Breast Cancer Stem Cell Population Research Articles

Probing the interaction between the histone methyltransferase/deacetylase subunit RBBP4/7 and the transcription factor BCL11A in epigenetic complexes

The transcription factor BCL11A has recently been reported to be a driving force in triple-negative breast cancer (TNBC), contributing to the maintenance of a chemoresistant breast cancer stem cell (BCSC) population. Although BCL11A was shown to suppress γ-globin and p21 and to induce MDM2 expression in the hematopoietic system, its downstream targets in TNBC are still unclear. For its role in transcriptional repression, BCL11A was found to interact with several corepressor complexes; however, the mechanisms underlying these interactions remain unknown. Here, we reveal that BCL11A interacts with histone methyltransferase (PRC2) and histone deacetylase (NuRD and SIN3A) complexes through their common subunit, RBBP4/7. In fluorescence polarization assays, we show that BCL11A competes with histone H3 for binding to the negatively charged top face of RBBP4. To define that interaction, we solved the crystal structure of RBBP4 in complex with an N-terminal peptide of BCL11A (residues 2-16, BCL11A(2-16)). The crystal structure identifies novel interactions between BCL11A and the side of the β-propeller of RBBP4 that are not seen with histone H3. We next show that BCL11A(2-16) pulls down RBBP4, RBBP7, and other components of PRC2, NuRD, and SIN3A from the cell lysate of the TNBC cell line SUM149. Furthermore, we demonstrate the therapeutic potential of targeting the RBBP4-BCL11A binding by showing that a BCL11A peptide can decrease aldehyde dehydrogenase-positive BCSCs and mammosphere formation capacity in SUM149. Together, our findings have uncovered a previously unidentified mechanism that BCL11A may use to recruit epigenetic complexes to regulate transcription and promote tumorigenesis.

Protein tyrosine phosphatase receptor-type δ acts as a negative regulator suppressing breast cancer

Protein tyrosine phosphatase receptor-type δ (PTPRD) is frequently inactivated in human cancers. This study investigated the role of PTPRD in the regulation of stemness, epithelial-mesenchymal transition (EMT), and migration and invasion in breast cancer cells. In vitro, PTPRD silencing using siRNA enhanced the stem cell-like properties of breast cancer cells, including their mammosphere- and holoclone-forming abilities, and it promoted tumorigenicity in vivo. PTPRD knockdown also increased the CD44+/CD24− breast cancer stem cell (BCSC) population and the expression of the stem cell markers ALDH1 and OCT4. It also promoted migration and invasion by breast cancer cell, EMT, and activation of signal transducer and activator of transcription 3 (STAT3). BCSCs expressed low levels of PTPRD, displayed mesenchymal phenotypes, and were more sensitive to IL-6-mediated STAT3 activation than non-BCSCs. PTPRD expression was upregulated by IL-6 in breast cancer cells, thereby establishing a negative feedback circuit by which IL-6 induced canonical STAT3 phosphorylation and transiently upregulated PTPRD, which in turn dephosphorylated STAT3 and prevented downstream signaling via the IL-6/STAT3 cascade. These data suggest that therapies aimed at restoring or enhancing PTPRD expression may be effective in controlling breast cancer progression and metastasis.

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.

Breast cancer stem cells are involved in Trastuzumab resistance through the HER2 modulation in 3D culture.

Breast cancer human cells culture as spheroids develop autophagy and apoptosis, which promotes Trastuzumab resistance in HER2 overexpressing cells. Our aim was to study the association of the hostile environment developed in 3D with the breast cancer stem cells population and the HER2 modulation. Human mammary adenocarcinoma cell lines were cultured as spheroids using the hanging drop method. We generated hypoxia conditions by using a hypoxic chamber and CoCl2 treatment. Breast cancer stem cells were measured with mammosphere assays, the analysis of CD44 + CD24low population by flow cytometry and the pluripotent gene expression by RT-qPCR. HER2 expression was evaluated by flow cytometry and Western blot. MTS assays were conducted to study cell viability. Hostil environment developed in spheroids, defined by hypoxia and autophagy, modulated the response to Trastuzumab. In HER2+ cells with acquired resistance, we observed an increase in the breast cancer stem cell population. In BT474 spheroids, Trastuzumab induced the acquisition of resistance, along with an increase in breast cancer stem cells. Also, in 3D culture conditions we determined a modulation in the HER2 expression. Moreover, breast cancer stem cells showed enhanced HER2 expression. Finally, cells without HER2 gene amplification cultured as spheroids were sensitive to Trastuzumab, diminishing HER2 expression and cancer stem cells. Our findings show that 3D architecture is able to modulate breast cancer stem cell population and HER2 distribution, modifying the cell response to Trastuzumab.

Abstract 5261: Tocopherols inhibit the estrogen-stimulated expansion of cancer stem cells via down-regulation of OCT4 and NFκB

Abstract Cancer stem cells have been suggested to play a role in tumor initiation, maintenance, resistance, and metastasis. Breast cancer stem cells are defined as CD44+/CD24- cell populations that have the properties of tumor-initiating ability, self-renewal and differentiation. Although the importance of estrogen in breast cancer is well established, the effects of estrogen on breast cancer stem cells are not fully understood. Vitamin E, which is consisted of different forms of tocopherols and tocotrienols, has anti-tumor effects in various cancers, including breast cancer. In the present study, we examined the effects of α-, γ-, and δ-tocopherol against estrogen-stimulated expansion of cancer stem cells. To determine the effects of tocopherols on breast cancer stem cells, the MCF-7 tumorsphere cell culture system, which enriches for mammary progenitor cells and putative breast cancer stem cells, was utilized. Treatment of MCF-7 tumorsphere with estrogen for four days resulted in an increase in CD44+/CD24- stem cell populations in tumorspheres as well as the number and size of tumorspheres. Tocopherols at 1 and 10 μM inhibited formation of estrogen-induced stem cell enriched tumorspheres, indicating that tocopherols reversed estrogen-induced expansion of breast cancer stem cells. Tocopherols decreased the expression of estrogen-related genes such as TFF/pS2, cathepsin D, progesterone receptor and SerpinA1 in estrogen-stimulated tumorspheres. In addition, the mRNA levels of stem cell markers, including OCT4, NFκB, SOX-2 and CD44, were upregulated by estrogen, which were downregulated by the treatment with tocopherols. Our results suggest that tocopherols repress the expansion of breast cancer stem cell population by regulating OCT4 and NFκB signaling. Tocopherols could be effective natural agents for the prevention and treatment of estrogen-induced breast cancer. (This work was supported in part by the National Institutes of Health grant R01 AT007036.) Citation Format: Min Ji Bak. Tocopherols inhibit the estrogen-stimulated expansion of cancer stem cells via down-regulation of OCT4 and NFκB [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 5261. doi:10.1158/1538-7445.AM2017-5261

Abstract 1461: miR-424-cdc42, key signaling axis in hyperglycemic regulation of stemness in triple negative breast cancer

Abstract Introduction: Meta-analysis shows women with diabetes have a 20% increased risk of breast cancer. Diabetes increases the risk of breast cancer mortality. Additionally, patients with breast cancer and preexisting diabetes have an increased risk for distant metastasis compared with non-diabetic counterparts. The molecular mechanisms for distant metastasis in breast cancer patients with diabetes are not very well understood. Our preliminary data suggested that hyperglycemia enhances stem cell activity in triple negative breast cancer (TNBC) cells. Here, we report miR-424 to be a key regulator of breast cancer stem cell pool dynamics under hyperglycemic environment. Materials and Methods: MDA-MB-231, TNBC cell line was maintained under euglycemic (5mM) and hyperglycemic (10mM) culture conditions. RT-PCR based microRNA array was performed followed by validation of significantly altered microRNA to confirm the effect of hyperglycemia on the global microRNA profile. Flow cytometric analysis for CD44+/CD24- was performed to assess the alterations in breast cancer stem cell population. In addition, sphere-forming assay were also performed. Mir-424 over-expressing or knocked down cell lines were established from parental MDA-MB-231 cells and maintained in hyperglycemic or euglycemic condition respectively. Promoter analysis for PR/SET domain 14 (prdm14) was done. Western blotting and immunofluorescence assays were performed for cdc42 (cell division control 42), pStat5 (phospho signal transducer and activator of transcription factor 5) and prdm14. Results: Loss of miR-424 in TNBC cells under euglycemic conditions led to enhanced stem cell activity; whereas over expression of miR- 424 under hyperglycemic conditions resulted in suppressed stem cell activity. Introduction of cdc42, (a miR-424 target gene) in miR-424 over expressing TNBC cells under hyperglycemic conditions leads to increased stem cell activity demonstrating the significance of miR-424-cdc42 signaling in hyperglycemia. Over-expression of miR-424 in TNBC dramatically reduced its metastatic abilities in vivo. Mechanistically, we found that miR-424-cdc42 signaling in hyperglycemia promotes Prdm14 activation, a stem cell regulator through increased phosphorylation of Stat5. Conclusions: Our findings establish a key molecular signaling cascade (miR-424→cdc42→prdm14) that promotes TNBC stem cell activity under hyperglycemic conditions. Citation Format: Sushmita B. Nandy, Alexis Orozco, Gautham Prabhakar, Viktoria Stewart, Stephanie Jones, Paloma Munoz, Ramadevi Subramani, Diego Pedroza, Rajkumar Lakshmanaswamy. miR-424-cdc42, key signaling axis in hyperglycemic regulation of stemness in triple negative 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 1461. doi:10.1158/1538-7445.AM2017-1461

Abstract 2885: Activation of IL1α signaling by HER2 overexpression promotes breast cancer stem cells

Abstract Breast cancer is a heterogeneous disease and only a small proportion of cells known as cancer stem cells (CSCs) have ability to generate tumor. Recently, it has been reported that HER2 overexpression increase breast cancer stem cells (BCSCs). However, the underlying mechanism is not clearly known. In this study, we explored the molecular mechanism how HER2 increase BCSCs. Our data has demonstrated HER2 induces BCSCs by indirectly activating NF-kB and STAT3 signaling. To figure out the direct target of HER2, we did the microarray and found that HER2 upregulates the expressions of IL1a and IL6, two major cytokines which directly activates NFkB and STAT3, respectively. Interestingly, we found that IL1a also indirectly activates STAT3. IL1α induced STAT3 was blocked when IL6 was knock out from cells. We found that HER2 enhances IL1α transcription by activating PU.1 which is known transcription factor of IL1a. Finally, we studied effect of IL1a on tumorigenesis, and found that IL1α KO dramatically attenuates the tumor growth in NOD/SCID mice. Taken together, we found that HER2 induces BCSCs by upregulating IL1a and IL6 expression. We for first time demonstrating that IL1α plays a critical role in HER2-induced expansion of BCSC population and may be a potential therapeutic target for HER2 positive breast cancer. Citation Format: Shou Liu, Ji shin Lee, Hexin Chen. Activation of IL1α signaling by HER2 overexpression promotes 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 2885. doi:10.1158/1538-7445.AM2017-2885

Evaluation of Melatonin Effect on Human Breast Cancer Stem Cells Using a Threedimensional Growth Method of Mammospheres.

The high rates of women's death from breast cancer occur due to acquired resistance by patients to certain treatments, enabling the recurrence and/or tumor growth, invasion and metastasis. It has been demonstrated that the presence of cancer stem cells in human tumors, as responsible for recurrence and resistance to therapy. Studies have identified OCT4 as responsible for self-renewal and maintenance of pluripotency of stem cells. Thus, it is interesting to study potential drugs that target this specific population in breast cancer. Melatonin, appears to have oncostatic effects on cancer cells, however, little is known about its therapeutic effect on cancer stem cells. Evaluate the viability and the expression of OCT4 in breast cancer stem cells, MCF-7 and MDA-MB- 231, after melatonin treatment. The cells were grown in a 3-dimensional model of mammospheres, representing the breast cancer stem cell population and treated or not with melatonin. The cell viability of mammospheres were evaluated by MTT assay and the OCT4 expression, a cancer stem cells marker, was verified by immunocitochemistry. Our results demonstrated that the melatonin treatment decreased the cell viability of MCF-7 and MDAMB- 231 mammospheres. Furthermore, it was observed that in both cell lines, the expression of OCT4 was decreased in melatonin-treated cells compared to the control group. This fact suggests that melatonin is effective against breast cancer stem cells inhibiting the cell viability via OCT 4. Based on that, we believe that melatonin has a high potential to be used as an alternative treatment for breast cancer.

The role of TFEB in tumor-associated macrophages

Abstract Alternatively activated (M2-like) tumor-associated macrophages (TAMs) have been shown to promote immune suppression, invasion, angiogenesis, the establishment of distant metastases, and maintain the breast cancer stem cell population. However, the mechanisms governing TAM polarization are not fully understood. Transcription factor EB (TFEB) has been recently investigated due to its role as the master regulator of autophagy, but little is known regarding its role in governing macrophage activation or other immune response pathways. We show here that TFEB transcription and activation are reduced in TAMs, and TGF-β-induced activation of ERK signaling is the driving force behind this phenomenon. siRNA knockdown of TFEB in macrophages significantly enhances their alternative activation. Co-inoculation of these macrophages with breast tumor cells results in an increase of both tumor growth and infiltration of M2-like TAMs. Further mechanistic studies reveal that TFEB knockdown regulates M2 polarization by decreasing activation of SOCS3, which acts as an inhibitor of STAT3. These findings led us to further investigate whether increasing TFEB activation in TAMs could decrease their alternative activation and their tumor promoting properties. We found that activation of macrophage TFEB suppresses alternative activation, and introduction of these macrophages at the time of tumor initiation decreases tumor growth in multiple murine models. Therefore, our data demonstrate that the downregulation of TFEB is an integral part of immune editing in the tumor microenvironment, and provides a rationale for a new cancer treatment strategy involving the modulation of TAM polarization through activating TFEB.

Abstract P5-05-01: Physico-biochemical regulation of EMT by microtubule associated protein 7 (MAP7)

Abstract Background: We previously reported about 500 cancer stem cell (CSC) specific gene signatures from patient tumor samples. After screening with shRNAs for the 500 genes affecting mammosphere forming ability, we identified microtubule-assoicated protein 7 (MAP7) as one of the top candidate genes, which may serve as a target for breast CSCs. Although MAP7 is a predominant epithelial microtubule binding protein, only limited number of reports suggests that MAP7 may be a regulator of microtubule dynamics during cell division and a cofactor of Kinesin-1 in compartment transport in cells. However, little is known about how MAP7 supports epithelial cancers, especially breast cancer. Previously, we have reported that the mammosphere forming cells exhibit treatment resistance and high metastatic potential, which are intrinsic characters for CSCs displaying epithelial mesenchymal transition (EMT). We hypothesize that MAP7 supports breast cancer progression by promoting CSC self-renewal and survival through regulation of EMT. Objectives: Here, we aim to show that MAP7 is an essential regulator of breast CSCs and to elucidate mechanism behind EMT regulation by MAP7 in breast CSCs. Methods and Results: On Oncomine database analysis, MAP7 was up-regulated in most epithelial cancers, when compared to the corresponding normal tissues. Similarly, its expression in breast cancer was 2-fold higher than in the normal breast tissue (p<0.05), but without significant variances in the expression across the breast cancer subtypes. Gene silencing of MAP7 significantly reduced CD44+/CD24- breast CSC populations and mammosphere forming efficiencies of MDA-MB-231, HCC1937, and MDA-MB-468 breast cancer cells. Furthermore, the silencing of MAP7 expression compromised invasive potential of MDA-MB-231 cells by 50% and significantly altered the cell membrane mechanics of MDA-MB-468 cells, as indicated by a high-content image analysis for cell shapes and cell adhesion efficiency. More importantly, delivery of siRNA in vivo inhibited the growth of BCM2147 patient-derived tumor, and limiting dilution assay demonstrated that the tumor initiation potential of BCM2147 can be eliminated by MAP7 silencing. Through confocal microscope analysis of images of fluorescent immunostaining and co-immunoprecipatation assays, MAP7 showed polarized-expressions in spindle-shaped cancer cells and was co-localized with Focal Adhesion Kinase (FAK). Moreover, MAP7 silencing inhibited the phosphorylation of FAK by inactivating p130CAS and JSAP1, the upper stream and the down-stream regulators of FAK. Conclusion: We have showed the ectopic expression of MAP7 in breast tumors and other epithelial tumors, suggesting MAP7 may be involved in tumorigenesis and critical for the survival of tumor cells. Moreover, our results suggest that MAP7 is a key element for survival and self-renewal of breast CSCs through polarization of cells and activation of FAK, required for the initiation of EMT. To that end, here we report that MAP7 is essential for breast cancer growth by supporting CSC survival and self-renewal. Citation Format: Choi DS, Dave B, Rosato RR, Chang JC. Physico-biochemical regulation of EMT by microtubule associated protein 7 (MAP7) [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 P5-05-01.

Abstract S4-05: Expression of the DEK oncogene promotes M2 polarization and iron recycling in tumor associated macrophages

Abstract The chromatin-remodeling DEK oncogene is highly expressed in over 60% of breast cancers, regardless of sub-type. DEK over-expression correlates with poor clinical outcome, including decreased survival and chemotherapy resistance. Previously, using human breast cancer cell lines and the MMTV-Ron mouse breast cancer model, we have demonstrated that DEK expression promotes proliferation and metastasis and supports the breast cancer stem cell population. These functions were due, in part, through upregulated Wnt ligand expression and β-catenin activation, leading to paracrine and autocrine effects promoting tumorigenesis. To investigate additional drivers of DEK-induced tumorigenesis, we performed RNA-Sequencing on control and DEK over-expressing immortalized MCF10A cells. Gene ontology analysis identified several immune signaling processes. This is in alignment with the well-characterized role of DEK expression in the promotion of inflammatory diseases. We investigated the immune cell infiltration of tumors from MMTV-Ron/Dek+/+ and MMTV-Ron/Dek-/- mice. Interestingly, we discovered that Dek-expressing tumors demonstrated decreased tumor associated macrophage (TAM) infiltration, where TAMs were largely limited to the periphery. Furthermore, these TAMs demonstrated an iron-recycling phenotype (M2 polarization), whereas TAMs from Dek-knockout tumors retained iron (M1 polarization). M1 macrophages are typically associated with inflammatory responses whereas M2 macrophages have been linked to tissue remodeling, angiogenesis, and tumor promotion. To investigate this further, we compared Dek+/+ and Dek-/- bone marrow-derived macrophages (BMDM) and found no inherent difference in polarization. However, BMDM demonstrated different gene expression profiles when cultured in conditioned media from Dek-expressing murine breast cancer cell lines compared to isogenic Dek-/- cells. BMDM exposed to conditioned media from Dek-expressing cancer cells demonstrated the same iron recycling phenotype observed in vivo, likely caused by increased expression of ferroportin, as well as increased expression of M2 markers CXCR4, VEGF, and an elevated ARG1hi/NOSlo population. We thus used a combination of genetic models and small molecule inhibitors to identify the mechanism of M2 polarization of BMDM exposed to Dek-expressing cancer cells. NRF2 transcription activity was eliminated as a possible mechanism for ferroportin regulation. However, small molecule inhibitors and gene expression analyses implicated secreted Wnt ligands and S100A8/A9 alarmins from the cancer cells, and downstream C/EBP transcriptional activity in the macrophages, as possible mechanisms of Dek-induced M2 polarization. Combined, this suggests that the poor clinical outcome of high DEK expressing breast cancers may be due to not only cell intrinsic factors, but also cell extrinsic factors, such as the tumor-promoting M2 polarization of tumor associated macrophages. Citation Format: Privette Vinnedge LM, Pease NA, Cheek J. Expression of the DEK oncogene promotes M2 polarization and iron recycling in tumor associated macrophages [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 S4-05.

FOXM1 recruits nuclear Aurora kinase A to participate in a positive feedback loop essential for the self-renewal of breast cancer stem cells

Substantial evidence suggests that breast cancer initiation, recurrence and drug resistance is supported by breast cancer stem cells (BCSCs). Recently, we reported a novel role of Aurora kinase A (AURKA) in BCSCs, as a transactivating co-factor in the induction of the c-Myc oncoprotein. However, the mode of action and transcriptional network of nuclear AURKA in BCSCs remain unknown. Here, we report that nuclear AURKA can be recruited by Forkhead box subclass M1 (FOXM1) as a co-factor to transactivate FOXM1 target genes in a kinase-independent manner. In addition, we show that AURKA and FOXM1 participate in a tightly coupled positive feedback loop to enhance BCSC phenotype. Indeed, kinase-dead AURKA can effectively transactivate the FOXM1 promoter through a Forkhead response element, whereas FOXM1 can activate AURKA expression at the transcriptional level in a similar manner. Consistently, breast cancer patient samples portrayed a strong and significant correlation between the expression levels of FOXM1 and AURKA. Moreover, both FOXM1 and AURKA were essential for maintaining the BCSC population. Finally, we demonstrated that the AURKA inhibitor AKI603 and FOXM1 inhibitor thiostrepton acted synergistically to inhibit cytoplasmic AURKA activity and disrupt the nuclear AURKA/FOXM1-positive feedback loop, respectively, resulting in a more effective inhibition of the tumorigenicity and self-renewal ability of BCSCs. Collectively, our study uncovers a previously unknown tightly coupled positive feedback signalling loop between AURKA and FOXM1, crucial for BCSC self-renewal. Remarkably, our data reveal a novel potential therapeutic strategy for targeting both the cytoplasmic and nuclear AURKA function to effectively eliminate BCSCs, so as to overcome both breast cancer and drug resistance.

H19/let-7/LIN28 reciprocal negative regulatory circuit promotes breast cancer stem cell maintenance

Long noncoding RNA-H19 (H19), an imprinted oncofetal gene, has a central role in carcinogenesis. Hitherto, the mechanism by which H19 regulates cancer stem cells, remains elusive. Here we show that breast cancer stem cells (BCSCs) express high levels of H19, and ectopic overexpression of H19 significantly promotes breast cancer cell clonogenicity, migration and mammosphere-forming ability. Conversely, silencing of H19 represses these BCSC properties. In concordance, knockdown of H19 markedly inhibits tumor growth and suppresses tumorigenesis in nude mice. Mechanistically, we found that H19 functions as a competing endogenous RNA to sponge miRNA let-7, leading to an increase in expression of a let-7 target, the core pluripotency factor LIN28, which is enriched in BCSC populations and breast patient samples. Intriguingly, this gain of LIN28 expression can also feedback to reverse the H19 loss-mediated suppression of BCSC properties. Our data also reveal that LIN28 blocks mature let-7 production and, thereby, de-represses H19 expression in breast cancer cells. Appropriately, H19 and LIN28 expression exhibits strong correlations in primary breast carcinomas. Collectively, these findings reveal that lncRNA H19, miRNA let-7 and transcriptional factor LIN28 form a double-negative feedback loop, which has a critical role in the maintenance of BCSCs. Consequently, disrupting this pathway provides a novel therapeutic strategy for breast cancer.

Electrospinning Parameters Selection to Manufacture Polycaprolactone Scaffolds for Three-dimensional Breast Cancer Cell Culture and Enrichment

Only a small cell population of a breast tumour presents stem cell characteristics, thus so-called breast cancer stem cells (BCSCs). BCSCs are tumour-initiating cells and chemoresistant and they can grow in non-adherent conditions as mammospheres. Study of BCSCs is a challenge due to their low representation and the inability to propagate them without inducing differentiation. Previous studies have demonstrated that three-dimensional (3D) cell culture models such as scaffolds enhance the BCSCs population.In this project, scaffolds were fabricated by electrospinning technology. Specific values of voltage and flow rate were fixed to electrospun 7.5 and 15% poly(ɛ-caprolactone) (PCL) solutions according to process stability. Both scaffolds were seeded with MCF-7 breast cancer cells and 7.5% meshes displayed lower cell proliferation compared with 15% scaffolds. Cells cultured in both scaffolds presented a significant Mammosphere Forming Index (MFI) increase, thus indicating a BCSCs enrichment.Results show that three-dimensional cell culture with electrospun 15% PCL scaffolds could be useful to expand BCSCs population facilitating the future development of new therapeutic strategies against this tumour subpopulation.

NF-kappaΒ-inducing kinase regulates stem cell phenotype in breast cancer.

Breast cancer stem cells (BCSCs) overexpress components of the Nuclear factor-kappa B (NF-κB) signaling cascade and consequently display high NF-κB activity levels. Breast cancer cell lines with high proportion of CSCs exhibit high NF-κB-inducing kinase (NIK) expression. The role of NIK in the phenotype of cancer stem cell regulation is poorly understood. Expression of NIK was analyzed by quantitative RT-PCR in BCSCs. NIK levels were manipulated through transfection of specific shRNAs or an expression vector. The effect of NIK in the cancer stem cell properties was assessed by mammosphere formation, mice xenografts and stem markers expression. BCSCs expressed higher levels of NIK and its inhibition through small hairpin (shRNA), reduced the expression of CSC markers and impaired clonogenicity and tumorigenesis. Genome-wide expression analyses suggested that NIK acts on ERK1/2 pathway to exert its activity. In addition, forced expression of NIK increased the BCSC population and enhanced breast cancer cell tumorigenicity. The in vivo relevance of these results is further supported by a tissue microarray of breast cancer samples in which we observed correlated expression of Aldehyde dehydrogenase (ALDH) and NIK protein. Our results support the essential involvement of NIK in BCSC phenotypic regulation via ERK1/2 and NF-κB.

TRIM28 multi-domain protein regulates cancer stem cell population in breast tumor development.

The expression of Tripartite motif-containing protein 28 (TRIM28)/Krüppel-associated box (KRAB)-associated protein 1 (KAP1), is elevated in at least 14 tumor types, including solid and hematopoietic tumors. High level of TRIM28 is associated with triple-negative subtype of breast cancer (TNBC), which shows higher aggressiveness and lower survival rates. Interestingly, TRIM28 is essential for maintaining the pluripotent phenotype in embryonic stem cells. Following on that finding, we evaluated the role of TRIM28 protein in the regulation of breast cancer stem cells (CSC) populations and tumorigenesis in vitro and in vivo. Downregulation of TRIM28 expression in xenografts led to deceased expression of pluripotency and mesenchymal markers, as well as inhibition of signaling pathways involved in the complex mechanism of CSC maintenance. Moreover, TRIM28 depletion reduced the ability of cancer cells to induce tumor growth when subcutaneously injected in limiting dilutions. Our data demonstrate that the downregulation of TRIM28 gene expression reduced the ability of CSCs to self-renew that resulted in significant reduction of tumor growth. Loss of function of TRIM28 leads to dysregulation of cell cycle, cellular response to stress, cancer cell metabolism, and inhibition of oxidative phosphorylation. All these mechanisms directly regulate maintenance of CSC population. Our original results revealed the role of the TRIM28 in regulating the CSC population in breast cancer. These findings may pave the way to novel and more effective therapies targeting cancer stem cells in breast tumors.

Enhancement of Radiation Response in Breast Cancer Stem Cells by Inhibition of Thioredoxin- and Glutathione-Dependent Metabolism.

The goal of this study was to determine if depletion of glutathione (GSH) and inhibition of thioredoxin (Trx) reductase (TrxR) activity could enhance radiation responses in human breast cancer stem cells by a mechanism involving thiol-dependent oxidative stress. The following were used to inhibit GSH and Trx metabolism: buthionine sulfoximine (BSO), a GSH synthesis inhibitor; sulfasalazine (SSZ), an inhibitor of xc- cysteine/glutamate antiporter; auranofin (Au), a thioredoxin reductase inhibitor; or 2-AAPA, a GSH-reductase inhibitor. Clonogenic survival, Matrigel assays, flow cytometry cancer stem cell assays (CD44+CD24-ESA+ or ALDH1) and human tumor xenograft models were used to determine the antitumor activity of drug and radiation combinations. Combined inhibition of GSH and Trx metabolism enhanced cancer cell clonogenic killing and radiation responses in human breast and pancreatic cancer cells via a mechanism that could be inhibited by N-acetylcysteine (NAC). Au, BSO and radiation also significantly decreased breast cancer cell migration and invasion in a thiol-dependent manner that could be inhibited by NAC. In addition, pretreating cells with Au sensitized breast cancer stem cell populations to radiation in vitro as determined by CD44+CD24-ESA+ or ALDH1. Combined administration of Au and BSO, given prior to irradiation, significantly increased the survival of mice with human breast cancer xenografts, and decreased the number of ALDH1+ cancer stem cells. These results indicate that combined inhibition of GSH- and Trx-dependent thiol metabolism using pharmacologically relevant agents can enhance responses of human breast cancer stem cells to radiation both in vitro and in vivo.

Abstract 1189: Combination treatments with retinoic acid and the synthetic retinoid ST1926 in 2D and 3D breast cancer models overcome retinoic acid resistance and eradicate breast cancer stem/progenitor cells

Abstract Despite recent advances in breast cancer therapy, achieving complete remission in metastatic breast cancer patients remains a challenge. Retinoids are crucial regulators of cellular proliferation, differentiation, and cell death, and have shown potent chemotherapeutic and chemopreventive properties. The major drawback of the use of all-trans retinoic acid (ATRA) in cancer therapy is acquired resistance. Therefore, synthetic retinoids such as ST1926 emerged as potent anti-cancer agents. Here, we investigated the anti-tumor activities of ATRA, ST1926, and their combination treatments in 2D and 3D human breast cancer models and their targeting of breast cancer stem cells (CSCs)/progenitor cells. We have shown that in 2D culture models, MCF-7 and MDA-MB-231 cells are resistant to ATRA while being sensitive to ST1926 at sub-micromolar (μM) concentrations in an irreversible manner. Importantly, ST1926 had no effect on the ‘normal-like’ MCF-10A breast epithelial cells. ST1926 induced massive apoptosis in MCF-7 cells and it resulted in S-phase arrest and necrosis in the triple negative and metastatic MDA-MB-231 cells. Furthermore, ST1926 caused early DNA damage, increased the expression of the tumor suppressors p53 and p21, downregulated the Wnt/β-catenin pathway, and modulated the expression levels of the different retinoid receptors. Interestingly, combination treatments as low as 0.1 μM ST1926 and 0.5 μM ATRA synergistically inhibited the proliferation in 2D models of MCF-7 and MDA-MB-231 cells, independently of retinoid receptor signaling, while sparing the normal breast epithelial cells. Anchorage-independent growth of MCF-7 and MDA-MB-231 cells was examined using the soft agar colony formation assay where sub-μM concentrations of ST1926 or μM concentrations of ATRA were shown to reduce the size and the number of breast cancer colonies. ST1926 drastically induced cell death in 3D Matrigel ‘on-top’ assay cultures of breast cancer cells while the lumen of the normal-like breast epithelial cell line S1 was maintained. Finally, treatment with 0.01 μM ST1926 alone or 0.001 μM ST1926 in combination with 0.1 μM ATRA abrogated sphere formation and the self-renewal ability of breast CSCs in the 3D sphere formation assay. In summary, ST1926, ATRA, and their combination treatments were shown to display more potent anti-tumor properties in 3D versus 2D human breast cancer models. Our results also demonstrate the therapeutic potential of ST1926 in sensitizing breast cancer cells to ATRA and in targeting the population of breast CSCs. As 3D culture models are more representative of the tumor microenvironment and serve as valid tools in drug discovery, our results highlight the promising use of ATRA/ST1926 combinations in metastatic and triple negative breast cancers. Citation Format: Patrick Aouad, Melody Saikali, Rana Abdel-Samad, Leeanna El–Houjeiri, Claudio Pisano, Rabih Talhouk, Nadine Darwiche. Combination treatments with retinoic acid and the synthetic retinoid ST1926 in 2D and 3D breast cancer models overcome retinoic acid resistance and eradicate breast cancer stem/progenitor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1189.

Enhanced anti-tumor activity and cytotoxic effect on cancer stem cell population of metformin-butyrate compared with metformin HCl in breast cancer.

Metformin, which is a drug commonly used to treat type 2 diabetes, has shown anti-tumor effects in numerous experimental, epidemiologic, observational, and clinical studies. Here, we report a new metformin derivative, metformin-butyrate (MFB). Compared to metformin-HCl, it more potently activates AMPK, inhibits mTOR, and impairs cell cycle progression at S and G2/M phases. Moreover, MFB inhibits the mammosphere formation of breast cancer cells and shows cytotoxic effects against CD44+CD24−/low populations in vitro and in vivo, indicating that it might have preferential effects on the cancer stem cell population. MFB showed synergistic cytotoxicity with docetaxel and cisplatin, and MFB pretreatment of breast cancer cells prior to their injection into the mammary fat pads of mice significantly decreased the obtained xenograft tumor volumes, compared with untreated or metformin-pretreated cells. Overall, MFB showed greater anti-neoplastic activity and greater efficacies in targeting the G2/M phase and breast cancer stem cell population, compared to metformin-HCl. This suggests that MFB may be a promising therapeutic agent against aggressive and resistant breast cancers.

Benzyl butyl phthalate promotes breast cancer stem cell expansion via SPHK1/S1P/S1PR3 signaling.

Understanding the regulatory mechanisms unique to breast cancer stem cells (BCSCs) is required to control breast cancer metastasis. We found that phthalates promote BCSCs in human breast cancer cell cultures and xenograft tumors. A toxic phthalate, benzyl butyl phthalate (BBP), activated aryl hydrocarbon receptor in breast cancer cells to stimulate sphingosine kinase 1 (SPHK1)/sphingosine 1-phosphate (S1P)/sphingosine-1-phosphate receptor 3 (S1PR3) signaling and enhance formation of metastasis-initiating BCSCs. BBP induced histone modifications in S1PR3 in side population (SP) cells, but not in non-SP cells. SPHK1 or S1PR3 knockdown in breast cancer cells effectively reduced tumor growth and lung metastasis in vivo. Our findings suggest S1PR3 is a determinant of phthalate-driven breast cancer metastasis and a possible therapeutic target for regulating BCSC populations. Furthermore, the association between breast carcinogenesis and environmental pollutants has important implications for public health.