Triple Negative Tumor Cells Research Articles

THU555 Stress Induced AIPB Expression In Triple Negative (ER-/PR-/Her2-) Breast Tumors Reduces Estradiol

Abstract Disclosure: M. Patrawala: None. H.S. Bose: None. In the United States, 1 in 8 women will develop invasive breast cancer in their lifetimes, and currently, more than 3.8 million individuals are survivors. Breast cancers are hormonally regulated and are categorized ER+/PR+/HER2-, ER-/PR-/HER2+, ER+/PR-/HER2-, and ER-/PR-/HER2-. In patients with Estrogen Receptor positive (Er+) and Progesterone Receptor positive (PR+), these receptors are used for targeted therapy. However, about fifteen percent of patients are diagnosed with triple-negative breast cancer. Currently, there are no molecular therapies for this cancer and only 20% of the cases respond to chemotherapy, so new prognostic markers and therapies are necessary. Aromatase, the rate limiting enzyme catalyzes testosterone to estradiol, is expressed in the ovaries of pre-menopausal women and increases 20-fold in peripheral tissues and fat bodies, like breast, in post-menopausal women contributing to tumor growth. We observed “Aromatase Interacting Partner in Breast (AIPB)” overexpression inhibits aromatase activity resulting by decreasing in estradiol. In triple-negative breast cancer, patients have a more than ten-fold decline in the expression of AIPB compared to ER+/PR+/Her2- breast tumors. Patients diagnosed with triple-negative breast cancer are profoundly stressed considering the low survival rate, which induces higher cortisol synthesis. We hypothesize the stressor will change the expression of AIPB in the tumor cells which will possibly reduce estradiol synthesis. The stress was emulated using a laboratory model in which a heat shock of 42° was delivered for varying periods (0,15,30,45 minutes). The model was used on engineered triple-negative tumor cells which conditionally overexpress AIPB with the addition of tetracycline. We observed MDA-MD-231 cells had a 50% reduction of estradiol from 160 ng/ml to 100 ng/ml following stress for 45 minutes. Interestingly MDA-MD-231-AIPB cells synthesized only 62 ng/ml, which is an 18.75% reduction. On addition of doxycycline, the estradiol level was further decreased to 47 ng/ml, which is about 29.4%. Western blot with a CT antibody of the MDA-MB-231-AIPB cells showed an increase in AIPB expression at the maximum level when stress was performed in combination with doxycycline. Aromatase is absent in triple-negative breast cancer, so we conclude aromatase independent action AIPB in these triple-negative tumorigenic cells plays a crucial role in estradiol synthesis. With our current data, we can suggest a model in which stimulating higher expression of AIPB in patients can provide them with better and longer lives. Presentation: Thursday, June 15, 2023

Advanced Cell Culture Models Illuminate the Interplay between Mammary Tumor Cells and Activated Fibroblasts.

The interaction between tumor cells and activated fibroblasts determines malignant features of desmoplastic carcinomas such as rapid growth, progression towards a metastatic phenotype, and resistance to chemotherapy. On one hand, tumor cells can activate normal fibroblasts and even reprogram them into CAFs through complex mechanisms that also involve soluble factors. Among them, transforming growth factor beta (TGF-β) and Platelet-Derived Growth Factor (PDGF) have an established role in the acquisition of pro-tumorigenic phenotypes by fibroblasts. On the other hand, activated fibroblasts release Interleukin-6 (IL-6), which increases tumor-cell invasiveness and chemoresistance. However, the interplay between breast cancer cells and fibroblasts, as well as the modes of action of TGF-β, PDGF, and IL-6, are difficult to investigate in vivo. Here, we validated the usage of advanced cell culture models as tools to study the interplay between mammary tumor cells and fibroblasts, taking mouse and human triple-negative tumor cells and fibroblasts as a case study. We employed two different settings, one permitting only paracrine signaling, the other both paracrine and cell-contact-based signaling. These co-culture systems allowed us to unmask how TGF-β, PDGF and IL-6 mediate the interplay between mammary tumor cells and fibroblasts. We found that the fibroblasts underwent activation induced by the TGF-β and the PDGF produced by the tumor cells, which increased their proliferation and IL-6 secretion. The IL-6 secreted by activated fibroblasts enhanced tumor-cell proliferation and chemoresistance. These results show that these breast cancer avatars possess an unexpected high level of complexity, which resembles that observed in vivo. As such, advanced co-cultures provide a pathologically relevant tractable system to study the role of the TME in breast cancer progression with a reductionist approach.

Retrospective comparisons of nanoparticle albumin-bound paclitaxel and docetaxel neoadjuvant regimens for breast cancer.

Aim: To compare the efficacy and safety of 2-weekly nanoparticle albumin-bound paclitaxel (nP) and 3-weekly docetaxel regimens as neoadjuvant systemic therapy (NST) for breast cancer. Materials & methods: Patients (n=201) received NST comprising either dose-dense epirubicin and cyclophosphamide followed by 2-weekly nP (n=104) or 3-weekly courses of epirubicin and cyclophosphamide followed by docetaxel (n=97). Results: Higher pathological complete responserates were achieved by the nP group. Subgroup analysis showed that the nP-based regimen achieved higher pathological complete response rates in patients with triple-negative tumor cells and high Ki67 levels. However, grades 3-4 peripheral sensory neuropathies were more frequent in the nP group. Conclusion: The 2-weekly nP-based regimen might be a better choice of NST for patients with breast cancer.

Caffeine and Cisplatin Effectively Targets the Metabolism of a Triple-Negative Breast Cancer Cell Line Assessed via Phasor-FLIM.

Triple-negative tumor cells, a malignant subtype of breast cancer, lack a biologically targeted therapy. Given its DNA repair inhibiting properties, caffeine has been shown to enhance the effectiveness of specific tumor chemotherapies. In this work, we have investigated the effects of caffeine, cisplatin, and a combination of the two as potential treatments in energy metabolism for three cell lines, triple-negative breast cancer (MDA-MB-231), estrogen-receptor lacking breast cancer (MCF7) and breast epithelial cells (MCF10A) using a sensitive label-free approach, phasor-fluorescence lifetime imaging microscopy (phasor-FLIM). We found that solely using caffeine to treat MDA-MB-231 shifts their metabolism towards respiratory-chain phosphorylation with a lower ratio of free to bound NADH, and a similar trend is seen in MCF7. However, MDA-MB-231 cells shifted to a higher ratio of free to bound NADH when cisplatin was added. The combination of cisplatin and caffeine together reduced the survival rate for MDA-MD231 and shifted their energy metabolism to a higher fraction of bound NADH indicative of oxidative phosphorylation. The FLIM and viability results of MCF10A cells demonstrate that the treatments targeted cancer cells over the normal breast tissue. The identification of energy metabolism alteration could open up strategies of improving chemotherapy for malignant breast cancer.

Abstract 2971: The prognostic relevance of inflammatory cytokines and growth factors elaborated from adipose-derived stem cells in breast cancer

Abstract Introduction: Given the high rates of obesity and aggressive breast cancers, it is paramount to understand how adipose tissue supports the tumor microenvironment. We previously found that adipose-derived stem cells cultured with triple-negative tumor cells enhanced the expression of numerous pro-inflammatory cytokines. This finding is significant as it could explain, in part, how obesity contributes to worse outcomes in breast cancer. To further expand this study, we probed the Cancer Genome Atlas (TCGA) breast invasive carcinoma dataset for the expression profiles of the inflammatory cytokines we found to be highly up-regulated in vitro. We predicted TCGA data would demonstrate a correlation between cytokine overexpression and worse clinical scenarios. Methods: Cytokines and growth factors were measured in the media of adipose stromal cells and MDA-MB-231 tumor cells via membrane-based antibody array. To validate clinical significance, we compared each cytokine’s mRNA expression profile, available from primary tumor samples of TCGA breast invasive carcinoma dataset (n = 1,005), to indices of tumor progression and survival status. For survival analysis, samples were dichotomized as high or low expression by individual cytokines (z score ≥ 1.5). Cytokine and growth factor expression was further stratified according to molecular subtype of breast cancer (luminal A, luminal B, HER2, triple negative) and receptor status. Ingenuity pathway analysis (IPA) was performed to identify candidate markers related to obesity or inflammation. Results: High levels of FGF7 and CCL5 protein were found in the conditioned media of tumor and adipose stem cell co-cultures and together they showed a significant impact on breast cancer survival within TCGA data. FGF7, CCL5, together with CCL2, IL6, and IL6R were further analyzed for expression across molecular subtypes of breast cancer. IL6R, CCL5, and CCL2 expression varied significantly between breast cancer subtypes, with mean expression highest in triple-negative tumors. After dichotomizing the samples for estrogen receptor status, IL6R, CCL5, and CCL2 showed significantly higher mean expression in ER negative tumors. Greater expression of these cytokines in tumors was correlated with reduced patient survival in triple-negative tumors and ER negative subtypes. IPA analysis indicated IL6, IL6R, CCL5, and CCL2, but not FGF7, cooperate through inflammatory pathways to promote tumor progression. Conclusions: Genomic data analysis supports in vitro findings that IL6R, CCL5, and CCL2 overexpression, either independently or synergistically, predict a worse prognosis in breast cancer. For adipose-driven, hormone receptor negative breast tumors, these cytokines and growth factors could serve as novel therapeutic targets. (Supported by NIH P20GM103434 and NIGMS U54GM104942) Citation Format: Nicole Werwie, Kelsey Sadlek, Eric Lundstrom, Daniel Berrebi, Gerald Hobbs, Linda Vona-Davis. The prognostic relevance of inflammatory cytokines and growth factors elaborated from adipose-derived stem cells in breast cancer [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 2971. doi:10.1158/1538-7445.AM2017-2971

Nuclear basic fibroblast growth factor regulates triple-negative breast cancer chemo-resistance.

IntroductionChemotherapy remains the only available treatment for triple-negative (TN) breast cancer, and most patients exhibit an incomplete pathologic response. Half of patients exhibiting an incomplete pathologic response die within five years of treatment due to chemo-resistant, recurrent tumor growth. Defining molecules responsible for TN breast cancer chemo-resistance is crucial for developing effective combination therapies blocking tumor recurrence. Historically, chemo-resistance studies have relied on long-term chemotherapy selection models that drive genetic mutations conferring cell survival. Other models suggest that tumors are heterogeneous, being composed of both chemo-sensitive and chemo-resistant tumor cell populations. We previously described a short-term chemotherapy treatment model that enriches for chemo-residual TN tumor cells. In the current work, we use this enrichment strategy to identify a novel determinant of TN breast cancer chemotherapy resistance [a nuclear isoform of basic fibroblast growth factor (bFGF)].MethodsStudies are conducted using our in vitro model of chemotherapy resistance. Short-term chemotherapy treatment enriches for a chemo-residual TN subpopulation that over time resumes proliferation. By western blotting and real-time polymerase chain reaction, we show that this chemotherapy-enriched tumor cell subpopulation expresses nuclear bFGF. The importance of bFGF for survival of these chemo-residual cells is interrogated using short hairpin knockdown strategies. DNA repair capability is assessed by comet assay. Immunohistochemistry (IHC) is used to determine nuclear bFGF expression in TN breast cancer cases pre- and post- neoadjuvant chemotherapy.ResultsTN tumor cells surviving short-term chemotherapy treatment express increased nuclear bFGF. bFGF knockdown reduces the number of chemo-residual TN tumor cells. Adding back a nuclear bFGF construct to bFGF knockdown cells restores their chemo-resistance. Nuclear bFGF-mediated chemo-resistance is associated with increased DNA-dependent protein kinase (DNA-PK) expression and accelerated DNA repair. In fifty-six percent of matched TN breast cancer cases, percent nuclear bFGF-positive tumor cells either increases or remains the same post- neoadjuvant chemotherapy treatment (compared to pre-treatment). These data indicate that in a subset of TN breast cancers, chemotherapy enriches for nuclear bFGF-expressing tumor cells.ConclusionThese studies identify nuclear bFGF as a protein in a subset of TN breast cancers that likely contributes to drug resistance following standard chemotherapy treatment.

Abstract IA22: Investigating breast cancer evolution and diversity with single-cell sequencing

Abstract Sequencing breast tumor cohorts has identified many prevalent mutations, but provides limited insight into the genomic diversity within tumors. Here, we developed a whole-genome and exome single-cell sequencing approach called Nuc-Seq that can achieve 83-97% coverage breadth with low error rates. We applied this method to sequence single normal and tumor cells from an estrogen-receptor positive breast cancer and a triple-negative ductal carcinoma, and performed copy number profiling in parallel. Our data shows that aneuploid rearrangements occurred early in tumor evolution and remained highly stable as the tumor masses expanded. In contrast, point mutations evolved gradually, generating extensive clonal diversity. Many of the diverse mutations were shown to occur at low frequencies (0.03 -10%) in the tumor mass by targeted single-molecule sequencing. Using mathematical modeling we found that the triple-negative tumor cells had an increased mutation rate (13.3X) while the ER+ tumor cells did not. These findings have important implications for the diagnosis, therapeutic treatment and evolution of chemoresistance in breast cancer. Citation Format: Nicholas E. Navin. Investigating breast cancer evolution and diversity with single-cell sequencing. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr IA22.

Abstract LB-310: Single cell genome sequencing reveals clonal stability and diversity in breast cancer

Abstract Human breast cancers often display intratumor genomic heterogeneity. This clonal diversity confounds the clinical diagnosis and basic research of cancer, because single samples may not represent that tumor as a whole. Sequencing breast tumor cohorts en masse has identified many prevalent mutations, but has limited ability for resolving subclonal diversity. Here, we developed a whole-genome and exome single-cell sequencing approach (Nuc-Seq) using G2/M cells. To validate our method, we applied Nuc-Seq to sequence the whole genomes of two single cells from a genetically monoclonal breast cancer cell line (SK-BR-3) at high coverage depth (61X ± 5 sem, n=2) and breadth (83.70% ± 3.40 sem, n=2) to detect somatic mutations. Our analysis suggests that Nuc-Seq generates low allelic dropout rates (9.73% ± 2.19%) and low false positive error rates for point mutations (FPR = 1.24e-6). We then applied this method to sequence single normal and tumor cells from an estrogen-receptor positive breast cancer and a triple-negative ductal carcinoma at base-pair resolution. In parallel, we performed single cell copy number profiling. In both tumors, we observed a large number of rare variants that were not detected by sequencing the bulk tumor en masse. In contrast, we find that single cell copy number profiles are highly similar. Our data suggest that aneuploid rearrangements occurred early in tumor evolution and remained highly stable as the tumor mass expanded. In contrast we find that point mutations evolved gradually, generating extensive clonal diversity. Many of the diverse mutations were shown to occur at low frequencies (0.03 -10%) in the tumor mass by targeted duplex sequencing. Mathematical modeling suggests that the triple-negative tumor cells have an increased mutation rate (13.3X), while the ER+ tumor cells do not. These findings have important implications for the diagnosis, therapeutic treatment and evolution of chemoresistance in breast cancer. Citation Format: Yong Wang, Nicholas Navin, Jill Waters, Marco Leung, Anna Unruh, Xiuqing Shi, Whijae Roh, Ken Chen, Paul Scheet, Selina Vattathil, Han Liang, Asha Multani, Hong Zhang, Funda Meric-Bernstam, Franziska Michor, Rui Zhao. Single cell genome sequencing reveals clonal stability and diversity in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-310. doi:10.1158/1538-7445.AM2014-LB-310

377 Selective Inhibition of AKT Protein Isoforms Augments Gefitinib Response in EGFR Mutated NSCLC Cells

Introduction: Genetic changes occurring in the triple negative (TN) subset of breast tumors (BT) contribute to different phenotypes in the tumor cells via deregulation of specific signaling-pathways. We identified a differential upregulation of the Wnt-beta-catenin pathway in TNBT (Barwick etal, 2008; Dey etal. 2009), and reported that in TNBT, the poor prognosis and the higher incidence of metastasis are associated with an activation of this pathway (Barwick etal., 2009). We also reported a role of the pathway in the control of migration and invasion of tumor cells (Dey etal. 2009, 2010). Since integrin-directed migration and invasion of the tumor cells are instrumental for the metastasis, and TN tumor cells are highly invasive in nature, we hypothesized that the upregulation of this pathway in TNBT is functionally connected to the control of metastasis-associated phenotypes. This hypothesis is further strengthened by our identification of MMP7 as a selective-biomarker of TNBT (Abramovitz etal, 2009). Purpose: We studied the involvement of the pathway in the control of metastasis-associated phenotypes in TN breast cancer (BC) cells. Methods: We tested the effects of (1) different chemical modulators of the Wnt signaling, and (2) downregulation of beta-catenin on the (a) cell signaling marker(s), (b) cell proliferation, (c) clonogenic growth, (d) migration, (e) invasion, and (f) vascular mimicry (VM) using a panel of six TNBT cells. Results: (1) CHIR99021 (selective GSK3b inhibitor which activates Wnt signaling) enhanced clonogenic growth, and VM while WntC59 (palmitylates Wnt proteins and blocks the Wnt secretion/activity), as well as XAV939 (tankyrase inhibitor that blocks beta-catenin-mediated transcription via axin stabilization) blocked clonogenic growth, cell proliferation, fibronectindirected migration, and VM in TNBT cells. (2) Sulindac sulfide (decreases cellular levels of total and active beta-catenin) inhibited migration, invasion, VM, actin cytoskeleton, podia-parameters, RAC1/Cdc42 activation, and MMP7 in TNBC cells. (3) SiRNA mediated downregulation of betacatenin protein in TNBC cells inhibited cell proliferation, clonogenic growth, migration, invasion, c-MYC, and VM. Conclusion: Wnt-beta-catenin pathway activates cellular signaling for the metastasis-associated phenotypes like proliferation, clonogenic growth, migration, invasion and VM in TNBT cells and this function is directly mediated by via cellular levels of beta-catenin. An inhibition of the Wntbeta-catenin pathway might be an effective combination treatment strategy in TNBC.

Abstract 5287: Role of Wnt-beta-catenin pathway in the control of tumor cell invasion in the triple negative subset of breast cancer

Abstract A plethora of genetic changes occurring in the triple negative (TN) breast tumors (BT) contribute to the different phenotypes exhibited by the tumor cells via deregulation of the specific signaling-pathways. Our group identified a differential upregulation of the Wnt-beta-catenin pathway in TN subset of BT (Barwick etal, 2008, BCS; Dey etal. 2009, AACR) and reported that the poor prognosis and higher incidence of metastasis is associated with this pathway in TNBT (Barwick etal., 2009; PNAS, revised). Of late, we demonstrated a role of the pathway in the control of integrin-mediated migration in TNBT cells (Dey etal. 2009, Adv. BCR). Since integrin-directed migration is instrumental for the invasion (directional movement across extracellular-matrix following matrix degradation) of the tumor cells and TN tumor cells are highly invasive in nature, we hypothesized that the upregulation of Wnt-beta-catenin pathway in TNBT is functionally connected to the control of invasion. Our hypothesis is strengthened by our recent identification of MMP7 (key target-gene of this pathway), as a selective-biomarker of TN subtype (Abramovitz etal, 2009, AACR). In an attempt to define the significance of the pathway in the regulation of invasion, we examined (a) the expression of MMP7 and CTNNB1 in tumor samples and 15 TNBT cell lines, (b) the activity of MMP7 in high MMP7-expressing TNBT cell lines, and (c) effects of pharmacological intervention of the pathway on the invasion of MMP7-expressing TNBT cells. We demonstrated that MMP7 is selectively upregulated in TNBT tumors (our data sets; Montreal-91, Georgia-137) and published data set (MSK-96). We validated the DASL-data by RT-PCR in tumors and cell lines. The expression of MMP7 and beta-catenin (mRNA) in tumor samples from TN patients was comparable to the pattern of expression of MMP7 and beta-catenin (protein) in TNBT cell lines. Zymography of MMP7 demonstrated that the secreted protein is enzymatically active. MDA-MB231, MDA-MB468 and SUM149 TNBT cell lines invaded more in vitro than non-TNBT cells. Administration of sulindac to attenuate the Wnt-beta-catenin pathway showed, 1) a decrease in the levels of beta-catenin (total and active) and MMP7, 2) an inhibition of invasion of cells which corroborated with the integrin-directed cell movement (real-time-video-microscopy), 3) an altered distribution-pattern of filamentous-actin, 4) over 50% attenuation of the activation of RAC1-GTPase and 5) a blockade of migration of TNBT cells. The status of activation of cofilin is directly related to cell migration, invasion, and metastasis in many tumor cells including mammary tumors. We are currently studying the state of phosphorylation of cofilin in the context of fibronectin-dependent activation of the RAC1 (upstream of cofilin) in TNBT cells following the intervention of Wnt-beta-catenin pathway, the results of which will be presented in the meeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5287.