Breast Cancer Cell Line SUM149 Research Articles

Abstract 2616: RhoC decreases tight junction stabilization in breast cancer cells, revealing a potential therapeutic target

Abstract RhoC, a member of the Rho GTPase family, has been shown to have a role specifically in facilitating metastasis of breast cancer cells via influencing motility, invasion, and chemokine secretion, but as yet there is no integrated model of the precise mechanism of how RhoC promotes metastasis. As these processes require disruptions in cell-cell junctions, we use wild-type and CRISPR RhoC and RhoA knockout lines of the breast cancer cell lines SUM-149 (inflammatory breast cancer, IBC) and MDA-MB-231 (triple-negative breast cancer, TNBC) in order to assess the influence of RhoC and RhoA on their cell-cell junctions. Protein lysates from these cell lines were blotted for the adherens junction markers E-cadherin and β-catenin, as well as the tight junction markers ZO-1 and Occludin. Cells were also fixed and stained for these junction markers to further assess expression and localization. In order to measure junction stability, we conducted FITC-Dextran assays, wherein cells were grown to confluency on Transwell plates, the apical chambers were incubated with FITC-Dextran solution for 24 hours, and the fluorescence of the basal chambers was subsequently measured. We demonstrate that CRISPR-Cas9 knockout of RhoC in SUM-149 IBC and MDA-MB-231 TNBC cells results in increased epithelial morphology, cell-cell connections, and tight junction marker colocalization, as measured via immunofluorescent staining. Furthermore, RhoC knockout cells have increased junctional proteins compared to wild-type cells, as measured by Western blot. In functional assessments of junction stability, RhoC knockout cells have increased barrier integrity compared to wild-type cells, by the FITC-Dextran leakage assay. These results suggest that high expression of RhoC in cancer cells destabilizes tight junctions, although loose clusters of cells are observed depending on cell type; taken together, upregulation of RhoC activation may contribute to increased metastatic potential, in part through destabilization of tight junctions. We will report on further invasion functional assays and RNA-seq data on the RhoC knockout cell lines. Taken together, these data support an important role for RhoC in tight junction modulation, thereby advancing the evidence for a potential highly specific new therapeutic target in RhoC-driven metastases control. Citation Format: Hannah Abraham, Laura Goo, Andrew Little, Joel Yates, Zhifen Wu, Sofia D. Merajver. RhoC decreases tight junction stabilization in breast cancer cells, revealing a potential therapeutic target [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2616.

Abstract 4363: Effects of the small GTPase RhoC on inflammatory breast cancer metabolism

Abstract Inflammatory breast cancer (IBC) is an extremely aggressive and rare form of cancer that disproportionally affects African American and younger women. While IBC represents 1-5% of breast cancers, it accounts for 10% of all breast cancer deaths annually in the United States. We have previously shown that the metabolic characteristics of IBC, specifically in the triple negative (TN) and inflammatory breast cancer cell line SUM149, are significantly altered from those of normal breast cancer cells. We have shown previously that RhoC, a member of the Ras superfamily of GTPases, contributes to the metastatic IBC phenotype and acts as a regulator of the metabolite N-acetyl aspartate (NAA) in SUM149 cells. NAA is the second most abundant metabolite in the brain (only exceeded by glutamate) and has been used for the diagnosis of neurodegenerative disorders such as the fatal genetic disorder Canavan’s disease, which is characterized by toxic NAA levels due to a deleterious mutation in aspartoacyclase(ASPA). ASPA is the enzyme involved in the catabolism of NAA into aspartate and acetate. NAA has previously been considered a brain specific metabolite and the fundamental role of NAA outside of the central nervous system, especially in the context of cancer, remains elusive but highly intriguing. We have previously shown that NAA levels are significantly higher in the IBC-derived TN SUM149 cells than TN cancer cell line MDA-MB-231. Interestingly, and independently, recent studies have also implicated high levels of tumoral NAA with significantly worse survival rates in ovarian cancer patients. The synthesis of NAA from acetyl-CoA and aspartate is catalyzed by the enzyme Asp-NAT, which is encoded for by the gene NAT8L. Knockdown of RhoC in SUM149 cells using shRNA significantly decreases both NAT8L expression and NAA metabolite levels. To further understand RhoC’s role in the modulation of NAA, we generated RhoC and RhoA knockout cell lines using CRIPSR-Cas9 in a set of inflammatory and noninflammatory breast cancer cell lines. RNA-seq analysis of these show that SUM149 RhoC knockout cells produce the largest amount of differentially regulated genes when compared to wild-type cells. The differentially regulated genes include many of the key genes involved in NAA associated pathways. Furthermore, metabolic studies reveal that the most downregulated metabolites in the SUM149 RhoC knockout cell line are N-acetyl derivates including NAA, N-acetylglutamate(NAG), and N-acetylaspartylglutamate (NAAG). Overexpression and siRNA cell lines for both NAT8L and ASPA have been generated to further elucidate the altered metabolism seen in these cells. Additional molecular studies are ongoing to determine the specific role of NAA in the adapted metabolic pathways of inflammatory breast cancer that may shed light on IBC’s increased metastatic potential and decreased survival. Citation Format: Laura E. Goo, Joel A. Yates, Andrew C. Little, Daniel Kremer, Ilya Kovalenko, Christopher R. Oliver, Trisha Westerhof, Zhifen Wu, Nathalie Vandecan, Liwei Bao, Peter J. Ulintz, Costas A. Lyssiotis, Sofia D. Merajver. Effects of the small GTPase RhoC on inflammatory breast cancer metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4363.

Experimental study on inhibiting invasion of inflammatory breast cancer cell lines SUM149 and SUM190 by silencing RhoC gene with the help of RNA interference

Objective In this study, we silence RhoC gene in inflammatory breast cancer (IBC) SUM149, SUM190 cell lines and explore its effects on cell invasion of IBC by RNA interference (RNAi) technology so as to provide experimental principal for target gene therapy of IBC. Methods Human IBC cells were divided blank group, control siRNA group, RhoC-siRNA group by balance randomization. RhoC-siRNA was used to transfect human IBC SUM149, SUM190 cell lines. Microscopy was applied to analyze transfection efficiency. After transfecting for 48 hours, reverse transcriptase-polymerase chain reaction (RT-PCR) was made to detect mRNA expression levels of RhoC. Western blotting was also used to analyze the expression level of RhoC protein. After transfecting for 48 hours, the ability to invasion was analyzed by transwell hole. Results We used siRNA to transfect human IBC SUM149, SUM190 cell lines and found that transfection efficiency can exceed to 80%. After we transfected for 48 hours, the expression levels of RhoC protein in IBC SUM149 cell lines 0.375±0.164 in RhoC siRNA group were significantly lower than those 0.897±0.376 in control siRNA group (t=6.274, P=0.045). The expression levels of RhoC protein in IBC SUM190 cell lines 0.381±0.166 in RhoC siRNA group were significantly lower than those 0.902±0.386 in control siRNA group (t=6.891, P=0.028). However, there were no obvious differences between in blank group and control siRNA group. After we transfected for 48 hours, in IBC SUM149 cell lines, the ability to invasion 85.6±12.7 in RhoC siRNA group was markedly lower than that 340.5±43.4 in control siRNA group (t=12.275, P=0.034). In IBC SUM190 cell lines, the ability to invasion 92.8±15.6 in RhoC siRNA group was markedly lower than that 345.9±47.7 in control siRNA group (t=11.769, P=0.022). Conclusion RhoC is known as an important role in the carcinogenesis of IBC. With the help of RNAi technology, we can greatly inhibit the invasion of IBC SUM149, SUM190 cell lines by silencing RhoC gene. RNAi technology perhaps becomes an important gene therapy tool for IBC. Key words: Inflammatory breast cancer; RhoC; RNA interference; Gene silence

Abstract 433: Glycogen accumulation in aggressive breast cancers during hypoxic exposure

Abstract Isolation of specific metabolic alterations in aggressive triple negative and inflammatory breast cancers represents a compelling avenue for the development of treatments for these diseases. The ability of cancer to utilize diverse metabolic pathways to modulate increased survival and proliferation is well established. Indeed, we have previously described a series of metabolic adaptations in the triple negative inflammatory breast cancer cell line SUM149 and demonstrated a role for the small GTPase RhoC in the metabolic phenotype of the cell line. In this work we seek to describe a survival mechanism for cancer cells that are subjected to hypoxic environments. Hypoxic survival or growth is often necessitated at the center of a growing tumor mass or if the tumor is growing in the lymphatic system, as is often the case for inflammatory breast cancers. Here we investigate the regulation of glycogen levels in breast cancer and normal-like cell lines in atmospheric and 1% oxygen environments. Reserves of glucose are typically stored in liver and muscle cells as the polysaccharide glycogen. Elevated levels of glycogen have previously been observed in various cancers, and recent studies have implicated the importance of glycogen metabolism in promoting cancer cell survival. We hypothesize that aggressive breast cancers utilize modifications of the glycogen synthesis and degradation pathways to provide nutritional flexibility as they proliferate and invade into diverse environments. MCF10A, SUM149, and SUM190 cell lines demonstrate a greater than 10-fold increase in glycogen accumulation when subjected to hypoxic growth conditions. MDA-MB-231 cells also increase glycogen storage, though at levels on the order of 2-3 times higher in hypoxia when compared to normoxic growth. Conditioned media for all cell lines in normoxic and hypoxic conditions were analyzed for glucose and lactate levels to determine changes in metabolite consumption. The data presented indicate an unexpected accumulation of glycogen in response to hypoxic conditions. Studies to determine the mechanisms through which these changes occur and why they are important for continued survival and growth of the cancer cells are ongoing and will be presented. Citation Format: Megan Altemus, Joel Yates, ZhiFen Wu, LiWei Bao, Sofia Merajver. Glycogen accumulation in aggressive breast cancers during hypoxic exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 433. doi:10.1158/1538-7445.AM2017-433

Abstract 1203: Metastasis-associated oncogene RhoC as a regulator of glutamine metabolism in the inflammatory breast cancer cell line SUM149

Abstract Metabolic reprogramming is increasingly recognized as a fundamental hallmark of cancer. While the Warburg effect and normal proliferative metabolism are similar, they are not equivalent. We hypothesize that there are key drivers of malignant metabolism that can be modulated to impede cancer proliferation without substantial effects on normal tissue growth. Using 13C-labeled glucose and glutamine tracers in combination with mass spectrometry and measurements of extracellular glucose, lactate, and glutamine flux, we have characterized system level differences in a series of breast cancer cell lines as well as normal-like breast epithelial cells. We observed an increase in the reductive carboxylation of glutamine-derived citrate and alpha-ketoglutarate in the triple-negative inflammatory breast cancer cell line SUM149. We also observed that the SUM149 exhibit high levels of HIF-1α and low levels of oxygen consumption under normoxia, suggesting that the cell line is highly adapted to hypoxia. Surprisingly, the stable depletion of HIF-1α via shRNA had no significant effect on the metabolic profile of these cells. Previous work by our lab and others has demonstrated that the GTPase RhoC is a driver of the metastatic phenotype exhibited by inflammatory breast cancer. Activation of RhoC is known to induce cytoskeletal rearrangements and increase invasive potential. The Rho GTPase family of proteins has also recently been linked to metabolism, specifically regulation of glutaminase activity. Here we show that stable knockdown of RhoC in SUM149 cells results in a marked decrease in the rate of both glutamine uptake and intracellular reductive carboxylation. This work reinforces the role of RhoC as an important driver of inflammatory breast cancer metastatic potential. We conclude that RhoC remains an important clinical target with the potential to alter patient outcomes. Citation Format: Joel A. Yates, Michelle L. Wynn, ZhiFen Wu, Charles R. Evans, Charles Burant, Santiago D. Schnell, Sofia D. Merajver. Metastasis-associated oncogene RhoC as a regulator of glutamine metabolism in the inflammatory breast cancer cell line SUM149. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1203. doi:10.1158/1538-7445.AM2015-1203

Abstract 1600: Simvastatin targets breast cancer stem-like cells by inhibiting intracellular signaling pathways leading to radiosensitization of inflammatory breast cancer cells.

Abstract Background: Inflammatory breast cancer (IBC) is responsible for 10% of breast cancer deaths although contributing to a small percentage (∼1%) of total breast cancer cases each year. The hallmarks of IBC are skin involvement and a high propensity to metastasize. It is thought that a small population of stem-like cancer cells is responsible for the metastatic potential and treatment resistance to therapies such as radiation therapy in IBC. In this study we explored the radiosensitization of several breast cancer cell lines in vitro with simvastatin and the mechanism for this. Methods: In order to determine cell proliferation, breast cancer cell lines SUM149, SUM159 and MCF-7 were grown in 96 well plates with different concentrations of simvastatin for one week. In order to determine the effect of simvastatin in stem-like cancer cells, cells were seeded in serum free, growth factor enriched, anchorage independent, treated with simvastatin for 24 hours and mammosphere formation was quantified a week later. In addition, SUM149, SUM159 and MCF-7 cells were cultured in standard monolayer cultures and in stem cell enriching anchorage independent cultures with simvastatin and treated with increasing concentrations of radiation. Survival curves were generated using Sigmaplot 8.0 and t-test was used to compare surviving fraction (SF) of groups. Finally, cell lysates were obtained from cultures untreated and treated with simvastatin and radiation and analyzed through western blot. Results: Simvastatin significantly decreased cancer cell proliferation in vitro in all cell lines (p<0.002). Cell lines SUM 159 and MCF 7 grown in mammosphere enriched media showed significant decreased mammosphere formation when grown in the presence of simvastatin (p<0.05). Moreover, simvastatin radiosensitized all cell lines in both monolayer and anchorage independent clonogenic assays. The triple-negative IBC cell line SUM149 had the greatest response to combined treatment regardless of the radiation dose used in monolayer cultures (SF2: 0.417 vs 0.319, SF4: 0.136 vs 0.075, SF6: 0.026 vs 0.018, in control vs treated respectively, all p<0.0001) and in stem cell enriching cultures (SF2: 0.880 vs 0.762, SF4: 0.863 vs 0.492, SF6: 0.920 vs 0.492, in control vs treated respectively, all p<0.0001). The triple-negative non-IBC cell line SUM159 was more sensitive to combined treatment in monolayer cultures (p<0.001) than in stem cell enriching cultures. Combination of simvastatin and radiation therapy decreased protein expression of phospho-rho A, VEGF-A, and phospho-ras. Conclusions: IBC remains a difficult disease to treat with a high rate of local recurrence after radiation therapy. Statins have been shown to increase tumor free survival in breast cancer. Herein we show simvastatin targets breast cancer stem-like cells resulting in radiosensitizing of IBC in vitro. Citation Format: Adam R. Wolfe, Lara Lacerda, Richard Larson, Bisrat G. Debeb, Wei Xu, Naoto T. Ueno, Wendy A. Woodward. Simvastatin targets breast cancer stem-like cells by inhibiting intracellular signaling pathways leading to radiosensitization of inflammatory breast cancer cells. [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 1600. doi:10.1158/1538-7445.AM2013-1600

Evaluating the HSP90 Inhibitor Ganetespib as a Radiosensitizing Agent in Breast Cancer Models In Vitro

• Experiments were conducted using the inflammatory breast cancer cell line SUM-149, and the triple negative cell line MDA-MB-231. The BT-474 (ER/PR positive, HER2 positive) and MCF-7 (ER/PR positive, HER2 negative) cell lines underwent XTT testing. • XTT Assays: • Cells were plated in 96 well plates, exposed to ganetespib in concentrations ranging from 50-250 nM for 24 hrs. Media was changed and cells were incubated for 72 hrs. XTT assays were then performed. • Clonogenic Assays: • Cells were plated on 6-well plates, ganetespib was added to the media at 10 or 50 nM and allowed to incubate 20 hours. Cells were then irradiated and incubated for 4 more hours. Media was changed, and 14 days later, plates were fixed, stained, and colonies counted. • Cell Cycle Analysis: • Cells were plated in flasks and treated with media or media + ganetespib at a concentration of 150 nM. The cells were incubated for 24 hours. They were then harvested, fixed in 70% ice cold ethanol, washed in 1xPBS, and re-suspended in 1 mL of PI solution containing DNAse-free RNAse. The cells were analyzed with a Beckman Coulter Flow Cytometer. • Western Blots: • Cells were treated with 100 nM ganetespib for 24 hours, harvested, then analyzed for expression of several marker proteins (HSP90, HSP70, AKT, p-AKT, pRb) Conclusions

Optimization of IGF-1R SPECT/CT Imaging Using 111In-Labeled F(ab′)2 and Fab Fragments of the Monoclonal Antibody R1507

The insulin-like growth factor 1 receptor (IGF-1R) is a potential new target for the treatment of breast cancer. Patients with breast cancer lesions that express IGF-1R may benefit from treatment with anti-IGF-1R antibodies. IGF-1R expression can be visualized using radiolabeled R1507, a monoclonal antibody directed against IGF-1R. However, antibodies clear slowly from the circulation, resulting in low tumor-to-background ratios early after injection. Therefore, we aimed to accelerate targeting of IGF-1R using radiolabeled R1507 F(ab')2 and Fab fragments. In vitro, immunoreactivity, binding affinity and internalization of R1507 IgG, F(ab')2 and Fab were determined using the triple negative IGF-1R-expressing breast cancer cell line SUM149. In vivo, pharmacokinetics of (111)In-labeled R1507 IgG, F(ab')2 and Fab were studied in mice bearing subcutaneous SUM149 xenografts. SPECT/CT images were acquired and the biodistribution was measured ex vivo. The in vitro binding characteristics of radiolabeled R1507 IgG and F(ab')2 were comparable, whereas the affinity of Fab fragments was significantly lower (Kd: 0.6 nM, 0.7 nM and 3.0 nM for R1507 IgG, F(ab')2 and Fab, respectively). Biodistribution studies showed that the maximum tumor uptake of (111)In-R1507 IgG, F(ab')2 and Fab was 31.8% ID/g (72 h p.i.), 10.0% ID/g (6 h p.i.), and 1.8% ID/g (1 h p.i.), respectively. However, maximal tumor-to-blood ratios for F(ab')2 (24 h p.i.: 7.5) were more than twice as high as those obtained with R1507 (72 h p.i.: 2.8) and Fab (6 h p.i.: 2.8). Injection of an excess of unlabeled R1507 significantly reduced tumor uptake, suggesting that the uptake of R1507 IgG and F(ab')2 was specific for IGF-1R, while the major fraction of the tumor uptake of Fab was nonspecific. IGF-1R-expressing xenografts were visualized with (111)In-F(ab')2 SPECT/CT as early as 6 h p.i., while with R1507 IgG, the tumor could be visualized after 24 h. No specific targeting was observed with (111)In-Fab. (111)In-F(ab')2 fragments showed improved targeting of IGF-1R expressing tumors. Tumor-to-blood ratios were twice as high as those obtained with (111)In-R1507, and adequate tumor targeting on SPECT/CT images was observed as early as 6 h p.i. For individualization and optimization of IGF-1R targeted therapy, (111)In-F(ab')2 may be the tracer of choice.

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 879: Developing a predictive in vitro 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 (< 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

Abstract P4-03-06: Selection of Rare and Novel Breast Cancer Cell Variants Based on Glutamine Metabolism

Abstract Background: Breast cancer is a heterogeneous disease composed of tumor cells that have different characteristics. Although there are several different ways to explain cellular heterogeneity, it is clear that only rare cancer cells out of the overall population succeed in metastasizing. The current approach of classifying subpopulations of cancer cells based on certain biomarkers derived from the bulk of primary tumor is unsatisfactory for identifying the rare cells that cause metastasis. The purpose of this study was to test a novel function-based, rather than biomarker-based, selection strategy using glutamine (Gln) metabolism (or Gln-dependence to be more specific) since it is a prominent feature of cancer cells. While the majority of cells present in metastatic breast cancer cell lines are predicted to be dependent on Gln and thus will die upon Gln withdrawal, we hypothesized that some rare cells would survive and grow without Gln as a nutrient, thus suggesting a simple robust selection method for “tumorigenic” cancer cells. Methods: We chose an aggressive inflammatory breast cancer cell line SUM149 and SUM149-FP76 cell line that was recently derived by “passaging” SUM149-Luc cells (luciferase transfected for imaging in mice) in nude mice in our laboratory, first selecting for growth subcutaneously and then in inguinal fat pad. Gln-independent variants were selected by growing cells in a medium without Gln, supplemented with dialyzed fetal bovine serum. We evaluated protein level of glutaminase (GLS), an enzyme that converts glutamine to glutamate and usually correlates with Gln addiction in cancer cells, by western blotting. Expression of COX-2, an enzyme associated with inflammation, metastasis, and with cellular hierarchy, was measured by western blotting and compared between parental and Gln-independent lines. Tests were repeated in triplicate and evaluated using paired T-test. Results: We were able to select rare variants (one in 35,000 cancer cells) from the SUM149 inflammatory breast cancer cell line that grow long-term in Gln-free medium. We found that Gln-independent variants produced a significantly lower level of GLS (1/5th) and a significantly higher level (5-fold) of COX-2 than did the parental cell line. To provide further support for a link between Gln metabolism and tumorigenesis, we were also able to select the rare Gln-independent variants from the SUM149-FP76 cell line. Further experiments involving siRNA-mediated COX-2 knockdown indicated that the high level of COX-2 protein present in Gln-independent variants was not a direct regulator of the reduced GLS level, but rather COX-2 may offer indirect selective advantage by influencing processes such as apoptosis and “developmental hierarchy”. Conclusions: Since COX-2 is very important in breast cancermetastasis, our results suggest the possibility of a strong link between a critical metabolic adaptation (decreased GLS to maintain a sufficient Gln pool) that may be essential for cell survival and growth, and metastatic ability (via increased COX-2) in cancer cells. Our study also revealed a deep connection between glutamine metabolism and metastasis that could be exploited for developing novel models of cancer cell function and for targeted therapy development against these cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-03-06.

ImmunoSPECT and ImmunoPET of IGF-1R Expression with the Radiolabeled Antibody R1507 in a Triple-Negative Breast Cancer Model

The insulinlike growth factor 1 receptor (IGF-1R) is a new target for the treatment of breast cancer. Patients with breast cancer lesions that express IGF-1R may benefit from treatment with anti-IGF-1R antibodies. Therefore, the aim of the present study was to develop a noninvasive, in vivo imaging method, using radiolabeled antibodies, to visualize IGF-1R expression. R1507 is a monoclonal antibody directed against the IGF-1R. In vitro, the affinity and internalization kinetics of (111)In-R1507 were determined using the IGF-1R-expressing triple-negative breast cancer cell line SUM149. In vivo, the pharmacodynamics of (111)In-R1507 and (125)I-R1507 were determined in mice with subcutaneous SUM149 tumors. (111)In-R1507 SPECT and (89)Zr-R1507 PET images of mice with subcutaneous SUM149 tumors were acquired at 1, 3, and 7 d after injection. (111)In-R1507 (concentration required to inhibit binding by 50%, 0.1 nM) was slowly internalized by SUM149 cells. (111)In-R1507 specifically and efficiently accumulated in the SUM149 xenografts: the tumor uptake was 20 percentage injected dose per gram (%ID/g), 33 %ID/g, and 31 %ID/g at 1, 3, and 7 d after injection, respectively. (125)I-R1507 accumulated in the tumor less efficiently. Small-animal SPECT and small-animal PET of mice clearly visualized the subcutaneous SUM149 xenograft, with increasing contrast at later time points. (111)In-R1507 and (89)Zr-R1507 are new tracers to noninvasively determine IGF-1R expression in vivo in breast cancer xenografts using SPECT and PET. In the future, these techniques may enable patient selection for IGF-1R-targeted therapy.

Differential regulation of the aggressive phenotype of inflammatory breast cancer cells by prostanoid receptors EP3 and EP4.

Although inflammatory breast cancer (IBC) is recognized as the most lethal variant of locally advanced breast cancer, few molecular signatures of IBC have been identified that can be used as targets to develop therapeutics that effectively inhibit the aggressive phenotype displayed by IBC tumors. Real-time polymerase chain reaction analysis, Western blot analysis, modified Boyden chamber invasion assays, vasculogenic mimicry (VM) assays, and gelatin zymography were used in the current studies. Agonists and antagonists of the prostanoid receptors EP3 and EP4 and of EP4 short-hairpin RNA (shRNA) knockdown approaches were used as tools to assess the role of prostanoid receptors EP3 and EP4 in the regulation of specific biologic activities of IBC cells. The current studies revealed that the IBC breast cancer cell lines SUM149 and SUM190 express high levels of cyclooxygenase-2 messenger RNA and protein, produce abundant levels of prostaglandin E(2), and produce both EP3 and EP4 receptor proteins. Studies using the EP4 antagonist GW627368X and shRNA molecular knockdown approaches revealed a role for EP4 in regulating invasion of IBC cells. EP3, but not EP4, regulated the ability of SUM149 cells to undergo VM, which is the ability to form capillary-like structures, a characteristic exhibited by very aggressive tumor types. Inhibition of VM by sulprostone was associated with an inhibition of matrix metalloprotease-2 (MMP-2) enzyme activity. The prostanoid receptors EP3 and EP4 differentially regulate activities exhibited by IBC cells that have been associated with the aggressive phenotype of this lethal variant of breast cancer. Whereas EP4 regulates invasion, EP3 regulates VM and the associated increased MMP-2 enzyme activity.

Characterization of the roles of RHOC and RHOA GTPases in invasion, motility, and matrix adhesion in inflammatory and aggressive breast cancers

The 2 closely related small GTPases, RHOC and RHOA, are involved in mammary gland carcinogenesis; however, their specific roles in determining cancer cell adhesion and invasion have not been elucidated. RHOA and RHOC are highly homologous, thereby posing a major challenge to study their individual functions in cancer cells. By selectively knocking down these proteins, we have been able to alternatively inhibit RHOC and RHOA, while preserving expression of the other rho protein. Quantitative analyses of the growth patterns and invasion in the aggressive estrogen receptor negative cell lines MDA-231 and SUM149 were carried out on collagen I and Matrigel substrates. RHOC, and not RHOA, modulates surface expression and colocalization of alpha2 and beta1 integrins in MDA-MB-231 on collagen I. Neither RHOC or RHOA affected integrin expression in the inflammatory breast cancer cell line SUM149, further highlighting the different regulation of adhesion and motility in inflammatory breast cancer. This work shows that RHOC and RHOA play different roles in cell-matrix adhesion, motility, and invasion of MDA-MB-231 and reaffirms the crucial role of RHOC-GTPase in inflammatory breast cancer cell invasion.

Abstract A36: ImmunoSPECT and ImmunoPET imaging of IGF-1R expression in a model for triple negative breast cancer

Abstract Introduction: Triple negative breast cancer has a poor prognosis and chemotherapy is currently the mainstay of treatment. A potential new target for the treatment of triple negative breast cancer is the insulin-like growth factor 1 receptor (IGF-1R). Patients with triple negative breast cancer lesions that express the IGF-1R (30 to 40%) may be most suited to be treated with IGF-1R antibodies. Therefore, the aim of the present study was to develop a noninvasive, in vivo imaging method, using radiolabeled R1507, to visualize IGF-1R expression. Material and methods: R1507, a monoclonal antibody directed against an epitope on the extracellular domain of the IGF-1R, was radiolabeled with 111Indium (111In), 125Iodine (125I) and 89Zirconium (89Zr). In vitro, the affinity and internalization kinetics of 111In-R1507 were determined using the IGF-1R expressing breast cancer cell line SUM149 (ER, PR and HER2 negative). In vivo studies were performed in BALB/c nude mice with subcutaneous SUM149 xenografts. To determine the pharmacodynamics of R1507, mice received an intravenous injection of 111In-R1507 and 125I-R1507. One, three and seven days post injection, mice were killed and ex vivo tumor uptake was measured. Non-specific uptake was determined by coinjection of an excess unlabeled R1507. A dose escalation study was performed with 111In-R1507 to determine the optimal protein dose of R1507 for in vivo imaging (dose range 1 – 1000 μg). Finally, SPECT and PET images were acquired of mice with subcutaneous SUM149 tumors one, three and seven days post injection of 111In-R1507 and 89Zr-R1507, respectively. Results: 111In-R1507 (IC50 = 0.1 nM) was slowly internalized by SUM149 cells. 111In-R1507 specifically and efficiently accumulated in the SUM149 xenografts: the specific tumor uptake was 20% ID/g, 33% ID/g and 31% ID/g at one, three and seven days post injection, respectively. The dose escalation study showed that the optimal dose for imaging experiments was ≤ 3 μg (tumor uptake: 35% ID/g). MicroSPECT and microPET imaging of mice clearly visualized the subcutaneous SUM149 xenograft with increasing contrast at later time points. Conclusion: 111In-R1507 SPECT and 89Zr-R1507 PET are excellent methods to visualize IGF-1R expression in vivo in triple negative breast cancer xenografts. In the future, these techniques may enable selection of patients with triple negative breast cancer for IGF-1R targeted therapy. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A36

Met and c-Src Cooperate to Compensate for Loss of Epidermal Growth Factor Receptor Kinase Activity in Breast Cancer Cells

Breast cancers are not responsive to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), although 30% of breast cancers overexpress EGFR. The mechanism of intrinsic resistance to EGFR TKIs in breast cancer is the focus of current studies. Here, we observed that EGFR remains tyrosine phosphorylated in breast cancer cells that proliferate in the presence of EGFR TKIs. In one such cell line, SUM229, inhibiting c-Src kinase activity with either a dominant-negative c-Src or a c-Src TKI decreased EGFR phosphorylation on Tyr(845), Tyr(992), and Tyr(1086) in the presence of EGFR TKIs. Conversely, overexpressing wild-type (wt) c-Src in the EGFR TKI-sensitive breast cancer cell line SUM149 increased EGFR kinase-independent EGFR tyrosine phosphorylation. In addition, in the presence of EGFR TKIs, inhibiting c-Src kinase activity decreased cell growth in SUM229 cells, and overexpressing wt-c-Src increased cell growth in SUM149 cells. We identified the receptor tyrosine kinase Met to be responsible for activating c-Src in SUM229 cells. Inhibiting Met kinase activity with a small molecule inhibitor decreased c-Src phosphorylation and kinase activation. In addition, inhibiting Met kinase activity in SUM229 cells decreased EGFR tyrosine phosphorylation and growth in the presence of EGFR TKIs. Stimulating Met kinase activity in SUM149 cells with hepatocyte growth factor increased EGFR tyrosine phosphorylation and cell growth in the presence of EGFR TKIs. These data suggest a Met/c-Src-mediated signaling pathway as a mediator of EGFR tyrosine phosphorylation and cell growth in the presence of EGFR TKIs.

Profiling YB-1 responsive genes in basal-like breast cancer cells by ChIP-on-chip reveals direct binding to PIK3CAs

Purpose: Basal-like breast cancers (BLBC) are typically very aggressive and they are also prone to a high rate of recurrence. We recently identified the transcription factor Y-box binding protein-1 (YB-1) as an important component of BLBC by screening breast tumour tissue microarrays. YB-1 is well known for its ability to confer chemotherapy resistance and to promote the growth of a wide range of cancer cell types. The breast cancer cell line SUM149 was characterized by us as having all the hallmarks of BLBC including a lack of the estrogen, progesterone, and Her-2 receptors. We also find that they express high levels of the epidermal growth factor receptor, a YB-1 responsive gene. 
 We performed a genome-wide promoter screen of the possible YB-1 responsive genes using the NimbleGen ChIP-on-chip (COC) platform to begin to understand how this oncogene regulates the growth of basal-like breast cancer cells. YB-1 was found to bind to ~6,700 human promoters. Several members of the phosphatidylinositol-3-kinase (PI3K) pathway were identified (p110a, p85, Akt-2, ILK). Of particular interest was the identification of PIK3CA, the gene that codes for the p110a catalytic subunit of PI3K. PI3K, a lipid kinase with important roles in neoplasia, phosphorylates membrane inositol lipids resulting in activation of downstream effectors that are involved in cellular responses such as cell proliferation, survival, metabolism, and cytoskeletal reorganization. Many studies have suggested importance of the role of PI3K signaling in various types of cancer. Approximately 20% of breast cancers harbour somatic activating mutations in PIK3CA, and about 8% have amplification of PIK3CA. The remainder of the cancers have elevated levels of PI3K via other mechanisms including increased transcription. 
 Methods & Results: Here we propose that YB-1 overexpression in cancer leads to increased PIK3CA transcription via direct binding to the promoter. To extend our COC findings, we knocked down YB-1 with siRNA and observed a concomitant loss in p110a by immunoblotting. We have recently identified two alternate PIK3CA transcriptional start sites (exon 1a and 1b) and two alternate PIK3CA promoters (promoter 1a and 1b). We mapped potential YB-1 binding sites on PIK3CA promoters 1a and 1b, and identified five putative YB-1 binding sites within promoter 1a and one putative binding site within promoter 1b. We further performed traditional ChIP and EMSA to confirm the interaction of YB-1 with the putative binding sites on PIK3CA promoter, and determined that YB-1 binds to promoter 1a, but not to promoter 1b. 
 Conclusion:The demonstration that YB-1 binds directly to the PIK3CA promoter and induces its activity identifies a novel mechanism whereby activation of YB-1 and subsequent up-regulation of PIK3CA may contribute to the pathophysiology, outcomes and therapeutic responsiveness of breast cancer.

P38MAPK-Activated AKT in HER-2 Overexpressing Human Breast Cancer Cells Acts as an EGF-Independent Survival Signal

HER-2 is an epidermal growth factor receptor (EGFR) family receptor tyrosine kinase that is overexpressed in about 30% of human breast cancers correlating with a poor prognosis. Previous work in our laboratory has found that HER-2 overexpression plays a role in growth factor independence, anchorage independence, motility, and invasion of naturally occurring basement membranes. We also found that AKT was activated by p38MAPK in these cells, but this activation did not play a role in invasion. Since AKT has been shown in other systems to be a survival factor, we hypothesized that HER-2 mediated activation of AKT is necessary for growth factor independence. Human mammary epithelial cells transduced to overexpress HER-2, HER-2, PTEN, and Myr-AKT and the primary breast cancer cell lines SUM-149 and SUM-225 were used to dissect the signaling pathways leading to growth factor independence and anchorage-independent growth in HER-2 overexpressing cells. We found that, in the absence of EGF, p38MAPK-activated AKT is necessary for HER-2 overexpressing cells to survive and to form colonies in soft agar. We show that EGF works as a survival signal in the absence of p38MAPK-mediated activation of AKT. We also show that human mammary epithelial cells expressing a constitutively active AKT do not require EGF for growth or colony formation in soft agar. The data presented here indicate that AKT activation can compensate for EGF-mediated cell survival signals leading to growth factor independence and anchorage-independent growth.