Selection of Metastatic Breast Cancer Cells Based on Adaptability of Their Metabolic State

A small subpopulation of highly adaptable breast cancer cells within a vastly heterogeneous population drives cancer metastasis. Here we describe a function-based strategy for selecting rare cancer cells that are highly adaptable and drive malignancy. Although cancer cells are dependent on certain nutrients, e.g., glucose and glutamine, we hypothesized that the adaptable cancer cells that drive malignancy must possess an adaptable metabolic state and that such cells could be identified using a robust selection strategy. As expected, more than 99.99% of cells died upon glutamine withdrawal from the aggressive breast cancer cell line SUM149. The rare cells that survived and proliferated without glutamine were highly adaptable, as judged by additional robust adaptability assays involving prolonged cell culture without glucose or serum. We were successful in isolating rare metabolically plastic glutamine-independent (Gln-ind) variants from several aggressive breast cancer cell lines that we tested. The Gln-ind cells overexpressed cyclooxygenase-2, an indicator of tumor aggressiveness, and they were able to adjust their glutaminase level to suit glutamine availability. The Gln-ind cells were anchorage-independent, resistant to chemotherapeutic drugs doxorubicin and paclitaxel, and resistant to a high concentration of a COX-2 inhibitor celecoxib. The number of cells being able to adapt to non-availability of glutamine increased upon prior selection of cells for resistance to chemotherapy drugs or resistance to celecoxib, further supporting a linkage between cellular adaptability and therapeutic resistance. Gln-ind cells showed indications of oxidative stress, and they produced cadherin11 and vimentin, indicators of mesenchymal phenotype. Gln-ind cells were more tumorigenic and more metastatic in nude mice than the parental cell line as judged by incidence and time of occurrence. As we decreased the number of cancer cells in xenografts, lung metastasis and then primary tumor growth was impaired in mice injected with parental cell line, but not in mice injected with Gln-ind cells

Abstract 1557: Developing a predictive cell-based assay for anti-cancer drug selection.

Abstract There is a pressing need of predictive cell-based models for selecting therapies that would have a high likelihood of success in the clinic. Cancer evolves through a clonal selection process, which involves the ability of a cancer cell to survive several rate-limiting steps, and give rise to progeny under favorable conditions. We hypothesized that the cancer cells that are endowed with high metabolic adaptability (ability to survive without critical nutrients) would have significant advantages in facing multiple challenges in the body, including resisting current therapies. To develop a realistic cell-based model of aggressive breast cancer, we reasoned that a robust selection of metabolic adaptability would enrich metastatic cells in cell culture. Investigating an aggressive human triple-negative breast cancer (TNBC) cell line SUM149 that harbors mutant p53 and BRCA1 genes, we recently reported that rare cells (0.01% in population) present in this cell line can be selected based on their ability to survive and proliferate without glutamine. These metabolically adaptable (MA) variants proved to be structurally adaptable as well (highly enriched in mesenchymal phenotype), resistant to chemotherapeutic drugs, and highly metastatic to multiple organs- lungs, liver, brain, and skin from fat pad xenografts in nude mice (Singh et al., PLoS ONE, 2012). Our function-based selection proved to be more powerful than the biomarker-based selections for isolating the type of rare cells that drive the disease. We are now investigating a variety of experimental therapies to identify those which may eradicate most adaptable breast cancer cells in patients with TNBC. It is noteworthy that our assessment of response versus resistance to therapies is based on long-term assays (1-2 months). Short-term assays, which are often used to assess proliferation and apoptosis, are not good at predicting response in the clinic. Our studies suggest that the MA cells are resistant to most drugs tested thus far, supporting the validity of our cell-based model. To illustrate with an example of evaluation of a specific drug, we found that a clinically-useful therapy against MET and ALK signaling in lung cancer, crizotinib, failed to eradicate SUM149-MA cells under the conditions it eradicated all parental cells. This result indicates that crizotinib is not suitable for clinical trials if the goal is to eradicate resistant cells in TNBC. However, if innovative clinical studies with crizotinib (such as trial of one) appear promising in a subset of breast cancer (better response than current therapies), our MA variants provide a suitable model to discover therapies that can be combined with crizotinib to increase its efficacy. In conclusion, our approach has a potential of improving therapeutic responses, but this potential can be best realized with close interaction with clinicians treating the disease. Supported by a State of Texas Grant for Rare and Aggressive Cancers. Citation Format: Balraj Singh, Anna Shamsnia, Milan R. Raythatha, Anthony Lucci. Developing a predictive cell-based assay for anti-cancer drug selection. [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 1557. doi:10.1158/1538-7445.AM2013-1557</jats:p

Abstract 879: Developing a predictive <i>in vitro</i> assay for evaluating therapies against metastatic breast cancer

Abstract The major challenge in breast cancer research pertains to tumor heterogeneity being a constant characteristic in the disease process. Although it is recognized that rare cancer cells drive the disease, there is a lack of suitable function-based assays to identify and focus on such cells for therapy development. Based on the hypothesis that only rare but highly adaptable cancer cells succeed in metastasizing, we have recently isolated rare (&amp;lt; 0.01% of population) highly metastatic cells present in an aggressive inflammatory breast cancer cell line SUM149. Using a variety of long-term adaptability assays we have found that such cells have adaptability to survive lack of glutamine, glucose, oxygen, and serum. Unlike the majority of cells, such cells have a high ability to dissociate their metabolic state from regulatory state, thus making them more resistant to chemotherapy drugs. These properties of cancer cells make them suitable for survival as resistant cells in the body, which often use quiescence as a way of surviving unfavorable conditions. As an initial step to optimize our in vitro system for evaluating potential personalized therapies, we compared the efficacy of several experimental targeted therapy drugs on metastatic Gln-independent variants and on parental SUM149 cell line. Since cell proliferation and apoptosis assays after a short exposure to experimental therapies are not good at predicting clinical response, we chose to determine therapeutic efficacy by analyzing residual/resistant cells after a relatively long exposure to therapy. Celecoxib, a COX-2 inhibitor (2-10 micromolar range), failed to inhibit resistant cells in both Gln-ind and parental SUM149 cell lines. At a 50 micromolar concentration, celecoxib eradicated all cells with a proliferative potential in the parental cell line but not in the Gln-ind subpopulation. Metformin, an anti-diabetes drug that activates AMPK and also inhibits “cancer stem cells,” was less effective in inhibiting Gln-ind cells than the parental cell line. BEZ235, a dual PI3K/mTOR inhibitor, sensitized Gln-ind and parental cells to the chemotherapy drugs doxorubicin and paclitaxel. However, Gln-ind cells yielded significantly more clonogenic (10-fold) cells after they were treated sequentially with BAZ235 and doxorubicin/paclitaxel. Salinomycin, a potassium ionophore and an inhibitor of “cancer stem cells,” was also less effective against Gln-ind cells. HDAC inhibitors, valproic acid, and butyrate, which are being evaluated as sensitizers to other therapies in various cancers, sensitized both Gln-ind and parental cell line to doxorubicin and paclitaxel. We conclude that the pair of cell lines used in this study will be useful in discovering strategies (e.g., novel therapies and ways to administer them) for eradicating metastatic breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 879. doi:1538-7445.AM2012-879</jats:p

Bimetallic Complexes of Nickel(II) with Tin(IV) Chloride & Tellurium(IV) Chloride

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Highly Adaptable Triple-Negative Breast Cancer Cells as a Functional Model for Testing Anticancer Agents

A major obstacle in developing effective therapies against solid tumors stems from an inability to adequately model the rare subpopulation of panresistant cancer cells that may often drive the disease. We describe a strategy for optimally modeling highly abnormal and highly adaptable human triple-negative breast cancer cells, and evaluating therapies for their ability to eradicate such cells. To overcome the shortcomings often associated with cell culture models, we incorporated several features in our model including a selection of highly adaptable cancer cells based on their ability to survive a metabolic challenge. We have previously shown that metabolically adaptable cancer cells efficiently metastasize to multiple organs in nude mice. Here we show that the cancer cells modeled in our system feature an embryo-like gene expression and amplification of the fat mass and obesity associated gene FTO. We also provide evidence of upregulation of ZEB1 and downregulation of GRHL2 indicating increased epithelial to mesenchymal transition in metabolically adaptable cancer cells. Our results obtained with a variety of anticancer agents support the validity of the model of realistic panresistance and suggest that it could be used for developing anticancer agents that would overcome panresistance

Abstract 878: <i>In vitro</i> selection of metastatic breast cancer cells based on their adaptability

Abstract The success of cancer depends on ongoing generation of genetic and epigenetic heterogeneity in cancer cells, and the selection of cells that are most adaptable at surviving numerous obstacles in the body. Current approaches of personalized therapy, mostly designed to inhibit cell proliferative function of a single protein, may not succeed unless we target adaptability of cancer cells as well. We reported recently that it is possible to isolate highly metastatic rare cancer cells (&amp;lt; 0.01% in SUM149 inflammatory breast cancer cell line) based on their adaptability to survive without glutamine in culture medium. These studies also provided evidence of a high COX-2 expression in such Gln-independent cells and in subpopulations that survived lack of serum and attachment. To determine the usefulness of our new function-based selections, we compared different COX-2 overexpressing subpopulations with the parental SUM149 cell line for their adaptability to survive lack of Gln- 1) COX-2 overexpressing cells that survive lack of serum and attachment (designated SWS), and 2) a pre-existing COX-2 overexpressing clone (clone 5) isolated by picking individual colonies. We found that the SWS variants yielded significantly more colonies (2-3 fold) than the parental cell line upon Gln withdrawal. In contrast, clone 5 failed to yield any Gln-ind colony. Further investigation into the adaptability of Gln-ind cells revealed that they are better able to survive exposure to hypoxia than the parental cell line. We obtained similar results with hypoxia caused by cell culture in 0.5% oxygen or with hypoxia caused by exposure to cobalt chloride. These results support a model in which rare cells that drive metastasis are proposed to be adaptable in multiple ways. Our studies suggest that the function-based selection of adaptable cancer cells is able to select unique cancer cells from a heterogeneous population that may not be selectable on the basis of specific biomarkers. We conclude that it is feasible to isolate rare metastatic breast cancer cells with a function-based robust selection of adaptable metabolic state. In vitro assays utilizing such adaptable and metastatic cancer cells will likely aid anti-cancer drug discovery efforts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 878. doi:1538-7445.AM2012-878</jats:p

Selection of Rare Cancer Cells That Survive Lack of Glutamine and Glucose.

<p>(A) Half a million SUM149-Luc cells were plated in a 10-cm dish. The next day, the medium was changed to a medium containing dialyzed FBS and lacking glutamine. After growth for 34 days in the Gln-deficient medium, we stained the colonies with crystal violet. (B) Half a million SUM149-FP76 cells were plated in a 10-cm dish. The next day, the medium was changed to a medium containing dialyzed FBS lacking glucose (left dish) or lacking both glucose and glutamine (right dish). After 15 days, the media were replaced with complete medium. We stained the colonies with crystal violet after 18 days in complete medium.</p

Low glutaminase level in Gln-ind cells.

<p>Selected proteins likely to be involved in Gln addiction (GLS, Myc) and in metastasis (COX-2, Myc) were analyzed by western blotting in Gln-ind and parental SUM149-Luc cells. Since the β-actin level was found to be lower in the Gln-ind cells than in the parental cell line by western blotting, we included additional gel loading controls: western blotting with Hsp90 and GAPDH antibodies, and Coomassie blue staining of a gel run in parallel.</p