Population Of Breast Cancer Cells Research Articles

Nicotine reduces effectiveness of doxorubicin chemotherapy and promotes CD44+CD24- cancer stem cells in MCF-7 cell populations.

Breast cancer is the most common type of cancer in females and the second most common cause of cancer mortality after lung cancer. Cancer stem cells represent a novel approach to target cancer and reduce cancer recurrence and metastasis. Many patients with breast cancer continue to smoke after receiving their diagnosis. Nicotine is a key factor in tobacco addiction and also changes some cellular functions, such as activation of mitogenic pathways, angiogenesis and cell proliferation. In the present study, the impact of nicotine was assessed in a population of MCF-7 human breast cancer cells. Cluster of differentiation (CD)44+CD24− cancer stem cell population of MCF-7 cells were evaluated using flow cytometry and scanning electron microscopy. Chemoresistance effects of nicotine were demonstrated in these cells. These findings demonstrated harmful effects of nicotine following metastasis of cancer, owing to the chemoresistance produced through uninterrupted smoking, which may impact the effectiveness of treatment.

Glioma-Associated Oncogene Homolog Inhibitors Have the Potential of Suppressing Cancer Stem Cells of Breast Cancer.

Overexpression of Sonic Hedgehog signaling (Shh) pathway molecules is associated with invasiveness and recurrence in breast carcinoma. Therefore, inhibition of the Shh pathway downstream molecule Glioma-associated Oncogene Homolog (Gli) was investigated for its ability to reduce progression and invasiveness of patient-derived breast cancer cells and cell lines. Human primary breast cancer T2 cells with high expression of Shh signaling pathway molecules were compared with breast cancer line MDA-MB-231 cells. The therapeutic effects of Gli inhibitors were examined in terms of the cell proliferation, apoptosis, cancer stem cells, cell migration and gene expression. Blockade of the Shh signaling pathway could reduce cell proliferation and migration only in MDA-MB-231 cells. Hh pathway inhibitor-1 (HPI-1) increased the percentages of late apoptotic cells in MDA-MB-231 cells and early apoptotic cells in T2 cells. It reduced Bcl2 expression for cell proliferation and increased Bim expression for apoptosis. In addition, Gli inhibitor HPI-1 decreased significantly the percentages of cancer stem cells in T2 cells. HPI-1 worked more effectively than GANT-58 against breast carcinoma cells. In conclusion, HPI-1 could inhibit cell proliferation, reduce cell invasion and decrease cancer stem cell population in breast cancer cells. To target Gli-1 could be a potential strategy to suppress breast cancer stem cells.

HER2 Overexpression Triggers an IL1α Proinflammatory Circuit to Drive Tumorigenesis and Promote Chemotherapy Resistance.

Systemic inflammation in breast cancer correlates with poor prognosis, but the molecular underpinnings of this connection are not well understood. In this study, we explored the relationship between HER2 overexpression, inflammation, and expansion of the mammary stem/progenitor and cancer stem-like cell (CSC) population in breast cancer. HER2-positive epithelial cells initiated and sustained an inflammatory milieu needed to promote tumorigenesis. HER2 induced a feedforward activation loop of IL1α and IL6 that stimulated NFκB and STAT3 pathways for generation and maintenance of breast CSC. In mice, Il1a genetic deficiency delayed MMTV-Her2-induced tumorigenesis and reduced inflammatory cytokine expression as well as CSC in primary tumors. In clinical specimens of human breast tumor tissues, tissue microarray analysis revealed a strong positive correlation between IL1α/IL6 expression and CSC-positive phenotype. Pharmacologic blockade of IL1α signaling reduced the CSC population and improved chemotherapeutic efficacy. Our findings suggest new therapeutic or prevention strategies for HER2-positive breast cancers.Significance: IL1α signaling driven by HER2 promotes chronic inflammation needed to support cancer stem-like cell maintenance in HER2-positive breast cancers. Cancer Res; 78(8); 2040-51. ©2018 AACR.

ElevatedCRB3 expression suppresses breast cancer stemness by inhibiting β‐catenin signalling to restore tamoxifen sensitivity

Tamoxifen is a first‐line drug for hormone therapy (HT) in oestrogen receptor‐positive breast cancer patients. However, 20% to 30% of those patients are resistant to tamoxifen treatment. Cancer stem cells (CSCs) have been implicated as one of the mechanisms responsible for tamoxifen resistance. Our previous study indicated that decreased expression of the CRB3 gene confers stem cell characteristics to breast cancer cells. In the current investigation, we found that most of the breast cancer patient tissues resistant to tamoxifen were negative for CRB3 protein and positive for β‐catenin protein, in contrast to their matched primary tumours by immunohistochemical analysis. Furthermore, expression of CRB3 mRNA and protein was low, while expression of β‐catenin mRNA and protein was high in tamoxifen resistance cells (LCC2 and T47D TamR) contrast to their corresponding cell lines MCF7 and T47D. Similarly, CRB3 overexpression markedly restored the tamoxifen sensitivity of TamR cells by the MTT viability assay. Finally, we found that CRB3 suppressed the stemness of TamR cells by inhibiting β‐catenin signalling, which may in turn lead to a decrease in the breast cancer cell population. Furthermore, these findings indicate that CRB3 is an important regulator for breast cancer stemness, which is associated with tamoxifen resistance.

The breast cancer antigen 5T4 interacts with Rab11, and is a target and regulator of Rab11 mediated trafficking.

5T4 is a transmembrane glycoprotein with limited expression in normal adult tissues and expression in some solid tumours. It is unclear whether 5T4 is preferentially expressed by stem or differentiated cell types. Modes of 5T4 regulation are unknown despite its ongoing development as a cancer immunotherapy target. Our aims were to clarify the differentiation status of 5T4 expressing cells in breast cancer and to understand the mechanism underlying 5T4 membrane presentation. We analysed 5T4 expression in breast cancer cell populations by flow cytometery and found that 5T4 is highly expressed on differentiated cells, where it localizes to focal adhesions. Using immunoprecipitation and mass spectrometry, we identified interactions between 5T4 and the membrane trafficking proteins Rab11, Rab18 and ARF6. Mechanistically we found that Rab11 and Rab18 have oppositional roles in controlling expression and surface presentation of 5T4. 5T4 depletion stabilizes Rab11 protein expression with a consequent stimulation transferrin surface labelling, indicating that 5T4 represses endocytic activity. Successful immunotherapeutic targeting of 5T4 requires surface presentation and different immunotherapy strategies require surface presentation versus endocytosis. While breast cancer cells with high 5T4 surface expression and rapid cell surface turnover would be susceptible to antibody-drug conjugates that rely on intracellular release, 5T4 positive cells with lower expression or lower turnover may still be responsive to T-cell mediated approaches. We find that endocytosis of 5T4 is strongly Rab11 dependent and as such Rab11 activity could affect the success or failure of 5T4-targetted immunotherapy, particularly for antibody-drug conjugate approaches. In fact, 5T4 itself represses Rab11 expression. This newly uncovered relationship between Rab11 and 5T4 suggests that breast tumours with high 5T4 expression may not have efficient endocytic uptake of 5T4-targetted immunotherapeutics. This should be considered when selecting amongst the different types of immunotherapies.

Post-translational modification of OCT4 in breast cancer tumorigenesis

Recurrence and drug resistance of breast cancer are still the main reasons for breast cancer-associated deaths. Cancer stem cell (CSC) model has been proposed as a hypothesis for the lethality of breast cancer. Molecular mechanisms underlying CSC maintenance are still unclear. In this study, we generated mammospheres derived from breast cancer MDA-MB231 cells and MCF7 cells to enrich CSCs and performed DNA microarray analysis. We found that the expression of carboxy terminus of HSP70-interacting protein (CHIP) E3 ubiquitin ligase was significantly downregulated in breast CSCs. CHIP depletion increased mammosphere formation, whereas CHIP overexpression reversed this effect. We identified interactomes by mass spectrometry and detected CHIP directly interacted with OCT4, a stemness factor. CHIP overexpression decreased OCT4 stability through proteasomal degradation. CHIP induced OCT4 ubiquitination, whereas H260Q, a catalytic CHIP mutant, did not. Interestingly, we determined that OCT4 was ubiquitinated at lysine 284, and CHIP overexpression did not degrade K284R mutant OCT4. CHIP overexpression decreased the proliferation and side population of breast cancer cells, but these were not occurred in K284R mutant OCT4 overexpressed cells. Only 1000 cells showing CHIP depletion or OCT4 overexpression sufficiently generated breast tumors and lung metastases in xenografted mice. Ubiquitination-defective mutant of OCT4(K284R) overexpressed cells drastically generated tumor burdens in mice. Patients with breast cancer who showed low CHIP expression had poor survival probability. Taken together, we suggest that CHIP-induced OCT4 ubiquitination is important in breast CSCs. Regulation of CHIP expression and OCT4 protein stability is a considerable approach for breast cancer therapy.

Apoptosis Reversal Promotes Cancer Stem Cell-Like Cell Formation

It has long been a puzzle in cancer treatment that despite the initial appearance of apoptosis, the process could be reversed in some cancer cells and often results in more aggressive tumors and metastasis. The mechanism for this recurrence is yet unknown. Here we report that human mammary carcinoma cells induced to undergo apoptosis could recover with increased tumorigenicity in vitro and in vivo, and induced lymph node metastasis. Specifically, the reversed cells underwent epithelial-to-mesenchymal transitions in the primary tumors in situ, and mesenchymal-to-epithelial transitions in the metastatic cells. Flow cytometry confirmed an elevated percentage of cells carrying cancer stem cells (CSCs) markers (CD44+/CD24-) in the reversed breast cancer cell population, with hypomethylated CD44 promoters and hypermethylated CD24 promoters. More importantly, CSCs were generated anew from non-stem cancer cells after apoptosis reversal possibly through epigenetic modifications. The results from this study can open doors to discovering more effective cancer treatments by suppressing apoptosis reversal.

Abstract CN08-02: Molecular detection and characterization of circulating tumor cells

Abstract Circulating tumor cells (CTCs) are shed from primary and metastatic tumor deposits into the bloodstream, where a small fraction may survive and ultimately give rise to distant lesions. As such, analysis of CTCs provides a noninvasive strategy to repeatedly sample tumor cells during the course of cancer progression and evolution, as well as to better understand the process of cancer metastasis. In addition, the early shedding of CTCs into the blood by localized but invasive cancers may present an opportunity for earlier detection and treatment of cancers, before metastases are established. We have previously described a microfluidic negative depletion platform, the CTC-iChip, which efficiently depletes blood specimens of normal hematopoietic cells, leaving behind untagged and unmanipulated CTCs. This technology, which is now automated, allows for enrichment of tumor cells from blood samples, without bias based on their expression of epithelial or other cell surface markers. Single-cell sequencing of CTCs captured in this way demonstrates high-quality RNA reads, enabling detailed molecular characterization, and some CTCs are viable, expanding in vitro to generate tumorigenic cell lines. In characterizing such cell lines derived from the blood of women with hormone receptor-positive breast cancer, we have identified both acquired treatment-associated mutations, as well as cell fate changes that may be of epigenetic origin. Among these, the acquisition of HER2 expression by initially HER2-negative breast cancer cells signals the emergence of a highly proliferative albeit chemotherapy-susceptible cell population, which appears to coexist and even interconvert with a less proliferative Notch1-dependent drug-resistant population. The interplay between these distinct cell populations in advanced hormone receptor-positive breast cancer and its impact on acquired drug resistance can be modeled using patient CTC-derived cultured cell lines. Finally, the intact CTCs isolated from patient blood samples using negative depletion microfluidic technologies also enable high-sensitivity, RNA-based digital PCR scoring of rare cancer cells within a blood specimen. Such highly quantitative and automatable analyses provide rapid measurements of tumor-specific burden from blood samples, before and after therapeutic intervention, and enable interrogation of intracellular signaling pathways that are targeted by drug treatment. In breast cancer, CTC-based scoring of estrogen receptor signaling pathways provides serial noninvasive monitoring of a major tumor driver, with implications for treatment selection. Taken together, technological advances in the isolation and analysis of CTCs present opportunities for cancer cell-based monitoring of breast cancer and other malignancies, in which distinct treatment options need to be molecularly targeted for individualized therapy. Citation Format: Daniel A. Haber. Molecular detection and characterization of circulating tumor cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr CN08-02.

Sox2 Communicates with Tregs Through CCL1 to Promote the Stemness Property of Breast Cancer Cells.

As an important component of the tumor microenvironment, CD4+ CD25+ Tregs reduce antitumor immunity, promote angiogenesis and metastasis in breast cancer. However, their function in regulating the "stemness" of tumor cells and the communication between Tregs and cancer stem cells (CSCs) remain elusive. Here, we disclose that the primarily cultured Tregs isolated from breast-tumor-bearing Foxp3-EGFP mouse upregulate the stemness property of breast cancer cells. Tregs increased the side-population and the Aldehyde dehydrogenase-bright population of mouse breast cancer cells, promoted their sphere formation in a paracrine manner, and enhanced the expression of stemness genes, such as Sox2 and so forth. In addition, Tregs increased tumorigenesis, metastasis, and chemoresistance of breast cancer cells. Furthermore, Sox2-overexpression tumor cells activated NF-κB-CCL1 signaling to recruit Tregs through reducing the binding of H3K27Me3 on promoter regions of p65 and Ccl1. These findings reveal the functional interaction between Tregs and CSCs and indicate that targeting on the communication between them is a promising strategy in breast cancer therapy. Stem Cells 2017;35:2351-2365.

IL6 blockade potentiates the anti-tumor effects of γ-secretase inhibitors in Notch3-expressing breast cancer.

Notch pathways have important roles in carcinogenesis including pathways involving the Notch1 and Notch2 oncogenes. Pan-Notch inhibitors, such as gamma secretase inhibitors (GSIs), have been used in the clinical trials, but the outcomes of these trials have been insufficient and have yielded unclear. In the present study, we demonstrated that GSIs, such as MK-0752 and RO4929097, inhibit breast tumor growth, but increase the breast cancer stem cell (BCSC) population in Notch3-expressing breast cancer cells, in a process that is coupled with IL6 induction and is blocked by the IL6R antagonist Tocilizumab (TCZ). IL6 induction results from inhibition of Notch3-Hey2 signaling through MK-0752. Furthermore, HIF1α upregulates Notch3 expression via direct binding to the Notch3 promoter and subsequently downregulates BCSCs by decreasing the IL6 levels in Notch3-expressing breast cancer cells. Utilizing both breast cancer cell line xenografts and patient-derived xenografts (PDX), we showed that the combination of MK-0752 and Tocilizumab significantly decreases BCSCs and inhibits tumor growth and thus might serve as a novel therapeutic strategy for treating women with Notch3-expressing breast cancers.

Abstract 2890: MicroRNA-mediated upregulation of the WNT signaling activities in human breast cancer stem cells

Abstract The canonical WNT signaling plays a critical role in many adult stem cells, including those of the breast and intestine. The fact that the canonical WNT signaling is implicated in both stem cell self-renewal and cancer suggests that normal physiological regulator of stem cell functions might be “hijacked” in cancer. Adenomatous polyposis coli (APC) is a component of the destruction complex that destabilizes β-catenin and suppresses the activity of the canonical WNT signaling. MicroRNAs (miRNAs) are important regulators of stem cell functions. We have previously reported a set of 37 miRNAs that are upregulated or downregulated in human breast cancer stem cells (BCSCs, a CD44+CD24-/lowlineage- population of human breast cancer cells) as compared to non-tumorigenic breast cancer cells (NTCs). Among them, miR-200c targets BMI1 that is a critical regulator of the stem cell maintenance, and strongly impairs the functions of human BCSCs in vivo. In this study, we compared the expression profiles of miRNAs, mRNAs and proteins between BCSCs and NTCs isolated from the patient specimens of human breast cancers and patient-derived tumor xenografts (PDXs) established by their transplantation. Luciferase assays were performed using the plasmid in which the 3’UTR region of candidate mRNA was cloned downstream of a luciferase minigene. The effect of miRNAs on the activity of WNT signaling was evaluated using a TCF reporter plasmid. Finally, the abilities to form organoids and to form tumors in immunodeficient mice were evaluated using the human BCSCs infected with the miRNA inhibitor expressing lentivirus. We found that miR-142 was highly upregulated in BCSCs, but was hardly expressed in NTCs in the patient breast cancer specimens. We confirmed that miR-142 targeted the sequence within the 3’UTR of APC mRNA and suppressed APC protein expression. Accordingly, miR-142 activated the canonical WNT signaling pathway in an APC-suppression dependent manner. The results of mRNA and protein expression profiling of the BCSCs isolated from human breast cancer PDXs suggested that the canonical WNT signaling was activated in BCSCs. Finally, inhibition of miR-142 in the BCSCs suppressed the tumor growth in vivo. These results suggest that the miR-142, a miRNA frequently upregulated in human BCSCs, could provide at least a part of the molecular mechanism for aberrant activation of the canonical WNT signaling in breast cancer in which APC mutations are much less frequent than colon cancer. Citation Format: Yohei Shimono, Taichi Isobe, Andrei Turtoi, Junko Mukohyama, Toru Mukohara, Akira Suzuki, Vincent Castronovo, Hironobu Minami. MicroRNA-mediated upregulation of the WNT signaling activities in human breast cancer stem cells [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 2890. doi:10.1158/1538-7445.AM2017-2890

Abstract 4074: Permeability changes and effect of chemotherapy in brain adjacent to tumor in an experimental model of brain metastases of breast cancer

Abstract Background: Brain metastases vasculature is compromised and more permeable than that of vasculature in normal brain. Unfortunately, little is known about vascular permeability changes in brain adjacent to tumor (BAT). It is critical to understand the potential changes as chemotherapy penetration and accumulation brain tumors positively correlate with breakdown of the brain vasculature. Herein, we evaluate quantitatively measure permeability, drug accumulation and cytotoxicity in normal brain that is adjacent (<500 microns) to a tumor. Methods: The subclone population of brain seeking metastatic breast cancer cells (MDA-MB-231Br) were injected into the left ventricle of female NuNu mice. Mice were given a chemotherapeutic regimen of vinorelbine (10mg/kg), erubilin (1.5mg/kg) or docetaxel (10mg/kg). Progression occurred until neurological compromise was noted, at which time animals were injected with texas red 625 Da(TR) and texas red dextran 3kDa (TRD). Dye concentrations (measuring passive permeability changes) were determined in lesions and the BAT using quantitative fluorescent microscopy. Chemotherapeutic induced cytotoxicity was observed by staining for activated astrocytes and DNA damage in neurons, glia and endothelia. Results: We observed permeability of TR is highly heterogeneous both within and between the tumors (normal brain Kin TR: 1.2 x10-5)with 2-40-fold increase in permeability over normal brain region. TR concentrations in BAT (100µm from tumor edge) is consistently 40% of the tumor but significantly higher than that of normal brain and TR permeability in BAT increased 1-2.5-fold over normal brain. The permeability of TRD in tumor increased 2.7- 8.4-fold over normal brain, whereas in BAT, the permeability increased 1-1.6-fold over normal brain. We also observed animals treated with chemotherapy showed activated astrocytes in BAT, along with some cellular death. In vehicle treated animals there were little activated astrocytes and little cell death in the BAT. Conclusions: Our data suggests that the tracer and drugs accumulate in regions around tumors in the brain which result in neurotoxic changes. Citation Format: Afroz Shareef Mohammad, Chris E. Adkins, Neal Shah, Rawaa Aljammal, Jessica Griffith, Paul R. Lockman. Permeability changes and effect of chemotherapy in brain adjacent to tumor in an experimental model of brain metastases of 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 4074. doi:10.1158/1538-7445.AM2017-4074

Abstract 1144: Dual inhibition of MEK1/2 and PLK1 specifically targets aggressive breast cancer cell population with CEP55 elevated expression

Abstract Triple negative breast cancers (TNBCs), lacking the expression of ER, PR and HER2 amplification, are the most aggressive form of breast cancer (BC). Due to their heterogeneity influenced by high level of genomic instability and aneuploidy, treatment of TNBC patients is one of the biggest challenges faced in the clinics. CEP55, discovered first by our laboratory, is a key regulator of cytokinesis malfunction of which has been shown to cause multinucleation. Furthermore, ERK2/PLK1 critically regulates the functional role of CEP55 via phosphorylation of CEP55 at particular stages of mitosis, allowing it to localize to the midbody for accurate cytokinesis. Research has demonstrated association of CEP55 with various cancers especially BC as over-expression of CEP55 mRNA is associated with worse BC prognosis and poor survival. We hypothesized that, CEP55 regulates the fate of aneuploid cell population, which are highly dependent on mitotic genes for tumor progression among aggressive BC, thus can be targeted for therapy development. We have performed a series of in vitro studies demonstrating that depletion of CEP55 sensitizes cells to anti-mitotic drugs like PLK1 inhibitor (BI2536) and leads to unscheduled CDK1/Cyclin B activation and favor CDK1-Caspase 3-dependent mitotic catastrophe. We also demonstrate that ERK1/2 transcriptionally controls CEP55 mRNA and due to lack of a specific small molecule inhibitor against CEP55, inhibition of MEK1/2 using the small molecule inhibitor Selumetinib, can mimic depletion of CEP55 in vivo. Thus, we rationalized the usage of a MEK1/2 inhibitor in combination with a PLK1 inhibitor across a series of BC cell lines. We observed synthetic lethality among the aggressive hormone receptor negative cell lines, which expressed higher expression of CEP55 compared to normal like and hormone receptor positive BC cell lines with lower CEP55 level. The combination synergistically increased apoptosis of aneuploid population via premature entry of these cells into mitosis in the presence of antimitotic drugs due to exhaustion of CEP55. We have also validated this synergistic effect of MEK1/2 and PLK1 inhibition using xenograft models, results of which imitated the in vitro findings. Therefore, we propose a novel treatment strategy of MEK1/2-PLK1 dual combination for selectively targeting CEP55 over-expressing BC in the clinics. Citation Format: Debottam Sinha, Murugan Kalimutho, J. Alejandro Lopez, Kum Kum Khanna. Dual inhibition of MEK1/2 and PLK1 specifically targets aggressive breast cancer cell population with CEP55 elevated expression [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 1144. doi:10.1158/1538-7445.AM2017-1144

Natural and synthetic progestins enrich cancer stem cell-like cells in hormone-responsive human breast cancer cell populations in vitro.

Clinical trials and studies have shown that combination estrogen/progestin hormone replacement therapy, but not estrogen therapy alone or placebo, increases breast cancer risk in postmenopausal women. Using animal models, we have previously shown that both natural and synthetic progestins (including medroxyprogesterone acetate [MPA], a synthetic progestin used widely in the clinical setting) accelerate the development of breast tumors in vivo and increase their metastasis to lymph nodes. Based on these observations, we have hypothesized that progestin-induced breast cancer tumor growth and metastasis may be mediated by an enrichment of the cancer stem cell (CSC) pool. In this study, we used T47-D and BT-474 hormone-responsive human breast cancer cells to examine the effects of progestin on phenotypic and functional markers of CSCs in vitro. Both natural and synthetic progestins (10 nM) significantly increased protein expression of CD44, an important CSC marker in tumor cells. MPA increased the levels of both CD44 variants v3 and v6 associated with stem cell functions. This induction of CD44 was blocked by the antiprogestin RU-486, suggesting that this process is progesterone receptor (PR) dependent. CD44 induction was chiefly progestin dependent. Because RU-486 can bind other steroid receptors, we treated PR-negative T47-DCO-Y cells with MPA and found that MPA failed to induce CD44 protein expression, confirming that PR is essential for progestin-mediated CD44 induction in T47-D cells. Further, MPA treatment of T47-D cells significantly increased the activity of aldehyde dehydrogenase (ALDH), another CSC marker. Finally, two synthetic progestins, MPA and norethindrone, significantly increased the ability of T47-D cells to form mammospheres, suggesting that enrichment of the CD44high, ALDHbright subpopulation of cancer cells induced by MPA exposure is of functional significance. Based on our observations, we contend that exposure of breast cancer cells to synthetic progestins leads to an enrichment of the CSC pool, supporting the development of progestin-accelerated tumors in vivo.

Low Intensity Ultrasound Induced Apoptosis in MCF-7 Breast Cancer Cell Lines

According to the World Cancer Research Fund International (WCRFI), breast cancer is the most common type of cancer in women worldwide with recorded 1.7 million new cases in 2012. The main line of treatments is still limited to chemotherapy, surgery and radiotherapy which could lead to a wide range of dangerous side effects. This study was conducted to evaluate the effect of low intensity ultrasound (LIUS) on cell proliferation, percentage of living and dead cells and the induction of apoptosis on the MCF-7 cell line with CHO cells as the control for non-cancerous group. In order to achieve the objective of this study, several methods of cell-bioguided assays were used including the MTT assay for cell proliferation, Live/Dead assay for the determination of both live and dead cells and gene expression study for the detection of apoptosis in the cells. The cytotoxicity and Live/Dead assays data provided preliminary data that the LIUS has potential to induce apoptosis in a wide population of breast cancer cells. Furthermore, the LIUS treatment induced the expression of p53-mRNA at a detectable level via qPCR analysis, indicating the activation of apoptosis. In short, our study suggested LIUS dosage used in this study could potentially show positive effects in the induction of apoptosis selectively on the MCF-7 with less harm to the control CHO cells.

Impact of Environmental Pollutant Cadmium on the Establishment of a Cancer Stem Cell Population in Breast and Hepatic Cancer.

Cadmium, a heavy metal pollutant, causes cancer. The existence of cancer stem cells (CSCs) in tumors is widely considered to be the reason for the recurrence and treatment failure of cancer. Increasing evidence has confirmed that under certain conditions non-CSCs could be converted into CSCs. The impact of cadmium on the development of CSC lineage in the bulk tumor cell population is not yet studied. The aim of this study was to evaluate the effect of cadmium on the conversion of non-CSCs to CSCs and the identification of CSCs based on the concurrent monitoring of multiple CSC markers. High-content monitoring of molecular markers was performed using quantum dot (QD) nanoprobes and an acousto-optical tunable filter (AOTF)-based imaging device. Cadmium treatment significantly increased the CSC population in MCF-7 and HepG2 cell lines. The cadmium-induced CSCs were identified by a concurrent analysis of stem-cell markers, namely, CD44, CD24, CD133, and ALDH1. Moreover, increased m-RNA expression of CD44, ALDH1, and CD133 and protein expression of p-Ras, p-Raf-1, p-MEK-1, and p-ERK-1 were observed in the cadmium-treated MCF-7 and HepG2 cells. This study demonstrates that cadmium induces the gene expression of CSC markers in the breast and liver cancer cell lineage and promotes the conversion of non-CSCs to CSCs.

Abstract P3-07-14: Targeting ERR-α regulated lactate metabolism eliminates drug-resistant breast cancer cells

Abstract Novel therapeutic strategies to eliminate chemo-resistant tumour cells responsible for the development of secondary lesions are essential in order to prevent breast cancer relapse. We have developed an in vitro model system enabling isolation of a putative metastasis-initiating breast cancer cell sub-population with a mitotically quiescent, drug-resistant phenotype. We hypothesise that this population is able to utilise oestrogen-related receptor-alpha (ERR-α)-regulated oxidative lactate metabolism and that inhibition of this critical survival pathway will result in elimination of tumour cells that have survived anti-cancer therapies. Flow cytofluormetric monitoring of Vybrant® DiD retention identified a mitotically quiescent sub-population (~0.05%) in MDA-MB-231 human breast cancer cells grown under high glucose (11mM) conditions. DiD-retaining cells accumulated in the G2/M phase of the cell cycle and were shown to be non-senescent following cytochemical analysis of β-galactosidase activity. Cytotoxicity assays with doxorubicin (0.40μM for 72 hours) demonstrated increased survival in the quiescent fraction (40.76%) compared with the rapidly dividing bulk cell population (1.63%, P ≤ 0.0001). Isolated drug-resistant cells contained a sub-fraction (~1%) that was able to form new clonal populations following cessation of treatment. Mitotically quiescent cells were cultured under high glucose (11mM) or glucose-depleted conditions with high lactate (22mM) in the presence of ERR-α inhibitors Cpd29 (10μM) or XCT790 (10μM). Colony formation was completely eliminated in Cpd29- and XCT790-treated samples compared to untreated controls. Drug-resistant sub-clones were isolated from the quiescent population following 72 hours treatment with doxorubicin (0.40μM) and were cultured under glucose-depleted conditions with high lactate (22mM) in the presence of ERR-α inhibitors Cpd29 (10μM) or XCT790 (10μM). In both instances, colony formation was completely prevented. We provide the first evidence that blocking cellular energy production through inhibition of ERR-α regulated oxidative lactate metabolism can eradicate a chemo-resistant, quiescent breast cancer cell population. Our data suggest that ERR-α inhibitors may be used in combination with chemotherapy to eliminate minimal residual disease and reduce breast cancer recurrence. Citation Format: Quayle L, Park S, McDonnell DP, Ottewell PD, Holen I. Targeting ERR-α regulated lactate metabolism eliminates drug-resistant breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-14.

Stress granule-associated protein G3BP2 regulates breast tumor initiation

Breast tumors contain tumorigenic cancer cells, termed "tumor-initiating cells" (TICs), which are capable of both replenishing themselves and giving rise to populations of nontumorigenic breast cancer cells (non-TICs). However, the molecular mechanisms responsible for breast tumor initiation remain poorly understood. Here we describe a chemical screening strategy to identify small molecules that enhance the effect of chemotherapeutic agents on TIC-enriched breast cancer cells. We identified proteins that interact with the lead compound C108, including the stress granule-associated protein, GTPase-activating protein (SH3 domain)-binding protein 2, G3BP2. G3BP2 regulates breast tumor initiation through the stabilization of Squamous cell carcinoma antigen recognized by T cells 3 (SART3) mRNA, which leads to increased expression of the pluripotency transcription factors Octamer-binding protein 4 (Oct-4) and Nanog Homeobox (Nanog). Our findings suggest that G3BP2 is important for the process of breast cancer initiation. Furthermore, these data suggest a possible connection between stress granule formation and tumor initiation in breast cancer cells.

Laminin Modulates the Stem Cell Population in LM05-E Murine Breast Cancer Cells through the Activation of the MAPK/ERK Pathway.

PurposeWe investigated the effects of laminin on the fraction of cells with self-renewing capacity in the estrogen-dependent, tamoxifen-sensitive LM05-E breast cancer cell line. We also determined whether laminin affected the response to tamoxifen.Materials and MethodsThe LM05-E breast cancer cell line was used as a model for all experiments. Aldehyde dehydrogenase (ALDH) activity, clonogenic and mammosphere assays were performed to measure the effects of laminin on modulation of the stem cell subpopulation. Pluripotent gene expression was analyzed by reverse transcriptase–polymerase chain reaction. The involvement of the mitogen-activated protein kinase (MAPK)/ERK pathway was determined using specific inhibitors. The effects of laminin on the response to tamoxifenwere determined and the involvement of α6 integrin was investigated.ResultsWe found that pretreatment with laminin leads to a decrease in cells with the ability to form mammospheres that was accompanied by a decrease in ALDH activity. Moreover, exposure of mammospheres to laminin reduced the capacity to form secondary mammospheres and decreased the expression of Sox-2, Nanog, and Oct-4. We previously reported that 4-OH-tamoxifen leads to an increase in the expression of these genes in LM05-E cells. Treatment with signaling pathway inhibitors revealed that the MAPK/ERK pathway mediates the effects of laminin. Finally, laminin induced tamoxifen resistance in LM05-E cells through α6 integrin.ConclusionOur results suggest that the final number of cells with self-renewing capacity in estrogen-dependent breast tumors may result from the combined effects of endocrine treatment and microenvironmental cues.

ErbB2 and p38γ MAPK mediate alcohol-induced increase in breast cancer stem cells and metastasis.

BackgroundBoth epidemiological and experimental studies suggest that excessive alcohol exposure increases the risk for breast cancer and enhances metastasis/recurrence. We have previously demonstrated that alcohol enhanced the migration/invasion of breast cancer cells and cancer cells overexpressing ErbB2/HER2 were more sensitive to alcohol exposure. However, the underlying mechanisms remain unclear. This study was designed to investigate the mechanisms underlying alcohol-enhanced aggressiveness of breast cancer. Cancer stem cells (CSCs) play a critical role in cancer metastasis and recurrence.MethodsWe evaluated the effect of chronic alcohol exposure on mammary tumor development/metastasis in MMTV-neu transgenic mice and investigated the cell signaling in response to alcohol exposure in breast cancer cells overexpressing ErbB2/HER2.Results and discussionChronic alcohol exposure increased breast cancer stem cell-like CSC population and enhanced the lung and colon metastasis in MMTV-neu transgenic mice. Alcohol exposure caused a drastic increase in CSC population and mammosphere formation in breast cancer cells overexpressing ErbB2/HER2. Alcohol exposure stimulated the phosphorylation of p38γ MAPK (p-p38γ) which was co-localized with phosphorylated ErbB2 and CSCs in the mammary tumor tissues. In vitro results confirmed that alcohol activated ErbB2/HER2 and selectively increased p-p38γ MAPK as well as the interaction between p38γ MAPK and its substrate, SAP97. However, alcohol did not affect the expression/phosphorylation of p38α/β MAPKs. In breast cancer cell lines, high expression of ErbB2 and p-p38γ MAPK was generally correlated with more CSC population. Blocking ErbB2 signaling abolished heregulin β1- and alcohol-stimulated p-p38γ MAPK and its association with SAP97. More importantly, p38γ MAPK siRNA significantly inhibited an alcohol-induced increase in CSC population, mammosphere formation and migration/invasion of breast cancer cells overexpressing ErbB2.Conclusionsp38γ MAPK is downstream of ErbB2 and plays an important role in alcohol-enhanced aggressiveness of breast cancer. Therefore, in addition to ErbB2/HER2, p38γ MAPK may be a potential target for the treatment of alcohol-enhanced cancer aggressiveness.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0532-4) contains supplementary material, which is available to authorized users.