Regulation Of Breast Cancer Stem Cells Research Articles

The SCRIB Paralog LANO/LRRC1 Regulates Breast Cancer Stem Cell Fate through WNT/β-Catenin Signaling.

SummaryTumor initiation, progression, and therapeutic resistance have been proposed to originate from a subset of tumor cells, cancer stem cells (CSCs). However, the current understanding of the mechanisms involved in their self-renewal and tumor initiation capacity remains limited. Here, we report that expression of LANO/LRRC1, the vertebrate paralog of SCRIB tumor suppressor, is associated with a stem cell signature in normal and tumoral mammary epithelia. Through in vitro and in vivo experiments including a Lano/Lrrc1 knockout mouse model, we demonstrate its involvement in the regulation of breast CSC (bCSC) fate. Mechanistically, we demonstrate that Lano/LRRC1-depleted cells secrete increased levels of WNT ligands, which act in a paracrine manner to positively deregulate the WNT/β-catenin pathway in bCSCs. In addition to describing the first function of LANO/LRRC1, our results suggest that its expression level could be used as a biomarker to stratify breast cancer patients who could benefit from WNT/β-catenin signaling inhibitors.

Immunoregulatory protein B7-H3 regulates cancer stem cell enrichment and drug resistance through MVP-mediated MEK activation.

B7-H3 is a tumor promoting glycoprotein that is expressed at low levels in most normal tissues but is overexpressed in various human cancers which is associated with disease progression and poor patient outcome. Although numerous publications have reported the correlation between B7-H3 and cancer progression in many types of cancers, mechanistic studies on how B7-H3 regulates cancer malignancy are rare, and the mechanisms underlying the role of B7-H3 in drug resistance are almost unknown. Here we report a novel finding that upregulation of B7-H3 increases the breast cancer stem cell population and promotes cancer development. Depletion of B7-H3 in breast cancer significantly inhibits the cancer stem cells. By immunoprecipitation and mass spectrometry, we found that B7-H3 is associated with the major vault protein (MVP) and activates MEK through MVP enhancing B-RAF and MEK interaction. B7-H3 expression increases stem cell population by binding to MVP which regulates the activation of the MAPK kinase pathway. Depletion of MVP blocks the activation of MEK induced by B7-H3 and dramatically inhibits B7-H3 induced stem cells. This study reports novel functions of B7-H3 in regulating breast cancer stem cell enrichment. The novel mechanism for B7-H3-induced stem cell propagation by regulating MVP/MEK signaling axis independent of the classic Ras pathway may have important implications in the development of strategies for overcoming cancer cell resistance to chemotherapy.

PO-107 A potential role for DELTA40P53 in the regulation of breast cancer stem cells

IntroductionBreast cancer is a heterogeneous disease that can be classified as a number of distinct molecular subtypes, however heterogeneity also exists between cancer cells. One class of breast cancer cells that have generated some controversy are breast cancer stem cells (BCSCs). This subpopulation of cells are thought to drive tumourigenesis through their abilities of self-renewal, multilineage differentiation, tumour regrowth, and resistance to chemotherapies. The tumour suppressor p53 is essential in the maintenance of genomic stability in somatic cells. More recently, p53 has been recognised in the maintenance, regulation and characterisation of stem cells, including cancer stem cells. The p53 isoforms, such as Δ40p53, are recognised to play an extensive role in the regulation and function of p53. We have shown that Δ40p53 is the most highly expressed isoform in breast cancer, and a high Δ40p53:p53 ratio was associated with worse prognosis. However, its role in regulating breast cancer stem cells is unknown.Material and methodsGene expression arrays were performed on 64 breast cancers and differentially expressed genes were identified. To investigate the expression of Δ40p53 in breast cancer, we have developed a cell line model that stably overexpresses (LeGO-Δ40p53) Δ40p53. LeGO-Δ40p53 cells were treated with different chemotherapy agents, key targets were assessed by qPCR and immunofluorescence, and DNA-damage measured using Annexin-V apoptosis assay.Results and discussionsDifferentially expressed genes were identified between breast cancer patients with high and low Δ40p53 expression. These genes significantly enriched for pathways in stem cell regulation and markers of migration/invasion. To confirm this in our cell line model, we found that LeGO-Δ40p53 cells showed significant upregulation of pluripotent markers Nanog, Oct4 and Sox2. In addition, following treatment with doxorubicin, LeGO-Δ40p53 cells showed a reduction in select pro-apoptotic genes and decreased apoptosis, suggesting that high Δ40p53 expression in breast cancer can selectively modulate the DNA-damage response pathway.ConclusionThis work suggests that Δ40p53 may play a role in the regulation of breast cancer stem cells and this may be essential for modulating the DNA-damage response in these cells. Further studies are needed to assess if high Δ40p53 expression can alter the functionality of breast cancer stem cells.

PO-290 ETV7 regulates breast cancer stem cells content and chemoresistance

IntroductionCancer stem cells (CSCs) are considered the population of cells within the tumour able to drive tumorigenesis and known to be highly resistant to conventional chemotherapy. ETV7 is a poorly studied transcription factor member of ETS large family, known to be an interferon-stimulated gene. It has been recently found over-expressed in breast cancer (BC), with higher expression levels in the more aggressive BC subtypes. In this work, we investigated the effects of ETV7 increased expression on breast CSCs population and resistance to chemotherapy in BC cells.Material and methodsWe generated MCF7 and T47D BC-derived cells stably over-expressing ETV7 and obtained ETV7 KO in MDA-MB-231 BC cells using CRISPR/Cas9 technology. We analysed breast CSCs content via CD44/CD24 staining and FACS analysis, as well as mammospheres formation assay. We measured expression of ABC transporters and anti-apoptotic proteins via RT-qPCR and western blot. We finally assessed sensitivity to Doxorubicin and 5-Fluorouracil (5-FU) via MTT assay and AnnexinV/PI staining at FACS.Results and discussionsWe observed that the expression of ETV7 could be induced by various stimuli, particularly by chemotherapeutic drugs able to induce DNA damage. We then analysed the impact of ETV7 expression on the sensitivity to Doxorubicin and 5-FU and we could observe a significantly decreased sensitivity to these drugs upon ETV7 over-expression. We could also appreciate an increase in ABC transporters and BCL2 anti-apoptotic protein expression following ETV7 over-expression. We further observed that alteration of ETV7 expression could significantly affect the population of breast cancer stem cells (CD44+/CD24low cells) in different BC cell lines.ConclusionWe propose a novel role for ETV7 in breast cancer stem cells plasticity and associated resistance to conventional chemotherapy. We finally suggest that an in-depth investigation of this mechanism could lead to novel breast CSCs targeted therapies and to the improvement of combinatorial regimens with the aim of avoiding resistance and relapse in breast cancer.

Diallyl Trisulfide inhibits breast cancer stem cells via suppression of Wnt/β-catenin pathway.

Cancer stem cells (CSCs) play a central role in the development of breast cancer. The canonical Wnt/β-catenin signal pathway is critical for maintaining CSCs characteristics. Diallyl trisulfide (DATS), a natural organosulfur compound from the garlic, exhibits effective antitumor properties. However, the role of DATS in regulating breast CSCs activity and the underlying molecular mechanisms remain obscure. In the present study, we reported that DATS efficiently inhibited the viability of breast CSCs as evidenced by reducing turmorspheres formation, decreasing the expression of breast CSCs markers (CD44, ALDH1A1, Nanog, and Oct4), as well as inhibiting proliferation and inducing apoptosis. Furthermore, we showed that DATS downregulated the activity of Wnt/β-catenin pathway, while LiCl-triggered Wnt/β-catenin activation diminished DATS inhibition on breast CSCs. Taken together, our results illustrated that DATS suppressed breast CSCs through inhibiting Wnt/β-catenin pathway activation. These novel findings could provide new insights into the molecular mechanisms of breast CSCs regulation as well as its target intervention and might provide new strategies for preventing and treating breast cancers.

MiR-200c/141 Regulates Breast Cancer Stem Cell Heterogeneity via Targeting HIPK1/β-Catenin Axis.

Increasing evidence demonstrates the existence of two inter-convertible states of breast cancer stem cells (BCSCs) with distinct behaviors in proliferation and mobility, and the BCSC heterogeneity is accurately regulated by sophisticated mechanisms including microRNAs. The microRNA-200 family including miR-200c/141 cluster was reported to affect cancer cell invasion and metastasis by regulating epithelial to mesenchymal transition (EMT). However, the effect of miR-200 family on BCSC heterogeneity is uncertain. Thus, we investigated whether the miR-200c/141 cluster had different effects on breast tumor growth and metastasis by switching the two states of BCSC.Methods: The spontaneous mammary tumor mouse model with miR-200c/141 conditional knockout was utilized for analyzing the role of miR-200c/141 cluster in vivo. The effect of miR-200c/141 cluster on BCSCs was performed by CD24/CD29 staining and ALDEFLUOR assay. miR-200c/141 target expression and EMT-related marker expression were verified in tumor sections, primary cells and breast cancer cell lines by qRT-PCR or western blotting. Statistical analysis was determined using two-way ANOVA and Student's t-test. All values were presented as the mean ± s.e.m.Results: The deletion of miR-200c/141 cluster regulated BCSC heterogeneity and promoted the EMT-like BCSC generation, which resulted in increased tumor metastasis and inhibited tumor growth by directly upregulating the target gene homeodomain-interacting protein kinase 1 (HIPK1) and sequential β-catenin activation.Conclusions: Our results indicated that miR-200c/141 played biphasic roles in breast tumor progression via affecting the BCSC heterogeneity, suggesting targeting BCSC heterogeneity to simultaneously restrict breast cancer initiation and metastasis could be a promising therapeutic strategy for breast cancer.

Expressions and clinical significances of aldehyde dehydrogenase 1 and transforming growth factor-β2 in triple negative breast cancer

Objective To investigate the expressions and clinical significances of breast cancer stem cell markers such as aldehyde dehydrogenase 1 (ALDH1) and transforming growth factor-β2 (TGF-β2) in patients with triple negative breast cancer. Methods Samples of 60 triple negative breast cancer tissues were investigated for the expressions of ALDH1 and TGF-β2 proteins by immunohistochemical staining. The correlation analysis, disease-free survival analysis and overall survival analysis were performed. Results The positive expressions of ALDH1 protein and TGF-β2 protein in the 60 breast cancer primary lesions were 23 cases (38.33%) and 38 cases (63.33%) respectively. The expression of ALDH1 protein was not correlated with tumor size (χ2=0.307, P=0.580), histological grade (χ2=4.244, P=0.120), clinical stage (χ2=0.982, P=0.612) or lymph node metastasis (χ2=1.111, P=0.292). The expression of TGF-β2 protein was not correlated with histological grade (χ2=4.651, P=0.098), lymph node metastasis (χ2=3.513, P=0.061), clinical stage (χ2=1.310, P=0.519) or tumor size (χ2=0.629, P=0.428). The disease-free survival time [(38.43±3.86) months vs. (53.38±2.58) months] and the overall survival time [(42.00±3.11) months vs. (53.84±2.19) months] of ALDH1-positive patients were significantly shorter than those of ALDH1-negative patients, and the differences were statistically significant (χ2=8.490, P=0.004; χ2=11.270, P=0.001). The disease-free survival time [(42.81±3.32) months vs. (54.72±2.50) months] and the overall survival time [(44.74±2.68) months vs. (57.18±1.55) months] of TGF-β2 positive patients were significantly shorter than those of TGF-β2-negative patients, and the differences were statistically significant (χ2=4.300, P=0.038; χ2=8.900, P=0.003). The expression of ALDH1 protein was positively correlated with the expression of TGF-β2 protein (r=0.360, P=0.005). Conclusion The ALDH1 phenotype is an indepen-dent predictor of poor prognosis. The activation of TGF-β2 signaling pathway may be involved in the regulation of triple-negative breast cancer stem cells. Key words: Breast neoplasms; Neoplastic stem cells; Transforming growth factor beta 2; Aldehyde dehydrogenase 1

MicroRNAs, a subpopulation of regulators, are involved in breast cancer progression through regulating breast cancer stem cells.

Cancer stem cells (CSCs; also known as tumor-initiating cells) are essential effectors of tumor progression due to their self-renewal capacity, differentiation potential, tumorigenic ability and resistance to chemotherapy, all of which contribute to cancer relapse, metastasis and a poor prognosis. Breast cancer stem cells (BCSCs) have been identified to be involved in the processes of BC initiation, growth and recurrence. MicroRNAs (miRNAs) are a class of non-coding small RNAs of 19-23 nucleotides in length that regulate gene expression at the post-transcriptional level through various mechanisms, and serve critical roles in cancer progression. miRNAs have been demonstrated to elicit effects on BCSCs characteristics via the targeting of oncogenes or tumor suppressor genes. The present study focused on the effect of miRNAs on BCSC, including BCSC formation, self-renewal and differentiation, by which miRNAs may inhibit BCSC invasion and metastasis, modulate clonogenicity and tumorigenicity of BCSCs as well as regulate chemotherapy resistance to BC. Through an improved understanding of the association between BCSCs and miRNAs, a novel and safer therapeutic target for BC may be identified.

Abstract 4768: A SHARP /Xist complex regulates breast cancer stem cells

Abstract Many cancers including breast cancer display hieratical organization and are driven by a cellular sub-population that displays stem cell properties. These cancer stem cells (CSC’s) mediate metastasis and contribute to treatment resistance highlighting the importance of elucidating CSC regulatory pathways. Nuclear proteins and lncRNAs involved in the maintenance of chromatin structure have been shown to play roles in CSC regulation. Among them the polycomb proteins bmi-1 and EZH2 are involved in epigenetic regulation of CSC’s in breast and other cancers. SHARP/SPEN, an RNA binding protein, has been previously been found to bind the lncRNA Xist which, by recruiting the PRC2 polycomb complex mediates transcriptional repression of the X chromosome. We utilized breast cancer cell lines and xenograft models to examine the role of the SHARP/Xist complex in the regulation of breast CSC’s. In a series of breast cancer cell lines including MCF7 (luminal )and SUM 159 (basal/claudin low) cells, SHARP mRNA and protein as well as the lncRNA Xist were more highly expressed in cells expressing aldehyde dehydrogenase as accessed by the Aldefluor assay than in ALDH -cells . Conditional knockdown of SHARP or Xist in SUM159 significantly decreased the ALDH+ CSC population without affecting cell growth in vitro. Furthermore, SHARP or Xist knockdown significantly reduced tumor initiating capacity and growth of SUM 159 cells in NOD/SCID mice. To determine the mechanism of breast CSC regulation by the SHARP complex we compared mRNA expression patterns of control and SHARP knockdown SUM 159 cells using RNAseq. There was significant overlap between the genes regulated by SHARP with genes regulated by the PCR1 and PRC2 polycomb complexes in addition to many histone genes. These studies suggest that the SHARP/Xist complex my regulate breast CSC’s through epigenetic regulatory histone and polycomb genes. Since SHARP expression is elevated in a number of cancers including breast cancer this pathway may represent a novel therapeutic target. Citation Format: Yongyou Zhu, Li Shang, Justin Colacino, Michael Brooks, Ramdane Harouaka, Chang-Ching Lin, Ming Luo, Max S. Wicha. A SHARP /Xist complex regulates 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 4768. doi:10.1158/1538-7445.AM2017-4768

Abstract 5152: Cancer stem cell targeted exosomes for the treatment of metastatic breast cancer

Abstract Introduction: Therapy resistance and metastasis remain the two biggest challenges of breast cancer that result in a majority of cancer deaths. Cancer stem cells (CSCs) are a subpopulation of cancer cells that can initiate new tumors, remain refractory to conventional therapies, and spread throughout the body. In order to effectively block metastasis, the present study aims to develop novel therapeutics that specifically target breast CSCs and spare normal cells. While nanomedicine represents one of the promising future directions, it is necessary to overcome the shortcomings of synthetic nanoparticles, including bio-instability and off-target toxicity. Here, we aim to bio-engineer the naturally produced cell-secreted small vesicles, exosomes (~100 nm diameter), for a novel therapeutic delivery system. Methods: Exosomes from the human mesenchymal stem cells (MSCs) were isolated by differential ultracentrifugation, and characterized by multiple approaches including transmission EM, Immunoblotting and immunofluorescence assays to confirm the size, shape and presence of exosomal markers CD63, CD81 and LAMP2B. Exosomes were engineered to express a peptide ligand on its surface to specifically recognize and target CSCs, and the presence of the ligand was confirmed by western blot, flow cytometry (FCM) and a super resolution confocal laser scanning microscopy (CLSM, Leica SP8). The CSC targeting efficiency of the exosomes was evaluated in vitro by FCM and CLSM. Further, the exosomes were loaded with microRNAs (miR-200, miR-30c), as one of the key factors that regulate breast CSC functions, by following a novel transfection technology. Results: The bio-engineered exosomes specifically target and internalize into the CSCs compared to normal cells with absent expression of the ligand-binding protein. The developed exosomes can effectively deliver candidate miRNAs into the recipient cells and knock down the genes important for functional CSCs. The in vivo targeting of CSCs and therapeutic applications of the exosomes using patient-derived human-in-mouse breast tumor xenograft models are underway. Conclusion: It is expected that exosome-based miRNA therapeutics will have high biocompatibility, low toxicity, high specificity, and promising efficiency in preclinical and clinical applications to reduce breast cancer metastasis and improve patient survival. Citation Format: Golam Kibria, Katelyn E. Lee, Erika K. Ramos, Simo Huang, Clifford V. Harding, Jan Lötvall, Huiping Liu. Cancer stem cell targeted exosomes for the treatment of metastatic 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 5152. doi:10.1158/1538-7445.AM2017-5152

Abstract P5-07-03: GD2-mediated FAK signaling regulates breast cancer stem cell function in triple negative breast cancer

Abstract Ganglioside GD2 identifies breast cancer stem cells (BCSCs, Battula et al., JCI, 2012) and expression of GD2 is tightly regulated by GD3 synthase (GD3S). GD3S is highly expressed in GD2+ cells and inhibition of GD3S inhibits tumor formation and metastasis of breast cancer cells. However, the mechanism of GD2-mediated regulation of BCSC function is not known. Here we hypothesize that GD2 regulates signaling pathways involved in cell adhesion, migration and invasion of breast cancer cells. To identify these signaling pathways, antibody micro-arrays were used with 850 validated antibodies specific to total or phosphorylated proteins. Interestingly, focal adhesion kinase (FAK) was the most significantly phosphorylated protein in GD2+ compared to GD2- cells (S910 and S722). In addition, expression of FAK downstream mediators including Csk, PKCq, PKCl/I, Pyk2, and p38MAPK, was up-regulated in GD2+ compared to GD2- cells. Western blot analysis of FACS sorted SUM159 cells also revealed increased phosphorylation of FAK >80% at Y397 and >25% at Y861 in GD2+ compared to GD2- cells. FAK downstream targets including paxillin, p130 Cas, pERK were also up-regulated in GD2+ cells compared to GD2- cells indicating definitive activation of FAK signaling in GD2+ BCSCs. To investigate the functional role of GD2 in FAK mediated functions, we genetically deleted GD3S using the CRISPR knock-out system in SUM159 cells. only <1% GD2expression compared to >20% in parental cells was observed. GD3S-KO cells grew 5-10% slower in cell culture mostly because of the reduction (15±5%) in adherence. Trans-well assays revealed 3-5 fold reduction in migration and invasion of GD3S-KO compared to parental cells. These data indicate that GD2 and GD3S are not only the markers of BCSCs but also regulators of their function. Finally, we tested the effect of FAK inhibitor (PF-573228) against GD2+ BCSCs and GD3S-KO SUM159 cells. PF-573228 treatment decreased the number of mammospheres generated by GD2+ cells 3-4 fold in a dose dependent manner (100nM-1µm). In addition, treatment of PF-573228, inhibited migration and invasion of GD2+ cells 2 and 3 fold, respectively. However, treatment with PF-573228 on GD3S-KO cells further reduced their ability to migrate and invade by over 70% compared to untreated cells. In addition, GD3S-KO cells failed to form any mammospheres when cultured under low adherence conditions (p<0.00001), whereas the parental cells formed 15-20 mammospheres per 1,000 cells plated. In conclusion, our data demonstrate that FAK signaling is activated in GD2+ cells but that FAK inhibition alone may not be sufficient to inhibit BCSC function. Combined FAK and GD3S inhibition may exert highly synergistic effects against BCSCs. Citation Format: Nguyen K, Sun JC, Hortobagyi GN, Andreeff M, Battula VL. GD2-mediated FAK signaling regulates breast cancer stem cell function in triple negative breast cancer [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 P5-07-03.

MiR-600 Acts as a Bimodal Switch that Regulates Breast Cancer Stem Cell Fate through WNT Signaling

Breast cancer stem cells (bCSCs) have been implicated in tumor progression and therapeutic resistance; however, the molecular mechanisms that define this state are unclear. We have performed two microRNA (miRNA) gain- and loss-of-function screens to identify miRNAs that regulate the choice between bCSC self-renewal and differentiation. We find that micro-RNA (miR)-600 silencing results in bCSC expansion, while its overexpression reduces bCSC self-renewal, leading to decreased invivo tumorigenicity. miR-600 targets stearoyl desaturase 1 (SCD1), an enzyme required to produce active, lipid-modified WNT proteins. In the absence of miR-600, WNT signaling is active and promotes self-renewal, whereas overexpression of miR-600 inhibits the production of active WNT and promotes bCSC differentiation. In a series of 120 breast tumors, we found that a low level of miR-600 is correlated with active WNT signaling and a poor prognosis. These findings highlight a miR-600-centered signaling network that governs bCSC-fate decisions and influences tumor progression.

Research progress in breast cancer stem cells

Many studies have shown that a small fraction of cells inside breast tumors possess the ability of self-renewal and differentiation; these cells are called breast cancer stem cells (BCSCs) and play a vital role in tumor progression and metastasis. The characteristics of BCSCs are regulated by multiple intracellular and extracellular factors, including some important signaling pathways (e.g., sonic hedgehog, Wnt, and Notch), transcription factors (e.g., Bmi-1), noncoding RNAs (e.g., miR-93 and miR-100 ), and cytokines (e.g., interleukin-6). In addition, the tumor microenvironment, including nontumor cells (e.g., mesenchymal stem cells and macrophages) and noncellular components (e.g., cytokines), is also a key factor regulating BCSCs. Because BCSCs are resistant to conventional therapies, these therapies may be more efficacious when used in combination with BCSC-targeted therapies. In this review, we focus on recent research progress regarding the regulation of BCSCs and BCSC-targeted therapies.

Genetic variations in the Hippo signaling pathway and breast cancer risk in African American women in the AMBER Consortium.

The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. Dysfunction of the Hippo pathway components has been linked with breast cancer stem cell regulation, as well as breast tumor progression and metastasis. TAZ, a key component of the Hippo pathway, is highly expressed in triple negative breast cancer; however, the associations of genetic variations in this important pathway with breast cancer risk remain largely unexplored. Here, we analyzed 8309 germline variants in 15 genes from the Hippo pathway with a total of 3663 cases and 4687 controls from the African American Breast Cancer Epidemiology and Risk Consortium. Odds ratios (ORs) were estimated using logistic regression for overall breast cancer, by estrogen receptor (ER) status (1983 ER positive and 1098 ER negative), and for case-only analyses by ER status. The Hippo signaling pathway was significantly associated with ER-negative breast cancer (pathway level P = 0.02). Gene-based analyses revealed that CDH1 was responsible for the pathway association (P < 0.01), with rs4783673 in CDH1 statistically significant after gene-level adjustment for multiple comparisons (P = 9.2×10(-5), corrected P = 0.02). rs142697907 in PTPN14 was associated with ER-positive breast cancer and rs2456773 in CDK1 with ER-negativity in case-only analysis after gene-level correction for multiple comparisons (corrected P < 0.05). In conclusion, common genetic variations in the Hippo signaling pathway may contribute to both ER-negative and ER+ breast cancer risk in AA women.

Abstract 4395: Tissue stiffness and hypoxia regulate breast cancer stem cells through ILK

Abstract The mechanical and chemical properties of the cellular microenvironment can influence the phenotype of cancer cells. The hypoxic conditions within solid breast tumors can contribute to invasiveness and the formation of breast cancer stem cells (CSCs). Integrin-linked kinase (ILK) is a critical mediator for mechanotransduction, a process that converts exogenous mechanical stimuli into biochemical signals. Yet, it is unclear how the physical properties of the tumor microenvironment, including matrix stiffness and hypoxia, integrate to direct the formation of the breast CSC niche. Using an innovative engineered culture model, we found that stiff and hypoxic microenvironments activate integrin signaling and a stem-like gene signature in breast cancer cells, suggesting that integrin signaling, matrix mechanics and oxygen tension cooperatively promote the formation of breast CSCs. Stiff and hypoxic microenvironments promote integrin signaling and the expression of CSC markers including CD44 and Nanog. Knocking down ILK reverts the CSC phenotype of invasive breast cancer cells on stiff matrix, and abrogates their ability to form mammospheres and colonies in soft agar. In contrast, ectopic expression of ILK enhances CSC properties. ILK promotes integrin signaling and the CSC phenotype through the PI3K/Akt pathway. Stiff microenvironments promote tumor formation and metastasis in ovo. ILK depletion significantly abrogates the tumorigenenic and metastatic potential of invasive breast cancer cells in ovo. Importantly, we found that breast cancer cells expressing ILK and the CSC marker CD44 were only present in the regions of tumors predicted to be stiff in breast cancer patient samples. Our data suggest that ILK act as an essential mechanosensor that regulates breast CSC phenotype in response to matrix mechanics and oxygen tension to regulate the formation of breast CSCs. Citation Format: Mei-Fong Pang, Melody Stallings-Mann, Michael J. Siedlik, Victor D. Varner, Siyang Han, Derek C. Radisky, Celeste M. Nelson. Tissue stiffness and hypoxia regulate breast cancer stem cells through ILK. [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 4395.

Abstract 2509: Determining the functional and mechanistic role of homeobox transcription factor Meox1 in breast cancer and breast cancer stem cells

Abstract Many mechanisms have been attributed to de novo and acquired trastuzumab drug resistance in patients with HER2-enriched breast cancer; common mediated resistance is inactivation of the tumor suppressor gene PTEN seen in approximately 40% of patients. Evidence show the HER2 gene plays a significant role in regulating breast cancer stem cells through PTEN deletion; increasing evidence show these cancer stem cells may be responsible for drug resistance, cancer recurrence and metastasis. Studies demonstrate how PTEN deletion in HER2-overexpressing breast cancer activates an IL6 inflammatory feedback loop expanding the cancer stem cell population displaying EMT properties, conferring trastuzumab drug resistance and metastasis. While new therapeutic approaches for targeting HER2-enriched tumors with PTEN deletion are in progress, further understanding of molecular systems involving EMT cancer stem cell expansion as well as of de novo and acquired trastuzumab drug resistance is important for improving therapeutic options. Our lab conducted RNA sequencing to further explore functional mechanisms behind this aforementioned molecular system and found the upregulation of mesenchyme homeobox 1 (Meox1) as a potential transcriptional factor regulator. Meox1 is a non-cluster homeobox transcription factor expressed during embryonic development, critical in somite morphogenesis. Recent evidence has linked Meox1 as a critical PBX1 cofactor in ovarian cancer. As of yet, the role of Meox1 has never been studied in the context of breast cancer in correlation with cancer stem cells. Therefore specifically, the purpose of this study is to elucidate the molecular role of Meox1 in HER2-amplified PTEN-deleted trastuzumab-resistant breast cancer in order to provide functional and mechanistic insight involving EMT cancer stem cell expansion as well as of de novo and acquired trastuzumab drug resistance. RT-qPCR analyses of Meox1 expression using different breast cancer cell lines show correlation between increase in Meox1 expression with PTEN deletion. In vitro studies show siRNA knockdown of Meox1 decreases cancer stem cell properties of both colony and mammosphere formation in the BT474 HER2-enriched breast cell line that is PTEN-deleted and long term trastuzumab treated. Furthermore, lentiviral stable overexpression of Meox1 in normal immortalized MCF10A breast cancer cell line shows an increase in mammosphere formation compared to control infections. These preliminary results suggest Meox1 may be a critical transcription factor responsible for cancer stem cell progression in PTEN-deleted trastuzumab-resistant breast cancer. As such, further functional and mechanistic investigation of this homeobox transcription factor may provide additional understanding for de novo and acquired drug resistance and disease recurrence, potentially offering advanced therapeutic options. Citation Format: Mari Gasparyan, Joseph P. Burnett, Lichao Sun, Duxin Sun. Determining the functional and mechanistic role of homeobox transcription factor Meox1 in breast cancer and breast cancer stem 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 2509.

Abstract 3311: Autocrine and paracrine IL-4 maintains breast cancer stem cells traits via RAS/MAPK/DUSP pathway

Abstract Background: Despite the advent of successful treatment of localized malignancies, metastatic cancer still lacks efficacious therapeutic approaches, including breast cancer. It is now well established that within malignant tumors Cancer Stem Cells (CSCs) constitute a unique cell subset that fuel and succeed at tumor growth and metastases formation. Tumor microenvironment sustains CSCs characteristics, making the molecular mechanisms driving tumor progression and recurrence more complex and difficult to elucidate. Furthermore, the interaction that occurs between CSCs and the nearby stroma has proven to enhance the aggressive behavior of several carcinomas through the secretion of microenvironmental cytokines. In this context, IL-4 has already been described to promote survival of cancer cells through the up-regulation of several anti-apoptotic factors. However, still little is known about its role in promoting breast cancer progression. Results: Here we show for the first time that autocrine and paracrine production of IL-4 regulates breast CSCs (BCSCs) features, including cell proliferation, motility and cytoskeletal organization via RAS/MAPK pathway. Interestingly, relief from IL-4 impaired BCSCs proliferation, colony forming efficiency and in vivo tumor formation, while it fostered the expression of the dual specificity phosphatase-4 (DUSP4) in triple-negative basal-like BCSCs, leading to the decrease of CD44+/CD24- population. DUSP4 is commonly deficient in the most aggressive breast cancers, such as the basal-like subtype. Likewise, we observed that the enforced expression of DUSP4 increases the CD24+ compartment in basal-like BCSCs, determining also a dramatic decrease of their proliferation, colony forming efficiency, invasiveness and metastases formation. Contrarily, in luminal-like BCSCs, DUSP4 suppression favors BCSCs cell traits, including their tumorigenic and metastagenic properties. Methods: Patient-derived BCSCs were obtained by digestion of breast cancer tissues and plated in serum-free media with bFGF and EGF. DUSP4 were inserted into the p-Lenti expression vector. Stable DUSP4 knockdown was produced by lentiviral transduction of the pGFP-C vector. IL-4 function was impaired by using a high affinity IL-4Rα antagonist. To assess tumorigenicity and metastases formation, BCSCs were suspended in matrigel and injected either orthotopically or intra caudal in NOD/SCID mice. Conclusions: These findings will shed light on the molecular basis of cancer progression and on the complex crosstalk occurring between tumor and its microenvironment. The identification of tumor-related molecular events, such as the IL-4 activated signaling, might be clinically exploited as therapeutic targets in the adjuvant setting and synergize the effect of conventional chemotherapy in patients affected by breast cancers with limited therapeutic options, such as triple-negative breast cancers. Citation Format: Alice Turdo, Miriam Gaggianesi, Tiziana Apuzzo, Antonina Benfante, Aurora Chinnici, Alessandro Giammona, Simone Di Franco, Giorgio Stassi, Matilde Todaro. Autocrine and paracrine IL-4 maintains breast cancer stem cells traits via RAS/MAPK/DUSP pathway. [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 3311.

P62/SQSTM1 enhances breast cancer stem-like properties by stabilizing MYC mRNA

Aberrant p62 overexpression has been implicated in breast cancer development. Here, we found that p62 expression was elevated in breast cancer stem cells (BCSCs), including CD44+CD24− fractions, mammospheres, ALDH1+ populations and side population cells. Indeed, short-hairpin RNA (shRNA)-mediated knockdown of p62 impaired breast cancer cells from self-renewing under anchorage-independent conditions, whereas ectopic overexpression of p62 enhanced the self-renewal ability of breast cancer cells in vitro. Genetic depletion of p62 robustly inhibited tumor-initiating frequencies, as well as growth rates of BCSC-derived tumor xenografts in immunodeficient mice. Consistently, immunohistochemical analysis of clinical breast tumor tissues showed that high p62 expression levels were linked to poorer clinical outcome. Further gene expression profiling analysis revealed that p62 was positively correlated with MYC expression level, which mediated the function of p62 in promoting breast cancer stem-like properties. MYC mRNA level was reduced upon p62 deletion by siRNA and increased with p62 overexpression in breast cancer cells, suggesting that p62 positively regulated MYC mRNA. Interestingly, p62 did not transactivate MYC promoter. Instead, p62 delayed the degradation of MYC mRNA by repressing the expression of let-7a and let-7b, thus promoting MYC mRNA stabilization at the post-transcriptional level. Consistently, let-7a and let-7b mimics attenuated p62-mediated MYC mRNA stabilization. Together, these findings unveiled a previously unappreciated role of p62 in the regulation of BCSCs, assigning p62 as a promising therapeutic target for breast cancer treatments.

Abstract P1-06-09: Proto-oncogene PELP1 signaling regulates breast cancer stem cells via G9a/EHMT2

Abstract BACKGROUND: Evolving evidence suggests that cancer stem cells (CSCs) evade hormonal therapy and therapy resistance occurs due to regrowth of tumor cells from cancer stem cells that escaped hormonal therapy or remained in the body after tumor resection. Recent studies suggest that estrogen stimulates breast cancer stem-cells and G9a/EHMT2 plays a critical role in stem cell maintenance. Proline, glutamic acid, and leucine rich protein (PELP1) is a proto-oncogene that functions as a critical coregulator of several nuclear receptors and other transcription factors. PELP1 is commonly overexpressed in hormone-related cancers, and is prognostically linked to shorter breast cancer survival. Recent studies from our lab discovered PELP1 interacts with G9a/EHMT2. However, it remains unknown whether PELP1-G9a signaling plays a role in breast cancer stem cell proliferation. The objective of this study is to develop small molecular inhibitors that block G9a/EHMT2 interactions and to test their utility. METHODS: We isolated CD44high/CD24low CSCs from three breast cancer cell lines (ZR75, MCF7, T47D) using FACS. To test the effect of PELP1 inhibitors on CSCs, we cultured CSCs in SFM in the presence or absence of PELP1 inhibitors for a period of 7-10 days. Cells were analyzed for spheroid formation, morphological changes, immunofluorescence for differentiation markers, protein (Western) and RNA (RT-qPCR) analysis. Expression of differentiation markers K19 and K14 and stem cell markers CD133, CD44, Id1, Nestin, Musashi-1, SOX2, Notch2, and OCT1 was determined. RESULTS: Using mapping studies, we identified a small peptide inhibitor (PIP1) that interferes PELP1 interaction with G9/EHMT2. Utilizing Hit-Ligand interaction site with the PELP1 hot spot residues based on 3D alignment and shape, we have identified 61 potential hits from Ligand-Based screening using a 10,000 Diverse Set. Screening of these 61 potential hits using MTT based cell viability assays identified three small organic molecule inhibitors (peptidomimetics) as leads. All three peptidomimetics (#20, #29, #34) showed activity similar to PELP1 peptide inhibitor 1 (PIP1) in assays using three different breast cancer cell lines. Further, PELP1 targeting peptidomimetic disrupted PELP1 interaction with G9a/EHMT2. Peptidomimetic treatment inhibited the proliferation of tamoxifen therapy resistant cells. In mechanistic studies, we found that knockdown of PELP1 inhibited stem cell maintenance. In FACS analysis of ZR75, ZR75-PELP1 and ZR75-PELP1KD cells, the percentage of CD44high/CD24low cells correlated with PELP1 status. Accordingly, in mammosphere formation assays, PELP1 targeting peptidomimetic significantly inhibited the formation of mammospheres and the size of the mammospheres was also substantially decreased. Further, in self-renewal assays, peptidomimetic-treated cells had decreased self-renewal capacity. CONCLUSIONS: Collectively, our studies have discovered an essential role for PELP1 in breast cancer stem cell maintenance and identified the PELP1- G9a/EHMT2 axis as a potential therapeutic target for reducing stemness. Further, the novel small molecule inhibitors of PELP1 could be used for therapeutic targeting of breast cancer stem cells and therapy resistance. Citation Format: Viswanadhapalli S, Mann M, Sareddy GR, Xaionan L, Vankayalapati H, Brann D, Vadlamudi RK. Proto-oncogene PELP1 signaling regulates breast cancer stem cells via G9a/EHMT2. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-06-09.

Estradiol, TGF-β1 and hypoxia promote breast cancer stemness and EMT-mediated breast cancer migration.

Breast cancer is one of the most common cancer types among women, acting as a distinct cause of mortality, and has a high incidence of recurrence. External stimuli, including 17β-estradiol (E2), transforming growth factor (TGF)-β1 and hypoxia, may be important in breast cancer growth and metastasis. However, the effects of these stimuli on breast cancer stem cell (CSC) regulation have not been fully investigated. In the present study, the proportion of cluster of differentiation (CD)44+/CD24-/low cells increased following treatment with E2, TGF-β1 and hypoxia in MCF-7 cells. The expression of CSC markers, including SOX2, KLF4 and ABCG2, was upregulated continually by E2, TGF-β1 and hypoxia. In addition, the expression levels of epithelial-mesenchymal transition-associated factors increased following treatment with E2, TGF-β1 and hypoxia. Therefore, the migration ability of E2-, TGF-β1- and hypoxia-treated MCF-7 cells was enhanced compared with control cells. In addition, the enhancement of apoptosis by 5-flurouracil or radiation was abolished following treatment with E2, TGF-β1 and hypoxia. These results indicate that E2, TGF-β1 and hypoxia are important for regulating breast CSCs, and that the modulation of the microenvironment in tumors may improve the efficiency of breast cancer therapy.