Tumor-initiating cells are not enriched in cisplatin-surviving BRCA1;p53-deficient mammary tumor cells in vivo

Although many breast cancers respond to chemotherapy or hormonal therapy, lack of tumor eradication is a central clinical problem preceding the development of drug resistant tumors. Using the K14cre;Brca1^F5-13/F5-13;p53^F2-10/F2-10mouse model for hereditary breast cancer, we have previously studied responses of mammary tumors generated in to clinically relevant anti-cancer drugs, including cisplatin. The BRCA1- and p53-deficient tumors generated in this model are hypersensitive to cisplatin and never become resistant to this agent due to the large, irreversible deletion in Brca1. We show here that even dose-dense treatment with a maximum tolerated dose of cisplatin does not result in complete tumor eradication. To explain this result we have addressed the hypothesis that the lack of eradication of drug-sensitive tumors is due to increased in vivo chemotherapy resistance of tumor-initiating cells (TICs). Using the CD24 and CD49f cell surface markers which detect normal mouse mammary stem cells, we have identified tumor-initiating cells in BRCA1- and p53-deficient tumors. In addition to the "OLE_LINK14">Lin^-/CD24⁺/CD49f⁺ subpopulation, we show that a larger population of Lin^-/CD24⁺/CD49f^- cells also has tumor-initiating capability in at least two serial orthotopic transplantations, suggesting that these are not more differentiated transit-amplifying cells. However, we did not find an enrichment of TICs in cisplatin-treated tumor remnants. We conclude that in this model the tolerance of the cisplatin-surviving cells cannot be attributed to special biochemical defense mechanisms of TICs.

     

Calcium channel α2δ1 subunit is a functional marker and therapeutic target for tumor-initiating cells in non-small cell lung cancer

It is hypothesized that tumor-initiating cells (TICs) with stem cell-like properties constitute a sustaining force to drive tumor growth and renew fully established malignancy. However, the identification of such a population in non-small cell lung carcinoma (NSCLC) has been hindered by the lacking of reliable surface markers, and very few of the currently available surface markers are of functional significance. Here, we demonstrate that a subpopulation of TICs could be specifically defined by the voltage-gated calcium channel α2δ1 subunit from non-small cell lung carcinoma (NSCLC) cell lines and clinical specimens. The α2δ1⁺ NSCLC TICs are refractory to conventional chemotherapy, and own stem cell-like properties such as self-renewal, and the ability to generate heterogeneous tumors in NOD/SCID mice. Moreover, α2δ1⁺ NSCLC cells are more enriched for TICs than CD133⁺, or CD166⁺ cells. Interestingly, α2δ1 is functionally sufficient and indispensable to promote TIC properties by mediating Ca²⁺ influx into cells, which subsequently activate Calcineurin/NFATc2 signaling that directly activates the expression of NOTCH3, ABCG2. Importantly, a specific antibody against α2δ1 has remarkably therapeutic effects on NSCLC xenografts by eradicating TICs. Hence, targeting α2δ1 to prevent calcium influx provides a novel strategy for targeted therapy against TICs of NSCLC.Subject terms: Cancer stem cells, Predictive markers     

EZN-2208 (PEG-SN38) Overcomes ABCG2-Mediated Topotecan Resistance in BRCA1-Deficient Mouse Mammary Tumors

BRCA1 dysfunction in hereditary breast cancer causes defective homology-directed DNA repair and sensitivity towards DNA damaging agents like the clinically used topoisomerase I inhibitors topotecan and irinotecan. Using our conditional K14cre;Brca1^F/F;p53^F/F mouse model, we showed previously that BRCA1;p53-deficient mammary tumors initially respond to topotecan, but frequently acquire resistance by overexpression of the efflux transporter ABCG2. Here, we tested the pegylated SN38 compound EZN-2208 as a novel approach to treat BRCA1-mutated tumors that express ABCG2. We found that EZN-2208 therapy resulted in more pronounced and durable responses of ABCG2-positive tumors than topotecan or irinotecan therapy. We also evaluated tumor-specific ABCG2 inhibition by Ko143 in Abcg2^−/− host animals that carried tumors with topotecan-induced ABCG2 expression. Addition of Ko143 moderately increased overall survival of these animals, but did not yield tumor responses like those seen after EZN-2208 therapy. Our results suggest that pegylation of Top1 inhibitors may be a useful strategy to circumvent efflux transporter-mediated resistance and to improve their efficacy in the clinic.     

CD49fhigh Cells Retain Sphere-Forming and Tumor-Initiating Activities in Human Gastric Tumors

Identification of gastric tumor-initiating cells (TICs) is essential to explore new therapies for gastric cancer patients. There are reports that gastric TICs can be identified using the cell surface marker CD44 and that they form floating spheres in culture, but we could not obtain consistent results with our patient-derived tumor xenograft (PDTX) cells. We thus searched for another marker for gastric TICs, and found that CD49f^high cells from newly-dissected gastric cancers formed tumors with histological features of parental ones while CD49f^low cells did not when subcutaneously injected into immunodeficient mice. These results indicate that CD49f, a subunit of laminin receptors, is a promising marker for human gastric TICs. We established a primary culture system for PDTX cells where only CD49f^high cells could grow on extracellular matrix (ECM) to form ECM-attaching spheres. When injected into immunodeficient mice, these CD49f^high sphere cells formed tumors with histological features of parental ones, indicating that only TICs could grow in the culture system. Using this system, we found that some sphere-forming TICs were more resistant than gastric tumor cell lines to chemotherapeutic agents, including doxorubicin, 5-fluorouracil and doxifluridine. There was a patient-dependent difference in the tumorigenicity of sphere-forming TICs and their response to anti-tumor drugs. These results suggest that ECM plays an essential role for the growth of TICs, and that this culture system will be useful to find new drugs targeting gastric TICs.     

Progesterone Receptor A Stability Is Mediated by Glycogen Synthase Kinase-3β in the Brca1-deficient Mammary Gland

Germ line mutations of the BRCA1 gene increase the risk of breast and ovarian cancer, but the basis of this tissue-specific tumor predisposition is not fully understood. Previously, we reported that the progesterone receptors are stabilized in Brca1-deficient mammary epithelial cells, and treating with anti-progesterone delays mammary tumorigenesis in Brca1/p53 conditional knock-out mice, suggesting that the progesterone has a critical role in breast carcinogenesis. To further explore how the stability of progesterone receptor is modulated, here, we have found that glycogen synthase kinase (GSK)-3β phosphorylation of progesterone receptor-A (PR-A) facilitates its ubiquitination. GSK-3β-mediated phosphorylation of serine 390 in PR-A regulates its subsequent ubiquitination and protein stability. Expression of PR-A^S390A mutant in the human breast epithelial cells, MCF-10A, results in enhanced proliferation and formation of aberrant acini structure in the three-dimensional culture. Consistently, reduction of phosphorylation of serine 390 of PR-A and GSK-3β activity is observed in the Brca1-deficient mammary gland. Taken together, these results provide important aspects of tissue specificity of BRCA1-mediated suppression of breast carcinogenesis.     

Induction of Ovarian Leiomyosarcomas in Mice by Conditional Inactivation of Brca1 and p53

Background

Approximately one out of every ten cases of epithelial ovarian cancer (EOC) is inherited. The majority of inherited cases of EOC result from mutations in the breast cancer associated gene 1 (BRCA1). In addition to mutation of BRCA1, mutation of the p53 gene is often found in patients with inherited breast and ovarian cancer syndrome.

Methodology/Principal Findings

We investigated the role of loss of function of BRCA1 and p53 in ovarian cancer development using mouse models with conditionally expressed alleles of Brca1 and/or p53. Our results show that ovary-specific Cre-recombinase-mediated conditional inactivation of both Brca1^LoxP/LoxP and p53^LoxP/LoxP resulted in ovarian or reproductive tract tumor formation in 54% of mice, whereas conditional inactivation of either allele alone infrequently resulted in tumors (≤5% of mice). In mice with conditionally inactivated Brca1^LoxP/LoxP and p53^LoxP/LoxP, ovarian tumors arose after long latency with the majority exhibiting histological features consistent with high grade leiomyosarcomas lacking expression of epithelial, follicular or lymphocyte markers. In addition, tumors with conditional inactivation of both Brca1^LoxP/LoxP and p53^LoxP/LoxP exhibited greater genomic instability compared to an ovarian tumor with inactivation of only p53^LoxP/LoxP.

Conclusions/Significance

Although conditional inactivation of both Brca1 and p53 results in ovarian tumorigenesis, our results suggest that additional genetic alterations or alternative methods for targeting epithelial cells of the ovary or fallopian tube for conditional inactivation of Brca1 and p53 are required for the development of a mouse model of Brca1-associated inherited EOC.     

High-throughput flow cytometry screening of human hepatocellular carcinoma reveals CD146 to be a novel marker of tumor-initiating cells

Hepatocellular carcinoma (HCC) remains a common and lethal cancer. Cancer stem cells, or tumor-initiating cells (TICs), are thought to contribute to the pathogenesis of HCC, but remain to be fully characterized. Unbiased screens of primary human HCC cells for the identification of novel HCC TIC markers have not been reported. We conducted high-throughput flow cytometry (HT-FC) profiling to characterize the expression of 375 CD antigens on tumor cells from 10 different human HCC samples. We selected 91 of these for further analysis based on HT-FC data that showed consistent expression in discrete, rare, sortable populations of HCC cells. Nine of these CD antigens demonstrated significantly increased expression in the EpCAM⁺ stem/progenitor fraction of a human HCC cell line and were further evaluated in primary human HCC tissues from 30 different patients. Of the nine tested, only CD146 demonstrated significantly increased expression in HCC tumor tissue as compared with matched adjacent non-tumor liver tissue. CD146⁺CD31^?CD45^? cells purified from HCC tumors and cell lines demonstrated a unique phenotype distinct from mesenchymal stem cells. As compared with other tumor cell fractions, CD146⁺CD31^?CD45^? cells showed significantly increased colony-forming capacity in vitro, consistent with TICs. This study demonstrates that HT-FC screening can be successfully applied to primary human HCC and reveals CD146 to be a novel TIC marker in this disease.     

Design and In Vitro Evaluation of Layer by Layer siRNA Nanovectors Targeting Breast Tumor Initiating Cells

Efficient therapeutics and early detection has helped to increase breast cancer survival rates over the years. However, the recurrence of breast cancer remains to be a problem and this may be due to the presence of a small population of cells, called tumor initiating cells (TICs). Breast TICs are resistant to drugs, difficult to detect, and exhibit high self-renewal capabilities. In this study, layer by layer (LBL) small interfering RNA (siRNA) nanovectors (SNVs) were designed to target breast TICs. SNVs were fabricated using alternating layers of poly-L-lysine and siRNA molecules on gold (Au) nanoparticle (NP) surfaces. The stability, cell uptake, and release profile for SNVs were examined. In addition, SNVs reduced TIC-related STAT3 expression levels, CD44⁺/CD24⁻/EpCAM⁺ surface marker levels and the number of mammospheres formed compared to the standard transfection agent. The data from this study show, for the first time, that SNVs in LBL assembly effectively delivers STAT3 siRNA and inhibit the growth of breast TICs in vitro.     

Preclinical evaluation of radiation therapy of BRCA1-associated mammary tumors using a mouse model

Although germline mutations in BRCA1 highly predispose women towards breast and ovarian cancer, few substantial improvements in preventing or treating such cancers have been made. Importantly, BRCA1 function is closely associated with DNA damage repair, which is required for genetic stability. Here, we examined the efficacy of radiotherapy, assessing the accumulation of genetic instabilities, in the treatment of BRCA1-associated breast cancer using a Brca1-mutant mouse model. Treatment of Brca1-mutant tumor-engrafted mice with X-rays reduced tumor progression by 27.9% compared with untreated controls. A correlation analysis of irradiation responses and biomarker profiles in tumors at baseline identified differences between responders and non-responders at the protein level (pERα, pCHK2, p53, and EpCAM) and at the SOX2 target expression level. We further demonstrated that combined treatment of Brca1-mutant mammary tumors with irradiation and AZD2281, which inhibits PARP, significantly reduced tumor progression and extended survival. Our findings enhance the understanding of DNA damage and biomarker responses in BRCA1-associated mammary tumors and provide preclinical evidence that radiotherapy with synthetic DNA damage is a potential strategy for the therapeutic management of BRCA1-associated breast cancer.     

Functional Heterogeneity within the CD44 High Human Breast Cancer Stem Cell–Like Compartment Reveals a Gene Signature Predictive of Distant Metastasis

The CD44^hi compartment in human breast cancer is enriched in tumor-initiating cells; however, the functional heterogeneity within this subpopulation remains poorly defined. We used a triple-negative breast cancer cell line with a known bilineage phenotype to isolate and clone CD44^hi single cells that exhibited mesenchymal/basal B and luminal/basal A features, respectively. Herein, we demonstrate in this and other triple-negative breast cancer cell lines that, rather than CD44^hi/CD24⁻ mesenchymal-like basal B cells, the CD44^hi/CD24^lo epithelioid basal A cells retained classic cancer stem cell features, such as tumor-initiating capacity in vivo, mammosphere formation and resistance to standard chemotherapy. These results complement previous findings using oncogene-transformed normal mammary cells showing that only cell clones with a mesenchymal phenotype exhibit cancer stem cell features. Further, we performed comparative quantitative proteomic and gene array analyses of these cells and identified potential novel markers of breast cancer cells with tumor-initiating features, such as lipolysis-stimulated lipoprotein receptor (LSR), RAB25, S100A14 and mucin 1 (MUC1), as well as a novel 31-gene signature capable of predicting distant metastasis in cohorts of estrogen receptor–negative human breast cancers. These findings strongly favor functional heterogeneity in the breast cancer cell compartment and hold promise for further refinements of prognostic marker profiling. Our work confirms that, in addition to cancer stem cells with mesenchymal-like morphology, those tumor-initiating cells with epithelial-like morphology should also be the focus of drug development.     

CD133(+)EpCAM(+) phenotype possesses more characteristics of tumor initiating cells in hepatocellular carcinoma Huh7 cells

Background: EpCAM or CD133 has been used as the tumor initiating cells (TICs) marker in hepatocellular carcinoma (HCC). We investigated whether cells expressing with both EpCAM and CD133 surface marker were more representative for TICs in hepatocellular carcinoma Huh7 cells.Methods: Four different phenotypes of CD133⁺EpCAM⁺, CD133⁺EpCAM^-, CD133^-EpCAM⁺ and CD133^-EpCAM^- in Huh7 cells were sorted by flow cytometry. Then cell differentiation, self-renewal, drug-resistance, spheroid formation and the levels of stem cell-related genes were detected to compare the characteristics of TICs. The ability of tumorigenicity was measured in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice to verify TICs.Results: CD133⁺EpCAM⁺ cells have many characteristics of TICs in Huh7 cells compared with CD133⁺EpCAM^-, CD133^-EpCAM⁺, CD133^-EpCAM^- cells, including enrichment in side population cells, higher differentiation capacity, increased colony-formation ability, preferential expression of stem cell-related genes, appearance of drug-resistant to some chemotherapeutics, more spheroid formation of culture cells and stronger tumorigenicity in NOD/SCID mice.Conclusion: CD133⁺EpCAM⁺ phenotype is precisely represented TICs in Huh7 cells. It might be useful for studying biology mechanism of TICs in hepatocellular carcinoma and screening new targets for cancer therapy.     

Conditionally Reprogrammed Normal and Transformed Mouse Mammary Epithelial Cells Display a Progenitor-Cell–Like Phenotype

Mammary epithelial (ME) cells cultured under conventional conditions senesce after several passages. Here, we demonstrate that mouse ME cells isolated from normal mammary glands or from mouse mammary tumor virus (MMTV)-Neu–induced mammary tumors, can be cultured indefinitely as conditionally reprogrammed cells (CRCs) on irradiated fibroblasts in the presence of the Rho kinase inhibitor Y-27632. Cell surface progenitor-associated markers are rapidly induced in normal mouse ME-CRCs relative to ME cells. However, the expression of certain mammary progenitor subpopulations, such as CD49f⁺ ESA⁺ CD44⁺, drops significantly in later passages. Nevertheless, mouse ME-CRCs grown in a three-dimensional extracellular matrix gave rise to mammary acinar structures. ME-CRCs isolated from MMTV-Neu transgenic mouse mammary tumors express high levels of HER2/neu, as well as tumor-initiating cell markers, such as CD44⁺, CD49f⁺, and ESA⁺ (EpCam). These patterns of expression are sustained in later CRC passages. Early and late passage ME-CRCs from MMTV-Neu tumors that were implanted in the mammary fat pads of syngeneic or nude mice developed vascular tumors that metastasized within 6 weeks of transplantation. Importantly, the histopathology of these tumors was indistinguishable from that of the parental tumors that develop in the MMTV-Neu mice. Application of the CRC system to mouse mammary epithelial cells provides an attractive model system to study the genetics and phenotype of normal and transformed mouse epithelium in a defined culture environment and in vivo transplant studies.     

A mammary repopulating cell population characterized in mammary anlagen reveals essential mammary stroma for morphogenesis

The cells with mammary repopulating capability can achieve mammary gland morphogenesis in a suitable cellular microenvironment. Using cell surface markers of CD24, CD29 and CD49f, mouse mammary repopulating unit (MRU) has been identified in adult mammary epithelium and late embryonic mammary bud epithelium. However, embryonic MRU remains to be fully characterized at earlier mammary anlagen stage. Here we isolated discrete populations of E14.5 mouse mammary anlagen cells. Only Lin⁻CD24^medCD29⁺ cell population was predicted as E14.5 MRU by examining their capacities of forming mammosphere and repopulating cleared mammary fat pad in vivo. However, when we characterized gene expressions of this E14.5 cell population by comparing with adult mouse MRU (Lin⁻CD24⁺CD29^hi), the gene profiling of these two cell populations exhibited great differences. Real-time PCR and immunostaining assays uncovered that E14.5 Lin⁻CD24^medCD29⁺ cell population was a heterogeneous stroma-enriched cell population. Then, limiting dilutions and single-cell assays also confirmed that E14.5 Lin⁻CD24^medCD29⁺ cell population possessed low proportion of stem cells. In summary, heterogeneous Lin⁻CD24^medCD29⁺ cell population exhibited mammary repopulating ability in E14.5 mammary anlagen, implying that only suitable mammary stroma could enable mammary gland morphogenesis, which relied on the interaction between rare stem cells and microenvironment.     

Circulating and Tumor-Infiltrating Myeloid-Derived Suppressor Cells in Patients with Colorectal Carcinoma

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous family of myeloid cells that suppress T cell immunity in tumor-bearing hosts. In patients with colon cancer, MDSCs have recently been described as Lin^−/lowHLA-DR⁻CD11b⁺CD33⁺ cells correlating with cancer stage, metastasis and chemotherapy response. To learn in more detail the dynamic change and clinical relevance of circulating and tumor-infiltrating Lin^−/lowHLA-DR⁻CD11b⁺CD33⁺ MDSC in colorectal cancer, we harvested the blood from 64 patients with varying stage of colorectal cancer and tumor and matched paraneoplastic tissues from 5 patients with advanced colorectal cancer, subjected them to multicolor flow cytometric analysis of percentage, absolute number and phenotype of MDSC and finally characterized their immunosuppressive functions. Our results demonstrate that peripheral blood from colorectal cancer patients contains markedly increased percentage and absolute number of Lin^−/lowHLA-DR⁻CD11b⁺CD33⁺ MDSCs compared with healthy individuals, and this increase is closely correlated with clinical cancer stage and tumor metastasis but not primary tumor size and serum concentrations of cancer biomarker. A similar increase of MDSCs was also observed in the tumor tissues. Phenotyping MDSCs shows that they express high CD13 and CD39, low CD115, CD117, CD124 and PD-L1, and devoid of CD14, CD15 and CD66b, reminiscent of precursor myeloid cells. MDSCs from cancer patients but not healthy donors have the immunosuppressive activity and were able to inhibit in vitro autologous T-cell proliferation. Collectively, this study substantiates the presence of increased immunosuppressive circulating and tumor-resident Lin^−/lowHLA-DR⁻CD11b⁺CD33⁺ MDSCs in patients with colorectal cancers correlating with cancer stage and metastasis, and suggests that pharmacologic blockade of MDSCs should be considered in future clinical trials.     

Conditional Inactivation of Brca1, p53 and Rb in Mouse Ovaries Results in the Development of Leiomyosarcomas

Epithelial ovarian cancer (EOC) is thought to arise in part from the ovarian surface epithelium (OSE); however, the molecular events underlying this transformation are poorly understood. Germline mutations in the BRCA1 tumor suppressor gene result in a significantly increased risk of developing EOC and a large proportion of sporadic EOCs display some sort of BRCA1 dysfunction. To generate a model in which Brca1-mediated transformation can be studied, we previously inactivated Brca1 alone in murine OSE, which resulted in an increased accumulation of premalignant changes, but no tumor formation. In this study, we examined tumor formation in mice with conditionally expressed alleles of Brca1, p53 and Rb, alone or in combination. Intrabursal injection of adenovirus expressing Cre recombinase to inactivate p53 resulted in tumors in 100% of mice. Tumor progression was accelerated in mice with concomitant inactivation of Brca1 and p53, but not Rb and p53. Immunohistologic analyses classified the tumors as leiomyosarcomas that may be arising from the ovarian bursa. Brca1 inactivation in primary cultures of murine OSE cells led to a suppression of proliferation that could be rescued by concomitant inactivation of p53 and/or Rb. Brca1-deficient OSE cells displayed an increased sensitivity to the DNA damaging agent cisplatin, and this effect could be modulated by inactivation of p53 and/or Rb. These results indicate that Brca1 deficiency can accelerate tumor development and alter the sensitivity of OSE cells to chemotherapeutic agents. Intrabursal delivery of adenovirus intended to alter gene expression in the ovarian surface epithelium may, in some strains of mice, result in more rapid transformation of adjacent cells, resulting in leiomyosarcomas.     

Wnt/β-catenin Signaling Inhibitors suppress the Tumor-initiating properties of a CD44+CD133+ subpopulation of Caco-2 cells

Tumor-initiating cells or cancer stem cells are a subset of cancer cells that have tumorigenic potential in human cancer. Although several markers have been proposed to distinguish tumor-initiating cells from colorectal cancer cells, little is known about how this subpopulation contributes to tumorigenesis. Here, we characterized a tumor-initiating cell subpopulation from Caco-2 colorectal cancer cells. Based on the findings that Caco-2 cell subpopulations express different cell surface markers, we were able to discriminate three main fractions, CD44^-CD133^-, CD44^-CD133⁺, and CD44⁺CD133⁺ subsets, and characterized their biochemical and tumorigenic properties. Our results show that CD44⁺CD133⁺ cells possessed an unusual capacity to proliferate and could form tumors when transplanted into NSG mice. Additionally, primary tumors grown from CD44⁺CD133⁺ Caco-2 cells contained mixed populations of CD44⁺CD133⁺ and non-CD44⁺CD133⁺ Caco-2 cells, indicating that the full phenotypic heterogeneity of the parental Caco-2 cells was re-created. Notably, only the CD44⁺CD133⁺ subset of Caco-2-derived primary tumors had tumorigenic potential in NSG mice, and the tumor growth of CD44⁺CD133⁺ cells was faster in secondary xenografts than in primary transplants. Gene expression analysis revealed that the Wnt/β-catenin pathway was over-activated in CD44⁺CD133⁺ cells, and the growth and tumorigenic potential of this subpopulation were significantly suppressed by small-molecule Wnt/β-catenin signaling inhibitors. Our findings suggest that the CD44⁺CD133⁺ subpopulation from Caco-2 cells was highly enriched in tumorigenic cells and will be useful for investigating the mechanisms leading to human colorectal cancer development.     

Genome-Wide Association Study in BRCA1 Mutation Carriers Identifies Novel Loci Associated with Breast and Ovarian Cancer Risk

BRCA1-associated breast and ovarian cancer risks can be modified by common genetic variants. To identify further cancer risk-modifying loci, we performed a multi-stage GWAS of 11,705 BRCA1 carriers (of whom 5,920 were diagnosed with breast and 1,839 were diagnosed with ovarian cancer), with a further replication in an additional sample of 2,646 BRCA1 carriers. We identified a novel breast cancer risk modifier locus at 1q32 for BRCA1 carriers (rs2290854, P = 2.7×10⁻⁸, HR = 1.14, 95% CI: 1.09–1.20). In addition, we identified two novel ovarian cancer risk modifier loci: 17q21.31 (rs17631303, P = 1.4×10⁻⁸, HR = 1.27, 95% CI: 1.17–1.38) and 4q32.3 (rs4691139, P = 3.4×10⁻⁸, HR = 1.20, 95% CI: 1.17–1.38). The 4q32.3 locus was not associated with ovarian cancer risk in the general population or BRCA2 carriers, suggesting a BRCA1-specific association. The 17q21.31 locus was also associated with ovarian cancer risk in 8,211 BRCA2 carriers (P = 2×10⁻⁴). These loci may lead to an improved understanding of the etiology of breast and ovarian tumors in BRCA1 carriers. Based on the joint distribution of the known BRCA1 breast cancer risk-modifying loci, we estimated that the breast cancer lifetime risks for the 5% of BRCA1 carriers at lowest risk are 28%–50% compared to 81%–100% for the 5% at highest risk. Similarly, based on the known ovarian cancer risk-modifying loci, the 5% of BRCA1 carriers at lowest risk have an estimated lifetime risk of developing ovarian cancer of 28% or lower, whereas the 5% at highest risk will have a risk of 63% or higher. Such differences in risk may have important implications for risk prediction and clinical management for BRCA1 carriers.     

CD271 Defines a Stem Cell-Like Population in Hypopharyngeal Cancer

Cancer stem cells contribute to the malignant phenotypes of a variety of cancers, but markers to identify human hypopharyngeal cancer (HPC) stem cells remain poorly understood. Here, we report that the CD271⁺ population sorted from xenotransplanted HPCs possesses an enhanced tumor-initiating capability in immunodeficient mice. Tumors generated from the CD271⁺ cells contained both CD271⁺ and CD271⁻ cells, indicating that the population could undergo differentiation. Immunohistological analyses of the tumors revealed that the CD271⁺ cells localized to a perivascular niche near CD34⁺ vasculature, to invasive fronts, and to the basal layer. In accordance with these characteristics, a stemness marker, Nanog, and matrix metalloproteinases (MMPs), which are implicated in cancer invasion, were significantly up-regulated in the CD271⁺ compared to the CD271⁻ cell population. Furthermore, using primary HPC specimens, we demonstrated that high CD271 expression was correlated with a poor prognosis for patients. Taken together, our findings indicate that CD271 is a novel marker for HPC stem-like cells and for HPC prognosis.