t