Abstract CN06-02: Breast cancer stem cells.
Authors: Chang Jenny C. , Dave Bhuvanesh , Granados-Principal Sergio , Mai Junhua , Gross Steven , Shen Haifa , Mittal Vivek , Ferrari Mauro 
Published: August 2014

Abstract
Even though recent trends suggest a turn downward in both incidence and mortality, breast cancer remains the most common female cancer in the industrialized world. Development of resistance to a wide variety of drugs and metastatic spread of cancer cells to distant organs pose major challenge in successful treatment of breast cancer. Virtually all the roughly 40,000 annual breast cancer related deaths in the United States can be said to have occurred because the conventional therapy has failed. It is the second-leading cause of deaths from cancer in women. The survival rate for those with advanced, metastatic breast cancer has not changed significantly for decades. Recent robust evidence indicates that treatment resistance and metastasis arise from a subpopulation with stem-like properties within a heterogeneous primary cancer. These observations are consistent with subpopulations of chemoresistant cancer stem cells within the bulk tumor cells having the potential for self-renewal and metastases through different adaptive mechanisms. Thus despite apparently successful chemotherapy, the persistence of this unique sub-population of cancer stem cells may undergo epithelial-mesenchymal transition and can serve to re-initiate tumor growth and seed metastases.
Breast cancer stem cells (BCSCs) constitute a subpopulation of tumor cells that express stem cell-associated markers and have a high capacity for tumor generation in vivo. Identification of BCSCs from tumor samples or breast cancer cell lines has been based mainly on CD44+/CD24−/low or ALDH+ phenotypes. BCSCs isolation has allowed the analysis of the molecular mechanisms involved in their origin, self-renewal, differentiation into tumor cells, resistance to radiation therapy and chemotherapy, invasiveness and metastatic ability. Over the past few years, candidate cancer stem cells have been identified in a variety of human malignancies including leukemias and a number of solid tumors such as glioblastomas, medulloblastomas and carcinomas. Breast cancer is the first human carcinoma for which a putative cancer stem cell subpopulation has been isolated (Al-Hajj et. al, PNAS, 2003). Using in vitro-separated tumorigenic cells from malignant human breast cancer-derived pleural effusions, Al Hajj and colleagues isolated a cell population characterized by high CD44 expression and low or undetectable levels of CD24 (CD44+CD24−/low). These cells were highly tumorigenic when injected into immunocompromised NOD/SCID mice and shared classic features of normal stem cells, including the capacity for self-renewal and generation of heterogeneous progeny. The stem/progenitor cell phenotype of these cells was further refined by the Daidone group, who were able to grow mammospheres from single-cell suspensions obtained from the dissociation of primary breast tumors. Mammospheres are non-adherent spherical cell clusters obtained in selective culture conditions, which have been shown to be enriched in mammary stem/progenitor cells. The vast majority of cells in culture were CD44+CD24−/low, and 10 to 20% of these retained the ability to self-renew. Recently, three groups have independently provided unequivocal direct and functional evidence for the presence of cancer stem cells by lineage tracing experiments in glioblastomas (GBM), squamous skin tumors, and intestinal adenomas, suggesting the hierarchical nature of cancer (Chen J et.al, Nature, 2012, Schepers AG et. al, Science 2012; Driessens G et. al, Nature 2012). By genetic “lineage retracing," these independent groups confirm that only a fraction of cells within the bulk tumor had clonogenic potential, giving rise to the hierarchical growth of the tumor. In the GBM model, this restricted subpopulation responsible for tumor initiation was intrinsically resistant to chemotherapy.
To combat the impact of this common cancer among women and the disparities of ouotcomes observed, efforts have been directed towards the development of new approaches to early detection, chemotherapeutics, surgical and radiation treatments with promising results. We are one of the first groups to describe the breast cancer stem cells are intrinsically resistant to conventional therapy (Li X et. al. J Natl Cancer Inst, 2008; Creighton CJ et. al, PNAS, 2009). Since these initial observations, other groups have confirmed these findings following conventional chemotherapy or radiation therapy, and have supported our findings that an increase in these BCSCs is associated with poorer prognosis. This CSC model has fundamental clinical implications. The current development of cancer therapeutics is largely based on identifying agents with the ability to cause tumor regression in animal models or in clinical trials. Since tumor stem cells comprise only a small percent of the tumor bulk, focus on tumor regression may have produced agents capable of killing actively cycling or fully differentiated cells while sparing the tumor stem cell population. Thus, targeting BCSCs in combination with chemotherapy will be necessary to eliminate the heterogeneous populations within a tumor, particularly the fraction capable of tumor initiation and metastasis.
We previously published a tumorigenic signature derived from patient biopsies (Creighton CJ et. al, PNAS, 2009). Subsequently, we used a functional approach to identify novel targets for cancer stem cells. This screen was performed by shRNA knockdown of the 477 genes in our tumorigenic signature using high-throughput mammosphere formation efficiency (MSFE) assay. We identified top candidates that significantly reduced MSFE. This stringent screening yielded two potential candidates, namely RPL39 and MLF2, as the top candidate genes that affect BCSC self-renewal. Selective siRNA knockdown of RPL39 and MLF2 in patient-derived breast tumorgrafts showed reduced tumor volume and lung metastases with a concomitant decrease in BCSC markers. Thus, targeting BCSCs in combination with chemotherapy should eliminate the heterogeneous populations within a tumor. Additionally, next generation RNA-deep sequencing confirmed mutations in RPL39 and MLF2 in approximately 40% of lung metastases from breast cancer patients (n=53). Allele specific PCR confirmed damaging mutations in RPL39 and MLF2. Gain-of-function of these mutations was demonstrated by increase in proliferation, invasion, and self-renewal assays. In vitro and in vivo siRNA knockdown of RPL39 and MLF2 showed decrease in nitric oxide synthase, suggesting that these genes are driven by nitric oxide signaling. Because of the effect of RPL39 and MLF2 siRNA in reducing lung metastases in vivo, we tested the possibility that BCSC may have escaped the primary cancer to evolve in distant metastatic sites. The presence of these damaging mutations was found only in the stem cell (CD44+/CD24 low/−) subpopulation but not in non-BCSCs. These gain-of-function mutations in RPL39 (AV14) and MLF2 (R158W and D12H) were present in both ERα-positive and ERα-negative tumors, suggesting that BCSCs with these mutations may be capable of multilineage differentiation. Most importantly, the presence of these mutations was significantly associated with worse relapse-free survival and a shorter median time to relapse.
The role of BCSCs in the clinical outcome of breast cancer patients has been analyzed using different strategies, including targeting of membrane markers and transporters, interruption of intracellular signaling pathways, and alteration of the BCSCs microenvironment. Despite the success of some of those therapies in preclinical models; toxicology and pharmacokinetic studies are still required before human tests. We have shown for the first time in human breast cancer patients that residual tumors after chemotherapy are 1) enriched for the tumorigenic CD44+/CD24−/low population, 2) show enhanced mammosphere-forming efficiency (MSFE), and 3) display an increase in outgrowths in xenograft transplants in immunocompromised SCID/Beige mice, thus suggesting their increased tumorigenicity. The preclinical data showed that pharmacological inhibition of the Notch pathway with gamma secretase inhibitors (GSI) reduced the breast cancer stem cells in breast tumorgraft models. GSI enhanced the efficacy of docetaxel in preclinical studies. The clinical trial on 30 patients with advanced breast cancer showed feasibility of the combination of GSI and chemotherapy (Schott AF et. al, Clin Cancer Res., 2013).
In conclusion, the identification of breast cancer stem cell markers and an improved understanding of the molecular mechanism of BCSC phenotypes should lead to progress in cancer therapy and improved prognoses for patients with advanced breast cancer. Such emerging picture of the biological properties of BCSCs would contribute for devising innovative therapies for breast cancer, targeting the intrinsic and extrinsic factors that maintain the BCSCs.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):CN06-02.
Citation Format: Jenny C. Chang, Bhuvanesh Dave, Sergio Granados-Principal, Junhua Mai, Steven Gross, Haifa Shen, Vivek Mittal, Mauro Ferrari. Breast cancer stem cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr CN06-02.