Non-metastatic Breast Cancer Cell Lines Research Articles

ANKRD1 Promotes Breast Cancer Metastasis by Activating NF-κB-MAGE-A6 Pathway.

Early detection and surgical excision of tumors have helped improve the survival rate of patients with breast cancer. However, patients with metastatic cancer typically have a poor prognosis. In this study, we propose that ANKRD1 promotes metastasis of breast cancer. ANKRD1 was found to be highly expressed in the MDA-MB-231 and MDA-LM-2 highly metastatic breast cancer cell lines compared to the non-metastatic breast cancer cell lines (MCF-7, ZR-75-30, T47D) and normal breast cancer cells (MCF-10A). Furthermore, high-grade tumors showed increased levels of ANKRD1 compared to low-grade tumors. Both in vitro and in vivo functional studies demonstrated the essential role of ANKRD1 in cancer cell migration and invasion. The previous studies have suggested a significant role of NF-κB and MAGE-A6 in breast cancer metastasis, but the upstream regulators of this axis are not well characterized. Our study suggests that ANKRD1 promotes metastasis of breast cancer by activating NF-κB as well as MAGE-A6 signaling. Our findings show that ANKRD1 is a potential therapeutic target and a diagnostic marker for breast cancer metastasis.

Abstract C069: Targeting the Bai1-driven signaling pathway as a therapeutic approach for breast cancer treatment

Abstract Breast cancer is the most prevalent cancer among women globally, and it remains the second leading cause of cancer-related mortality in this group. Notably, breast cancer exhibits a survival disparity between black and white women, with black women experiencing higher mortality rates. While the reasons for this disparity are multifaceted, differences in molecular mechanisms and signaling pathways driving disease progression may contribute to the observed survival gap. In this study, we investigate the role of cancer cell fusion-driven tumor heterogeneity and metastasis as a potential explanation for the survival disparity in breast cancer. We hypothesize that the mechanism of breast cancer fusion involves Bai1 activation-driven induction of the Elmo1/Dock 180/Rac 1 signaling pathway. Elmo1 and Dock 180 are overexpressed in breast cancer cell lines and Elmo1/Dock 180 activation is associated with breast cancer metastasis. Targeting this pathway could prevent cancer cell fusion-driven metastasis and death. To test our hypothesis, we used non-metastatic and metastatic breast cancer cells isolated from black (HCC 1806, MDA-MB-157) and white (T47D, MDA-MB-231) women, as well as mesenchymal stem cells (MSCs) (hMSC4099, hMSC39334) as fusion partners. We used Elmo1 and Dock 180 shRNAs and a Rac1 inhibitor to analyze the role of Elmo1/Dock 180/Rac 1 signaling in breast cancer cell fusion. Fusion products were scored using the Cre/loxP system. We observed that breast cancer cells isolated from black women are more prone to fuse with MSCs than cells isolated from white women (P<0.05). knocking down Bai1 significantly reduced the frequency of fusion across all cell lines (P<0.05) and more significantly in the non-metastatic breast cancer cell line isolated from black women (P<0.001). A notable reduction in the frequency of fusion between breast cancer cells and MSCs was observed when Elmo1, Dock180, or Rac1 was inhibited (P<0.05). These results suggest that the Bai1/Elmo1/Dock180/Rac1 signaling plays an important role in cancer cell fusion. Targeting this mechanism could lead to new drug development tactics for cancer treatment. Citation Format: Oluwatoyin V. Odubanjo, Paul B. Tchounwou, Brenda M. Ogle, Felicite K. Noubissi. Targeting the Bai1-driven signaling pathway as a therapeutic approach for breast cancer treatment [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C069.

The Binomial "Inflammation-Epigenetics" in Breast Cancer Progression and Bone Metastasis: IL-1β Actions Are Influenced by TET Inhibitor in MCF-7 Cell Line.

The existence of a tight relationship between inflammation and epigenetics that in primary breast tumor cells can lead to tumor progression and the formation of bone metastases was investigated. It was highlighted how the induction of tumor progression and bone metastasis by Interleukin-1 beta, in a non-metastatic breast cancer cell line, MCF-7, was dependent on the de-methylating actions of ten-eleven translocation proteins (TETs). In fact, the inhibition of their activity by the Bobcat339 molecule, an inhibitor of TET enzymes, determined on the one hand, the modulation of the epithelial-mesenchymal transition process, and on the other hand, the reduction in the expression of markers of bone metastasis, indicating that the epigenetic action of TETs is a prerequisite for IL-1β-dependent tumor progression and bone metastasis formation.

MiRNA‑490‑3p promotes the metastatic progression of invasive ductal carcinoma.

MicroRNA (miRNA/mir)-490-3p has been defined as a tumor suppressor in different types of cancer, including breast cancer. However, miR-490-3p has been shown to function as a tumor suppressor and promoter in a context-dependent manner in hepatocellular and lung cancer. Contrary to previous studies, the present study revealed that miR-490-3p expression was significantly higher in invasive ductal carcinoma (IDC) tissue specimens, the most common form of breast cancer, compared to tumor-adjacent normal tissue specimens (n=20). Its expression was also higher in the more metastatic breast cancer cell line, MDA-MB-231, compared to the non-metastatic breast cancer cell line, MCF7, and the moderately metastatic breast cancer cell line, MDA-MB-468. The expression of miR-490-3p was induced following transforming growth factor (TGF)-β-induced epithelial-to-mesenchymal transition (EMT) in MCF10A cells. Gain-and loss-of-function assays revealed that the expression of miR-490-3p regulated the proliferation, colony formation, EMT, migration and invasion in vitro, but not the apoptosis of MDA-MB-468 and MDA-MB-231 cells. The knockdown of miR-490-3p expression in MDA-MB-231 cells inhibited experimental metastasis in a tumor xenograft assay. As in lung cancer, miR-490-3p was found to target and downregulate the expression of the tumor suppressor RNA binding protein poly r(C) binding protein 1 (PCBP1). PCBP1 protein and miR-490-3p expression inversely correlated in patients with ductal carcinoma in situ (DCIS; n=10; no nodal involvement) and IDC (n=10; different stages of metastatic progression) with a significantly higher miR-490-3p expression in patients with IDC compared to those with DCIS. The expression of miR-490-3p was negatively associated with both overall and disease-free survival in the patients with breast cancer included in the present study. On the whole, the results confirm a pro-metastatic role of miR-490-3p in IDC, establishing it as a biomarker for disease progression in these patients.

Fluidic substrate as a tool to probe breast cancer cell adaptive behavior in response to fluidity level

Although the roles of elastic components in breast cancer progression have been widely studied, the importance of matrix dissipative elements in regulating breast cancer behavior is still poorly understood. In this study, we designed viscosity-tunable fluidic substrates to investigate the effects of matrix viscosity on the alteration of breast cancer cellular fate using a hydrophobic molten polymer of poly(e-caprolactone-co-D,L-lactide) [P(CL-co-DLLA)] with different levels of fluidity. The high- and low-fluidity substrates used in this study were shown to behave as viscoelastic liquids at physiological temperature. A nonmetastatic breast cancer cell line (MCF-7) was cultured at the interface of the fibronectin-coated substrate, and its behavior towards the substrate fluidity level was thoroughly characterized. Despite fibronectin-mediated cell-substrate interactions, MCF-7 cells show sensitivity to substrate fluidity levels by forming types aggregates of different sizes and structures over time. Accordingly, MCF-7 cells were undergoing senescence on fluidic substrates, as shown by high metabolic activity over time, suppressed proliferation ability, and positive expression of senescence markers. Moreover, senescence implies more resistance towards anticancer drug treatment. This indicates that a fluidic substrate, as a two-dimensional synthetic matrix system, could demonstrate the importance of mechanical cues in redefining cellular function and cellular fate by changing the viscosity of the pure substrate. A fluidic substrate behaving as a hydrophobic viscoelastic liquid was designed as a tool to investigate the role of matrix viscosity on the alteration of breast cancer cellular fate. The fluidity level of fluidic substrate was tuned by modulating the molecular weight of poly(e-caprolactone-co-D,L-lactide). MCF-7 cells responded to the change in fluidity level of fluidic substrates by forming weak attachment. On high-fluidity substrate, MCF-7 cells formed 3D aggregates, while coalesced on low-fluidity substrate. More importantly, the fluidic substrate mechanically promoted senescence of MCF-7 cells regardless the fluidity level of substrate.

Differential Response to 1,25-dihydroxyvitamin D in Metastatic and Non-Metastatic Breast Cancer Cell Lines in Hypoxia (P05-006-19)

ObjectivesPrevention of metastasis is of utmost importance for increasing survival in breast cancer patients. Oxygen tension is variable throughout tumors, creating regions of hypoxia that have been linked with poor cancer prognosis. Hypoxia increases glycolytic flux via hypoxia-inducible factor-1α (HIF1α), and can therefore alter growth and survival of cancer cells. Our objectives are to (1) characterize changes in metabolism and survival that occur when metastatic and non-metastatic mammary cancer cell lines are cultured in hypoxia, and (2) determine whether 1,25-dihydroxyvitamin D (1,25(OH)2D) reduces overall survival in hypoxia. MethodsWe utilized Wnt oncogene-driven murine mammary cancer cells that are non-metastatic (M-Wnt) or that preferentially metastasize to the lung in vivo (metM-Wntlung). Viability of M-Wnt and metM-Wntlung cells treated with 10 nM 1,25(OH)2D and/or 20 mM 2-deoxyglucose (2DG, an inhibitor of glycolysis) was measured with MTT. Expression of HIF1α protein was determined by Western blotting. ResultsWe show that 1,25(OH)2D treatment significantly decreased viability of metastatic metM-Wntlung cells grown in hypoxia by 41%, whereas viability of M-Wnt cells was not significantly impacted by 1,25(OH)2D treatment. Furthermore, treating cells with 2DG significantly decreased viability of both cells lines in hypoxia, with metM-Wntlung cells being more sensitive to 2DG. Interestingly, 1,25(OH)2D treatment partially rescued M-Wnt cells by 22% and metM-Wntlung cells by 24% when treated with 2DG in hypoxia. Finally, we show that M-Wnt cells have 1.9-fold increased expression of HIF1α protein compared to metM-Wntlung cells when grown in hypoxia. ConclusionsOur results collectively suggest that non-metastatic M-Wnt cells are less sensitive to treatment with 1,25(OH)2D and 2DG in hypoxia than metastatic metM-Wntlungcells. These data may be explained, in part, by elevated expression of HIF1α in M-Wnt cells, which may contribute to their improved survival in hypoxia. Funding SourcesNational Institute of Health and USDA.

Abstract PD9-10: Circulating CAF/cancer stem cell co-clusters bolster breast cancer metastasis

Abstract Background: Metastatic disease is the primary cause of breast cancer (BC) mortality. Cancer associated fibroblasts (CAFs) are the majority of stroma in BC and critical players in BC malignancy. For example, CAFs are the main source of SDF-1, a prominent chemokine in the tumor microenvironment (TME) that also imparts stem cell-like characteristics to BC cells. Metastasis occurs due to the transport of circulating tumor cells (CTC) and clusters of CTCs through the vasculature. Stem-like CTCs and clusters have a greater propensity to establish metastasis. We recently identified circulating CAFs (cCAFs) in blood from patients with BC and in spontaneous, syngeneic, and xenograft mouse models of BC. cCAFs not only circulate individually, but are also found in clusters with CTCs. In this study, we examine the role of CAFs in promoting egress of stem-like CTCs (cCSCs), determine the capacity of stem-like CTCs to cluster with CAFs, and evaluate the involvement of CTC/cCAF clustering in augmenting BC metastasis. Methods: Our model employs NSG mice with orthotopic xenograft implantation of BC cells, primary CAF cell lines, or co-implantation of BC and CAF cell lines. We used two different BC cell lines: the non-metastatic BC cell line, MCF-7, and the highly metastatic primary BC cell line, DT28. We also employed the MMTV-PyMT spontaneous model of BC metastasis, and we used BALB/c mice injected with syngeneic 4T1 or 67nR cells to evaluate cCAFs, CTCs, and cluster egress in preclinical models. Mice were sacrificed at specific time points, and cardiac blood was collected. Blood was filtered using the faCTChecker microfluidic filtration instrument (Circulogix). Filters were stained for IF and cCAFs, CTCs, cCSCs, and clusters were enumerated. Tumors from CAF co-injected mice were evaluated for their stem cell-like phenotype and re-implanted in mice to evaluate tumorigenicity and metastasis. Results: In spontaneous, syngeneic, and orthotopic xenograft models of BC, cCAFs, CTCs, and cCAF/CTCs co-clusters appear early in tumor development. cCAF/CTC clusters increase in correlation with tumor burden and metastasis. Co-inoculation of CAFs with BC cells resulted in a significant increase in tumor progression, metastasis, and in a substantially higher number of both individual cells and clusters in circulation. Dissociated tumor cells from CAF co-injected tumors had a higher proportion of CD44+stem cell-like cells (CSCs), enhanced ALDH-1 expression, and enhanced mammosphere formation. CD44+ CSCs, individually and in clusters, are found early on in the circulation of mice injected with dissociated tumor cells from CAF co-injected tumors. Upon re-implantation of CAF co-injected dissociated tumor cells without CAFs, dissociated tumor cells showed enhanced tumorigenicity and malignancy. Conclusion: CAFs are highly motile and cCAFs precede CTCs into circulation and can do so independently of tumor cells. CAFs sustain egress of tumor cells by augmenting malignancy and stemness of BC cells. cCAF clusters with the highly metastatic stem cell-like subset of CTCs bolster metastatic colonization. Targeting primary CAF function and/or cCAF/cCSC co-clusters may provide novel avenues to abrogate BC metastasis. Citation Format: Sharma U, Miller P, Medina Saenz K, Picon-Ruiz M, Morata-Tarifa C, Spartz A, Troness B, Park DN, Seagroves TN, Slingerland JM, Lippman ME, El-Ashry D. Circulating CAF/cancer stem cell co-clusters bolster breast cancer metastasis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-10.

Abstract 5062: CAFs in breast cancer circulation: The influence of cancer cell intrinsic mechanisms

Abstract Background: Metastatic disease is the primary cause of breast cancer (BC) mortality. Tumor-stromal cell interactions play a pivotal in tumor initiation, progression and metastasis. Cancer-associated fibroblasts (CAFs) are the majority of stroma in BC and are critical to BC tumorigenicity and malignancy. Metastasis occurs due to the transport of circulating tumor cells (CTC) and clusters of CTCs through the vasculature. We recently identified circulating CAFs (cCAFs) as a novel circulating biomarker associated with metastatic BC. cCAFs not only circulate individually, in BC patient blood and in blood from both spontaneous and xenograft murine BC models, but cCAFs are also found in clusters with CTCs. In this study, we examine the egress of CAFs and follow them through to metastatic sites, and evaluate the ability of BC cell subtype/metastatic propensity to influence cCAF and cCAF/CTC cluster egress. Methods: We used NSG mice with orthotopic xenograft implantation of BC cells, primary CAF cell lines, or co-implantation of BC and CAF cell lines. We used two different BC cell lines, the nonmetastatic BC cell line, MCF-7, and the highly metastatic primary BC cell line, DT28. We also employed the MMTV-PyMT spontaneous model of BC metastasis to evaluate cCAFs and CTCs in a preclinical model. Mice were sacrificed at specific time points, and cardiac blood was collected to ascertain the temporal dynamics of cCAF and CTC presence. Blood was filtered using the faCTChecker microfluidic filtration instrument (Circulogix). Filters were enumerated by IF for cCAFs, CTCs, and cCAF/CTC co-clusters. To explore BC-intrinsic factors that influence cCAF and cCAF/CTC cluster egress, we modeled egress from the primary tumor in vitro using transendothelial cell migration assays, where the chemoattractant for CAF cells was conditioned media from the different BC cells lines. Results: In both spontaneous and orthotopic xenograft models of BC, cCAFs, CTCs, and cCAF/CTCs appear early in tumor development. cCAF/CTC clusters increase in correlation with tumor burden and metastasis. CAFs injected alone in orthotopic xenografts are able to egress independently of BC cells and cCAFs and cCAF clusters were seen; however, co-inoculation with BC cells resulted in substantially higher numbers of both individual cCAFs and cCAF clusters, and now co-clusters were seen. cCAFs appear about 4 days post-injection, and precede appearance of CTCs and CAF/CTC clusters. CAFs co-injected with BC cells into the MFP appear at metastatic sites. In in vitro transendothelial assays, BC secreted factors that potentiate CAF egress are identified as well as the if cCAF/CTC clusters egress as clusters or cluster in the circulation. Conclusion: Although CAFs are highly motile and cCAFs precede CTCs into the circulation, BC subtype influences the ability of CAFs to egress. Targeting cCAF egress and/or cCAF/BC cell clusters provides novel avenues to prevent or treat BC metastasis. Citation Format: Utsav Sharma, Philip Miller, Kelsie Medina Saenz, Angela Spartz, Marc Lippman, Dorraya El-Ashry. CAFs in breast cancer circulation: The influence of cancer cell intrinsic mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5062.

High-Dose Deferoxamine Treatment Disrupts Intracellular Iron Homeostasis, Reduces Growth, and Induces Apoptosis in Metastatic and Nonmetastatic Breast Cancer Cell Lines.

Mounting evidence suggest that iron overload enhances cancer growth and metastasis; hence, iron chelation is being increasingly used as part of the treatment regimen in patients with cancer. Now whether iron chelation depletes intracellular iron and/or disrupts intracellular iron homeostasis is yet to be fully addressed. MCF-7 and MDA-MB-231 breast cancer cells treated with increasing concentrations of the iron chelator deferoxamine were assessed for intracellular iron status, the expression of key proteins involved in iron metabolism, cell viability, growth potential, and apoptosis at different time points following treatment. Treatment with deferoxamine at 1, 5, or 10 μM for 24 or 48 hours, while not leading to significant changes in intracellular labile iron content, upregulated the expression of hepcidin, ferroportin, and transferrin receptors 1 and 2. In contrast, deferoxamine at 30, 100, or 300 μM for 24 hours induced a significant decrease in intracellular labile iron, which was associated with increased expression of hepcidin, ferritin, and transferrin receptors 1 and 2. At 48 hours, there was an increase in intracellular labile iron, which was associated with a significant reduction in hepcidin and ferritin expression and a significant increase in ferroportin expression. Although low-dose deferoxamine treatment resulted in a low to moderate decrease in MCF-7 cell growth, high-dose treatment resulted in a significant and precipitous decrease in cell viability and growth, which was associated with increased expression of phosphorylated Histone 2A family member X and near absence of survivin. High-dose deferoxamine treatment also resulted in a very pronounced reduction in wound healing and growth in MDA-MB-231 cells. These findings suggest that high-dose deferoxamine treatment disrupts intracellular iron homeostasis, reduces cell viability and growth, and enhances apoptosis in breast cancer cells. This is further evidence to the potential utility of iron chelation as an adjunctive therapy in iron-overloaded cancers.

Abstract PR06: Conditional reprogramming is successful in generating patient-derived stable cell cultures from rare aggressive tumors

Abstract Purpose/Objectives: The lack of authenticated cell lines from rare tumors, such as adenoid cystic carcinoma (ACC), have compromised studies on basic biology and drug development. ACC of the salivary gland is a rare human cancer and is classified as a rare or “orphan” disease. It has the propensity for invasion of peripheral nerves or blood vessels early in the disease course resulting in a high incidence of both local and distant metastases to lung and bone. The primary course of treatment is surgical removal of tumor, which is usually combined with postoperative radiotherapy. To date no targeted therapy exists for this tumor type. Patient-derived mouse xenografts (PDX) have been shown to maintain the histology and gene expression profile of the primary tumor, making them a valid model system. However, PDX models suffer from high cost, expensive maintenance, low take rate (30-50%), lack of manipulation and high throughput capability. In order to study these tumor types, a reliable model system is critical that can maintain the key genetic features of the primary tumor. Materials/Methods: A new cell culture method, conditional reprogramming (CR), combines the use of irradiated mouse fibroblasts and a ROCK inhibitor to induce the rapid, efficient, and long-term growth of normal and tumor cells without requiring exogenous viral or cellular gene expression. We used this technology to establish cell cultures from rare tumor types such as ACC, neuroendocrine, anal cancer and recurrent respiratory papillomatosis. Herein, we present data collected on recently established stable cultures for ACC using PDX tissue materials. We tested the maintenance of metastasis in the cell lines by establishing a rapid and robust in-vivo zebrafish xenograft model system for ACCs, using ACC11 as a prototype. Results: We generated stable cultures for ACC from eight individual PDX tumors using CRC technology. We demonstrate that these cultures maintain the key genetic features of the primary tumors. One ACC cell line, ACC11, has shown to maintain the MYB-NFIB translocation, oncogenic mutations in FGFR2 and ATM genes and overexpression of Myb-NF1B fusion protein similar to the tumor of origin (PDX tumor). In the zebrafish in-vivo system, these cells are migratory and possess metastatic potential demonstrated by migration of tumor cells from the 2-day post fertilized yolk sac to the tail including vascular invasion within 3 days post injection. Similar results were obtained when PDX tissue material for ACC11 as well as a conventional metastatic breast cancer cell line, MDA-MB231. In contrast, injection of a patient-derived normal breast cell line established using the CR technology and a conventional non-metastatic breast cancer cell line, MCF7, did not show any movement of cells even after 7 days of post injection. Conclusions: We have successfully established a tool-kit consisting of an in-vitro (cell line) and a rapid in-vivo (zebrafish) metastasis model system for biological and translational research of rare tumor types. The use of this novel CR method has great potential in rapidly generating patient-derived normal and cancer primary cells for a wide variety of uses including biobanking, basic tumor biology, drug target identification, and drug discovery, with an ultimate goal of personalized cancer medicine. This abstract is also being presented as Poster B27. Citation Format: Chen Chen, Sujata Choudhury, Devan Wilkins, Eric Glasgow, Richard Schlegel, Seema Agarwal. Conditional reprogramming is successful in generating patient-derived stable cell cultures from rare aggressive tumors. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr PR06.

Melatonin decreases breast cancer metastasis by modulating Rho-associated kinase protein-1 expression.

The occurrence of metastasis, an important breast cancer prognostic factor, depends on cell migration/invasion mechanisms, which can be controlled by regulatory and effector molecules such as Rho-associated kinase protein (ROCK-1). Increased expression of this protein promotes tumor growth and metastasis, which can be restricted by ROCK-1 inhibitors. Melatonin has shown oncostatic, antimetastatic, and anti-angiogenic effects and can modulate ROCK-1 expression. Metastatic and nonmetastatic breast cancer cell lines were treated with melatonin as well as with specific ROCK-1 inhibitor (Y27632). Cell viability, cell migration/invasion, and ROCK-1 gene expression and protein expression were determined invitro. In vivo lung metastasis study was performed using female athymic nude mice treated with either melatonin or Y27832 for 2 and 5wk. The metastases were evaluated by X-ray computed tomography and single photon emission computed tomography (SPECT) and by immunohistochemistry for ROCK-1 and cytokeratin proteins. Melatonin and Y27632 treatments reduced cell viability and invasion/migration of both cell lines and decreased ROCK-1 gene expression in metastatic cells and protein expression in nonmetastatic cell line. The numbers of 'hot' spots (lung metastasis) identified by SPECT images were significantly lower in treated groups. ROCK-1 protein expression also was decreased in metastatic foci of treated groups. Melatonin has shown to be effective in controlling metastatic breast cancer invitro and invivo, not only via inhibition of the proliferation of tumor cells but also through direct antagonism of metastatic mechanism of cells rendered by ROCK-1 inhibition. When Y27632 was used, the effects were similar to those found with melatonin treatment.

Abstract P6-03-11: Erk5 as a therapeutic target in triple negative breast cancer (TNBC)

Abstract TNBC is a subtype of breast cancer known for its aggressive behavior and poor prognosis. TNBC accounts for 17-24% of all invasive breast cancers and is a subtype that lacks the expression of hormone receptors; oestrogen [ER], progesterone [PR]) and epidermal growth factor receptor 2 (HER2) receptors. Currently there has been a shift from using empirically derived agents that inhibit tumor cell growth and/or survival, to molecular therapies that target specific molecules that regulate these and other important biological processes. Despite the success of some new-targeted therapies to treat breast cancer, the outlook for the majority of patients with TNBC remains poor. Therefore we have utilized the KinexTM antibody array to resolve potential new targetable "nodes". We have screened 43 primary breast cancer biopsies (16 TNBC, 16 ER/PR positive and 11 HER2-positive) and 16 breast cancer cell lines for protein/phosphoprotein levels. Extracellular-signal-regulated kinase 5 (Erk5) is a member of the MAPK family. Erk5 has a large, unique C-terminal-half not found in other MAPK family members and because of this unique 400-amino acid extension, ERK5 is also called big MAP kinase 1 (BMK1). By extensive in silico analyses, we identified that the Erk5 metagene is prognostic and identifies TNBC/BLBC with poor prognosis. Our kinome study of primary breast cancer has further confirmed that the MEK5-Erk5 pathway is upregulated in the TNBC subgroup compared to the other TNBC subgroups and that pharmacological inhibition of Erk5 is therapeutic against primary MDA-MB-231 TNBC xenografts (1). Our preliminary data has shown that Erk5 is overexpressed in the TNBC subtype at the protein level. We have also shown that silencing Erk5 by siRNA or shRNA inhibits migration of TNBC cell lines in vitro. Expression of Erk5 (total and phosphoproteins) will be investigated in primary breast tumours and metastases samples from patient by immunohistochemistry. We will also overexpress Erk5 in non-metastatic breast cancer cell lines to study the effect of Erk5 overexpression on metastases. In an elegant study, site directed mutagenesis was used to modify Erk5 and show that phosphorylations of the C-terminal domain of kinase activated Erk5 is required for the transcriptional activity of Erk5. Using in vitro and in vivo models we will determine if the transcriptional activity of activated Erk5 is a major player to drive metastatic phenotype. Moreover, whether kinase-dependent or transcription-dependent activity of Erk5 on several pathways is sufficient to drive metastasis will also be determined. Our results strongly suggest the MEK5-Erk5 pathway as a prognostic and therapeutic pathway in a poor-prognosis subset of TNBC/BCLC. Characterization of this pathway in clinical cohorts particularly in TNBC tumors, mechanistic understanding of its contribution to metastasis and therapeutic targeting in vivo would help develop a personalised approach to identify and treat TNBC tumors and metastases with activated Erk5 pathway. 1. Al-Ejeh F, Miranda M et al. Kinome profiling reveals breast cancer heterogeneity and identifies targeted therapeutic opportunities for triple negative breast cancer. Oncotarget. March 19, 2014. Citation Format: Mariska Sauzine Miranda, Fares Al Ejeh, Wei Shi, Peter Simpson, Sunil Lakhani, Kum Kum Khanna. Erk5 as a therapeutic target in triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-03-11.

Loss of LKB1 disrupts breast epithelial cell polarity and promotes breast cancer metastasis and invasion.

BackgroundLKB1, also known as STK11, is a master kinase that serves as an energy metabolic sensor and is involved in cell polarity regulation. Recent studies have indicated that LKB1 is related to breast tumorigenesis and breast cancer progression. However, little work has been done on the roles of LKB1 in cell polarity and epithelial-mesenchymal transition in breast cancer. In this study, we tried to prove that loss of LKB1 disrupts breast epithelial cell polarity and causes tumor metastasis and invasion.MethodsThe relationships of LKB1 expression to clinic-pathological parameters and epithelial markers E-cadherin and high-molecular-weight -cytokeratin (HMW-CK) were investigated in 80 clinical breast cancer tissue samples and their paired normal control breast tissue samples by using immunohistochemistry. Then, the LKB1 expressions in metastatic and non-metastatic breast cancer cell lines were compared. The roles of LKB1 in cell polarity and epithelial-mesenchymal transition in breast cancer were determined by using immunofluorescence, western blot assay, and cell migration and invasive assays. Finally, the non-transformed human breast cell line MCF-10A was cultured in three dimensions to further reveal the role of LKB1 in breast epithelial cell polarity maintenance.ResultsHistopathological analysis showed that LKB1 expression level was significantly negatively correlated with breast cancer TNM stage, and positively correlated with ER/PR status and expression levels of E-cadherin and HMW-CK. Immunofluorescence staining showed that LKB1 was co-localized with E-cadherin at adheren junctions. In vitro analysis revealed that loss of LKB1 expression enhanced migration, invasion and the acquisition of mesenchymal phenotype, while LKB1 overexpression in MDA-MB-435 s cells, which have a low basal level of LKB1 expression, promoted the acquisition of epithelial phenotype. Finally, it was found for the first time that endogenous LKB1 knockdown resulted in abnormal cell polarity in acini formed by non-transformed breast epithelial cells grown in 3D culture.ConclusionOur data indicated that low expression of LKB1 was significantly associated with established markers of unfavorable breast cancer prognosis, such as loss of ER/PR, E-cadherin and HMW-CK. Knockdown of endogenous LKB1 gave rise to dysregulation of cell polarity and invasive phenotype of breast cancer cells.

Cell type-specific reciprocal regulation of HIF1A gene expression is dependent on 5′- and 3′-UTRs

In the present study, we demonstrated the reciprocal regulation of hypoxia-inducible factor 1 alpha (HIF1A) gene expression via untranslated region-(UTR) dependent mechanisms. A 151 nucleotide sequence found in the HIF1A 5′-UTR is sufficient for significant translational up-regulation. On the other hand, the 3′-UTR of HIF1A has been implicated in mRNA degradation. In the non-metastatic breast cancer cell line MCF7, the 3′-UTR-dependent down-regulatory machinery predominates over the 5′-UTR-dependent up-regulation of HIF1A. However, 5′-UTR-dependent up-regulation is dominant among metastatic cell lines (MDA-MB453, U87MG). It is therefore likely that the predominance of 5′-UTR-dependent translational enhancement of HIF1A is critical for the malignant phenotype of cancer cells. PTBP-1, but not HuR, is a candidate RNA binding protein for the translational control of HIF1A.

Loss of LKB1 disrupts breast epithelial cell polarity and promotes breast cancer metastasis and invasion

LKB1, also known as STK11, is a master kinase that serves as an energy metabolic sensor and is involved in cell polarity regulation. Recent studies have indicated that LKB1 is related to breast tumorigenesis and breast cancer progression. However, little work has been done on the roles of LKB1 in cell polarity and epithelial-mesenchymal transition in breast cancer. In this study, we tried to prove that loss of LKB1 disrupts breast epithelial cell polarity and causes tumor metastasis and invasion. The relationships of LKB1 expression to clinic-pathological parameters and epithelial markers E-cadherin and high-molecular-weight -cytokeratin (HMW-CK) were investigated in 80 clinical breast cancer tissue samples and their paired normal control breast tissue samples by using immunohistochemistry. Then, the LKB1 expressions in metastatic and non-metastatic breast cancer cell lines were compared. The roles of LKB1 in cell polarity and epithelial-mesenchymal transition in breast cancer were determined by using immunofluorescence, western blot assay, and cell migration and invasive assays. Finally, the non-transformed human breast cell line MCF-10A was cultured in three dimensions to further reveal the role of LKB1 in breast epithelial cell polarity maintenance. Histopathological analysis showed that LKB1 expression level was significantly negatively correlated with breast cancer TNM stage, and positively correlated with ER/PR status and expression levels of E-cadherin and HMW-CK. Immunofluorescence staining showed that LKB1 was co-localized with E-cadherin at adheren junctions. In vitro analysis revealed that loss of LKB1 expression enhanced migration, invasion and the acquisition of mesenchymal phenotype, while LKB1 overexpression in MDA-MB-435 s cells, which have a low basal level of LKB1 expression, promoted the acquisition of epithelial phenotype. Finally, it was found for the first time that endogenous LKB1 knockdown resulted in abnormal cell polarity in acini formed by non-transformed breast epithelial cells grown in 3D culture. Our data indicated that low expression of LKB1 was significantly associated with established markers of unfavorable breast cancer prognosis, such as loss of ER/PR, E-cadherin and HMW-CK. Knockdown of endogenous LKB1 gave rise to dysregulation of cell polarity and invasive phenotype of breast cancer cells.

Abstract 1678: Metastatic breast cancer cell phenotype is regulated by autophagy.

Abstract Autophagy is a catabolic process that is tightly regulated during normal cell growth, development, and homeostasis. Although it may be death inducing, it is also an important survival mechanism for cells in stressful environments, including hypoxia, nutrient deprivation, chemical or physical pressure, or detachment from colonies. Considering the stress associated with the process of metastasis, we hypothesized that autophagy may play a role in the ability of breast cancer cells to survive and metastasize. To test this, we first compared cell proliferation after autophagic induction between metastatic breast cancer cell lines (MDA-MB-231 and -435), a non-metastatic breast cancer cell line (MDA-MB-436), and a ‘normal’ breast epithelial cell line (MCF10A). Both metastatic cell lines continued to proliferate following induction of autophagy; however, the non-metastatic and normal cell lines were growth-inhibited. Autophagy was also more rapidly induced by rapamycin in the metastatic cell lines as demonstrated by an increase in LC3II expression. To test the different survival capabilities between metastatic and normal cells, we selected cells through five rounds of starvation with Earle's balanced salt solution (EBSS) and compared their ability to induce autophagy. Although both the normal and metastatic cells maintained their ability to proliferate under normal conditions, the MDA-MB-231 cells that survived starvation (231-EB5) proliferated more rapidly than the parental population following induction of autophagy. In contrast, both MCF10A and MCF10A-EB5 cells were growth-inhibited. The 231-EB5 cells induced autophagy more rapidly than the parental population as demonstrated by increased expression of LC3II (analyzed by immunoblot) and increased LC3 puncta (analyzed by immunofluorescence). Migration of parental 231 cells was inhibited by rapamycin; however, there was no change in the ability of 231-EB5 cells to migrate after treatment with rapamycin. Growth in three dimensions also was significantly altered. MCF10A-EB5 cells were more extended compared to the spherical growth of the parental MCF10A cells. Protrusions emitted from the 231-EB5 cells were more rounded compared to the extended spikes of the parental 231 cells. Altogether, these results demonstrate that select populations derived from metastatic breast cancer cells have different abilities to survive, proliferate, and migrate following induction of autophagy, suggesting that autophagy may be a key mechanism for tumor progression and metastasis. Citation Format: Yi Li, Monica J. Lewis, Jianzhong Liu, James J. Cody, Douglas R. Hurst. Metastatic breast cancer cell phenotype is regulated by autophagy. [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 1678. doi:10.1158/1538-7445.AM2013-1678

Abstract P6-01-01: Metastatic and non-metastatic isogenic breast cancer cells lines show different metabolic signatures in response to intermittent hypoxia and transient glutamine/glucose deprivation

Abstract INTRODUCTION. Cancer cells quickly adapt to their microenvironment by reprogramming their metabolism. Specific metabolic responses to defined stress conditions, mimicking tumor microenvironmental changes, may therefore unveil signatures of cancer phenotype, not detected in standard tissue culture. PURPOSE. To identify distinct metabolic signatures associated with metastatic ability, we investigated in the two isogenic breast cancer cell lines, 4T1 (highly metastatic) and 67NR (non-metastatic), how glutamine supply/deprivation affects cellular glucose metabolism, and vice-versa, in aerobic or hypoxic conditions, and the effects on cell growth. METHODS. 4T1 and 67NR cells were cultured in medium A, i.e. DME containing 1% P/S, 10% FBS, 25 mM glucose (Glc), and 6 mM glutamine (Gln). Cell growth was determined in tissue culture from cells cultured for 48 h in various media A-D: A, all nutrients available; B: A but no Gln; C: A but 2 mM Gln; D: A but no Glc. Cellular mitochondrial function was also assessed in both 4T1 and 67NR cells using an XF96 Analyzer (Sea Horse Bioscience, Billerica, MA) and the specific inhibitors: oligomycin, FCCP, antimycin and rotenone. Metabolism was studied in cells grown on microcarriers in the various media A-D under aerobic (∼21% O2) and hypoxic conditions (∼1% O2), using our magnetic resonance (MR)-compatible cell perfusion system on a 500 MHz spectrometer. For the cell perfusion studies, Glc or Gln were exchanged for 1–13C-Glc or 3–13C-Gln respectively. The metabolic fate of 13C-labeled nutrients was followed under the various environmental conditions by 13C MR spectroscopy (MRS), while energy metabolism was observed by 31P MRS. RESULTS. Deprivation of either Glc or Gln for 48 h reduced significantly the cell growth of 4T1 and 67NR cells, resulting in a similar growth rate for the nutrient-deprived 4T1 and 67NR cells. As determined from the MR experiments, each cell line adopted different metabolic strategies to cope when exposed to specific metabolite stress conditions, including on the level of oxygen availability. Unlike in 67NR cells, glycolysis is glutamine-dependent in the more aggressive 4T1 cell line (∼2-fold higher glucose-derived lactate synthesis rate was observed in the presence of glutamine), which decreases when oxygen becomes available to fuel TCA cycle activity, as monitored by glucose-derived glutamate synthesis. The studies further indicate an impaired TCA cycle activity in 67NR cells, reflected in a high accumulation rate of glucose- or glutamine-derived succinate (significantly higher compared to 4T1 cells). This is consistent with the mitochondrial functional analysis, showing higher mitochondrial TCA activity in 4T1 cells than in 67NR, most likely due to impairment of succinate dehydrogenase (SDH, respiratory Complex II) in the latter. CONCLUSIONS. Our results support the association between increased mitochondrial metabolism and metastatic potential, observed recently. We are investigating the SDH status in these cells lines and in additional metastatic and non-metastatic breast cancer cell lines, to establish whether SDH, and glutamine metabolism, could be potential targets for therapy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-01-01.

The lectin Helix pomatia agglutinin recognizes O-GlcNAc containing glycoproteins in human breast cancer

There has been considerable interest in understanding the epitopes that bind the lectin Helix pomatia agglutinin (HPA) in breast cancer as the lectin has been shown to identify glycosylation changes associated with the development of metastatic disease. HPA has previously been shown to recognize aberrant O-linked α-N-acetylgalactosamine (GalNAcα)/mucin glycosylation in cancer, including exposed Tn epitopes. However, recent glycan-array analysis reported that diverse epitopes are also recognized by the lectin, e.g. consortium for functional glycomics (CFG) data: GalNAcα1,3Gal; β-GalNAc; GlcNAcβ1,4Gal. The intriguing observations from the CFG array led to this study, in which HPA-binding epitopes were localized and characterized in an in vitro model of breast cancer metastasis. HMT3522 (benign disease), BT474 (primary cancer) and T47D/MCF7 (metastatic cancer) cells were assessed in confocal microscopy-based co-localization studies and a glycoproteomic analysis based on 2-dimensional electrophoresis (2DE), western blotting and mass spectrometry was adopted. HPA binding correlated with levels of integrin α6, transcription factors heterogeneous nuclear ribonuclear protein (HnRNP) H1, HnRNP D-like, HnRNP A2/B1 as well as heat shock protein 27 (Hsp27), glial fibrillary acidic protein and enolase 1 (ENO1). These glycoproteins were non-detectable in the non-metastatic breast cancer cell lines. The recognition of HnRNPs, Hsp27 and ENO1 by HPA correlated with O-GlcNAcylation of these proteins. Integrin α6 was the most abundant HPA glycoprotein in the breast cancer cells with a metastatic phenotype; this concurred with previous findings in colorectal cancer. This is the first report in which HPA has been shown to bind O-GlcNAcylated transcription factors. This class of proteins represents a new means by which HPA differentiates cancer cells with an aggressive metastatic phenotype.

Expression of metastasis suppressor gene maspin in breast cancer brain metastases

20057 Background: Recently our demonstrated that the expression of several Metastasis Suppressor Genes (MSG) is reduced in breast cancer brain metastases. Current data suggest that MSG Maspin (serpin B5) is a promising candidate for further treatment options. So, we examined the mRNA and protein expression of Maspin in normal breast tissue, breast cancer primaries, -brain metastases and breast cancer cell lines. Methods: Maspin mRNA expression was examined by real time reverse transcription polymerase chain reaction (RT-PCR) in fresh frozen samples from normal breast tissue, breast cancer primaries and -brain metastases. Furthermore maspin was examined in poorly invasive and non-metastatic breast cancer cell lines MCF-7, T47-D, in highly invasive and metastatic MDA-MB-231 breast cancer cells (231-parental) and in brain- and bone-selective metastatic clones (231-brain, 231-bone). Maspin protein was detected by immunohistochemistry in paraffin-embedded sections from 16 patients with breast cancer primaries and brain metastases using a specific monoclonal mouse antibody. Results: In relation to normal breast tissue, maspin mRNA expression was decreased in primary tumors and again decreased in brain metastases. Normalized ΔCT values were 1 (normal tissue), 0.3 (primary tumors) and 0.13 (brain metastases). Immunohistochemistry revealed the same tendency. 3 of 16 (19%) breast cancer primaries were positive whereas none of the brain metastases showed positive maspin staining. In comparison to poorly invasive breast cancer cell lines, maspin mRNA expression was decreased in 231-parental, increased in 231-brain and not detectable in 231-bone. Conclusions: Maspin expression is reduced in breast cancer brain metastases. It is differentially expressed in brain- and bone-selective metastatic cell lines. These results suggest an important role for maspin in breast cancer brain metastasis. No significant financial relationships to disclose.

Soyasaponin-I-modified invasive behavior of cancer by changing cell surface sialic acids

Objective Sialylation involving tumor formation and invasive behavior goes along with altered sialyltransferase (ST) activity. A potent ST inhibitor, soyasaponin I (SsaI), was discovered to selectively inhibit the cellular α2,3-sialyltranserase activity. In this study, we further test the effects of SsaI on modifying the metastatic and invasive behaviors of cancer cell lines. Methods Nonmetastatic breast cancer cell line, MCF-7, and highly metastastic breast cancer cell line, MDA-MB-231, were used to investigate the effects of SsaI on tumor cells. Results SsaI did not affect cell growth cycle and also failed to inhibit cell growth in this study (the concentration of SsaI ≤100 μM). SsaI was as predicted to successfully inhibit cellular α2,3-ST activity and depressed the dose-dependent tumor cell surface α2,3-sialic acid expression. In addition, different concentrations of SsaI did stimulate MCF-7 cell adhesion to collagen type I linearly and significantly enhanced cell adhesion to the Matrigel-matrix. Furthermore, SsaI significantly decreased MDA-MB-231 cell migration. Reverse transcriptase polymerase chain reaction for evaluating mRNA expression of ST3Gal I, III and IV showed that SsaI also down-regulated the expression of ST3Gal IV but did not affect the other two. Conclusions The results showed that SsaI was implicated in the invasive behavior of tumor cells, suggesting that altered α2,3-sialylation pathway played a crucial role in the adhesion and tumor metastases. SsaI is a good candidate for studying the biological roles of ST, and might provide a new preventive strategy in tumor metastasis.