EMT Transition Research Articles

361 FRA1 as the regulator of psoriasis-associated hyperproliferation and EMT transition of keratinocytes

361 FRA1 as the regulator of psoriasis-associated hyperproliferation and EMT transition of keratinocytes

MAD2B promotes tubular epithelial-to-mesenchymal transition and renal tubulointerstitial fibrosis via Skp2

Renal fibrosis activates MAD2B expression in renal tubules of human and mouse. TGF-β1 contributes to MAD2B enhancement in rat tubular epithelial cells. MAD2B depletion alleviates renal tubulointerstitial fibrosis in vivo and in vitro. MAD2B promotes EMT transition in rat tubular epithelial cells by inducing Skp2.

Abstract LB-239: Induction of therapeutic soluble decoy EGFR variants by antisense manipulation of RNA processing

Abstract Activation of the receptor tyrosine kinase (RTK) signaling pathways represents a key aspect of tumorigenesis in a broad range of human cancers. Thus, targeting of pathogenic RTKs, mainly with small molecule inhibitors and antibodies, has become an attractive therapeutic approach for cancer therapy. However, resistance by multiple mechanisms, including mutational re-activation of RTK signaling, activation of alternative RTK pathways, EMT transition and others, inevitably occurs over time. We recently identified a number of novel secreted soluble decoy RTKs isoforms (sdRTKs) generated by an U1-snRNP-dependent alternative intronic polyadenylation mechanism. We therefore developed a novel antisense-based method to effectively activate the expression of these sdRTKs, both in vitro and in vivo. This leads to the generation of endogenous dominant negative variants of RTKs, which can specifically affect the signaling of pathogenic RTKs and therefore provides a novel cancer therapy approach. Employing specific antisense compounds designed to activate intronic polyA sites, we are able to induce natural truncated EGFR variants that encode its extracellular ligand-binding domain but lack the intracellular signaling domain. These compounds thus can inhibit EGFR signaling by a three fold-mechanism: 1) removal of FL receptor, 2) sequestration of ligands and 3) non-productive dimerization with residual receptors. They are therefore designed to affect signaling both of targeted cells and of bystanders. Here we will show that, using this approach, we can target EGFR signaling both in vitro and in tumors. Importantly, with this method we can bypass the emergence of resistance typically seen with targeted drug treatments. Further development of this methodology will therefore provide the basis for an exciting novel approach to targeting and inhibiting oncogenic EGFR signaling and other pathways in a variety of cancers. Citation Format: Jeong Eun Park, Lee Spraggon, Luca Cartegni. Induction of therapeutic soluble decoy EGFR variants by antisense manipulation of RNA processing. [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 LB-239.

An improved strategy to detect the epithelial-mesenchymal transition process in circulating tumor cells in hepatocellular carcinoma patients.

We adopted a new strategy to explore the relationship between the EMT process of CTCs and hepatocellular carcinoma (HCC). Furthermore, we intend to illustrate the potential diagnostic value of CTCs of distinct phenotypes in HCC. The clinical data of 33 HCC patients and 10 healthy volunteers were collected retrospectively. By using the optimized CanPatrol CTC enrichment technique, patient blood samples of about 5ml were collected, and CTCs were identified and characterized. The first step of this detection process was to isolate CTCs via a filter-based method; then, an RNA in situ hybridization (RNA-ISH) technique based on the branched DNA signal amplification technology was used to classify the CTCs according to EMT markers. The relationships between HCC CTCs and clinical characteristics were analyzed. The number of epithelial CTCs was related to tumor size (r=0.456, p=0.008), epithelial-mesenchymal-mixed CTCs were related to tumor number (r=0.421, p=0.015), and mesenchymal CTC was associated with metastasis (r=0.375, p=0.032). There was no significant correlation between CTC number and other clinicopathological factors, such as age, serum AFP level or cirrhosis. Epithelial-mesenchymal-mixed CTCs seem to play an important role in EMT transition in HCC, mixed CTCs might be a vital factor for intrahepatic metastasis, and mesenchymal CTCs had the potential to be a predictor of extrahepatic metastasis.

The Nuclear Receptor, RORγ, Regulates Pathways Necessary for Breast Cancer Metastasis

We have previously reported that RORγ expression was decreased in ER−ve breast cancer, and increased expression improves clinical outcomes. However, the underlying RORγ dependent mechanisms that repress breast carcinogenesis have not been elucidated. Here we report that RORγ negatively regulates the oncogenic TGF-β/EMT and mammary stem cell (MaSC) pathways, whereas RORγ positively regulates DNA-repair. We demonstrate that RORγ expression is: (i) decreased in basal-like subtype cancers, and (ii) inversely correlated with histological grade and drivers of carcinogenesis in breast cancer cohorts. Furthermore, integration of RNA-seq and ChIP-chip data reveals that RORγ regulates the expression of many genes involved in TGF-β/EMT-signaling, DNA-repair and MaSC pathways (including the non-coding RNA, LINC00511). In accordance, pharmacological studies demonstrate that an RORγ agonist suppresses breast cancer cell viability, migration, the EMT transition (microsphere outgrowth) and mammosphere-growth. In contrast, RNA-seq demonstrates an RORγ inverse agonist induces TGF-β/EMT-signaling. These findings suggest pharmacological modulation of RORγ activity may have utility in breast cancer.

Abstract 3079: Mechanism of hsa-miR-15a in EMT and chemoresistance in colorectal cancer

Abstract Epigenetic alterations such as non-coding microRNAs are critical factors in EMT and chemoresistance. Hsa-miR-15a, as one of the first miRNA identified to be associated with cancer, has been investigated for its role in several types of cancer. We are interested in understanding the molecular and cellular mechanism of hsa-miR-15a in colorectal cancer as hsa-miR-15a is down-regulated in colon cancer and associated with poor patient prognosis. In this study, we have identified several direct key targets of hsa-miR-15a that impact EMT transition, cell cycle and apoptosis. Our results show that the expression of BMI-1, an important regulator of EMT, was suppressed by hsa-miR-15a. The reduction of hsa-miR-15a in colorectal cancer patient samples was significantly inversely correlated with BMI-1 over-expression. Hsa-miR-15a also suppresses the expression of anti-apoptotic protein BCL-2. The suppression of BMI-1, BCL-2 as well as other key targets by hsa-miR-15a has potential for increasing chemosensitivity as well as disrupting cancer stem cell self renewal. In addition, hsa-miR-15a also has potential as a prognostic biomarker in colorectal cancer. This work will provide a further understanding of the mechanism of hsa-miR-15a loss in colorectal cancer and its clinical significance in relationship with its targets and pathways. The functional significance of hsa-miR-15a in regulating key targets and pathways establishes the foundation for hsa-miR-15a as a potential novel therapeutic strategy for treating colorectal cancer. Citation Format: Andrew Fesler, Haiyan Zhai, Jingfang Ju. Mechanism of hsa-miR-15a in EMT and chemoresistance in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3079. doi:10.1158/1538-7445.AM2015-3079

Abstract 588: Lrig1 suppresses breast cancer stem cell expansion and promotes mesenchymal to epithelial transition

Abstract Leucine-rich repeats and immunoglobulin-like domains-1 (Lrig1) is known as a negative regulator of oncogenic receptor tyrosine kinases (ErbB, Met and Ret); and low Lrig1 expression correlates with poor patient prognosis in breast cancer. Lrig1 has been found to maintain stem cell quiescence and marks a subset of quiescent intestinal stem cells where its loss leads to heightened ErbB expression and duodenal adenomas. Lrig1 is defined as a tumor suppressor in healthy tissue such as intestine and epidermis where Lrig1 loss leads to stem cell expansion and epidermal hyperplasia. However, the role of Lrig1 in cancer stem cells (CSCs) and as a metastatic suppressor is unknown. Lrig1 expression is lowest in the highly invasive breast cancer subtype MDA-MB-231, and Lrig1 expression is highest in the weakly invasive luminal A breast cancer subtype. In patients, high Lrig1 expression exhibits longer relapse-metastatic free survival suggesting that Lrig1 expression protects against metastatic relapse. We hypothesized that Lrig1 suppresses breast CSC expansion and promotes mesenchymal to epithelial transition. Our results indicated that restoration of Lrig1 in highly aggressive cell lines promoted mesenchymal to epithelial transition (MET) and a loss of invasive morphology in 3D matrigel cell culture. These results demonstrated that Lrig1 suppressed cell proliferation in both 2D and 3D matrigel cultures and suppressed tumorsphere formation 10-fold. Importantly, we have shown that Lrig1 is down-regulated in the hTERT-immortalized mammary epithelial cell line-twist ER, (HMLE-twist ER) which undergoes EMT. When Lrig1 is knocked-down in HMLE-twist ER by shRNA, the results showed that loss of Lrig1 enhanced mesenchymal and stem cell expansion. These observations suggest that Lrig1 may play a role in EMT transition in metastasis, as well as providing a whole new area of therapeutic targets in breast cancer along with an improved basic understanding in cancer stem cell biology. Note: This abstract was not presented at the meeting. Citation Format: Nucharee Yokdang, Colleen Sweeney. Lrig1 suppresses breast cancer stem cell expansion and promotes mesenchymal to epithelial transition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 588. doi:10.1158/1538-7445.AM2014-588

Abstract 3044: Cancer associated fibroblasts-derived HGF regulates cancer stem cell-like properties in hepatocellular carcinoma

Abstract Cancer stem cells (CSCs) play pivotal roles in tumor growth and therapeutic response; hence it is of fundamental importance to understand how CSCs is regulated inside the tumor mass. Similar to the regulation of normal stem cells by their ‘niche’, CSCs are also regulated by cells within the tumor microenvironment. Although most cases of HCC develop within a background of cirrhosis in which liver tissue is enriched in activated myofibroblasts, the role of cancer associated fibroblasts (CAFs) on liver CSCs within the tumor microenvironment has not yet studied. In order to achieve the goal of developing an effective therapy for HCC by specifically eliminating liver CSCs, we aim to dissect the cross-talks between HCC cells and CAFs. To study this, CAFs from fresh HCC tissues was successfully isolated, cultured and characterized by α-SMA immunoreactivity. Conditioned medium from CAFs enriched the populations of liver CSCs as reflected by an increased ability of self-renewal, chemoresistance, invasiveness and expression of stemness-associated genes and markers CD44 and CD47; and its CSC- stimulating effect was further enhanced when CAFs was stimulated with the conditioned medium of HCC cells. Using cytokine antibody array, hepatocyte growth factor (HGF) was found to be preferentially secreted by CAFs and increased upon stimulation by HCC cells. To verify whether the effect of CAFs on regulation of liver CSCs is due to HGF, the effect of recombinant HGF on regulation of liver CSCs was examined. Recombinant HGF at the physiological levels of CAFs (2ng/mL and 10ng/mL) promoted stem-like properties of HCC cells, and the effect of CAFs on CSC properties was alleviated with addition of HGF neutralizing antibody and c-met inhibitor (PHA665-752). In a cohort of HCC patient’s samples, HGF expression was significantly correlated with α-SMA (p=0.0003), suggesting the paracrine effect of CAFs on HCC cells. Further experiments showed that HCC cells induced proliferation and migration of CAFs to tumor-associated stroma; and CAFs in turn secretes HGF to regulate the CSC plasticity by activating NF-κB-mediated EMT transition. This study potentially dissects the signaling cross-talks between HCC cells and CAFs within the tumor microenvironment which may potentially open a new avenue to develop targeted therapy against this deadly disease. Citation Format: Yuen Ting Lau, Jessica Lo, Irene Oi Lin Ng, Terence Kin Wah Lee. Cancer associated fibroblasts-derived HGF regulates cancer stem cell-like properties in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3044. doi:10.1158/1538-7445.AM2014-3044

IL-7 splicing variant IL-7δ5 induces EMT and metastasis of human breast cancer cell lines MCF-7 and BT-20 through activation of PI3K/Akt pathway.

Our previous study has confirmed that IL-7δ5 (an IL-7 variant lacking exon 5) promotes breast cancer growth. However, whether IL-7δ5 is involved in tumor cell EMT and metastasis remains unclear. In this study, we investigated the preclinical effects and molecular mechanisms of IL-7δ5 on EMT and metastasis in human MCF-7 and BT-20 breast cancer cells in vitro and in vivo. The results showed that IL-7δ5 induced EMT and invasion in tumor cells, associated with up-regulation of N-cadherin and the down-regulation of E-cadherin. Furthermore, we found that IL-7δ5 induced the activation of Akt. Inhibition of PI3K/Akt pathway by LY294002 reversed the EMT transition in breast cancer cell lines MCF-7 and BT-20 induced by IL-7δ5. In addition, IL-7δ5 enhanced cancer metastasis and shortened survival time, with increased level changes of activated Akt in nude mice with breast cancer. In conclusion, our findings demonstrate that IL-7δ5 induces human breast cancer cell lines EMT and metastasis via activation of PI3K/Akt pathway. Thus, IL-7δ5 may be a potential target against human breast cancer.

Over-Expression of Platelet-Derived Growth Factor-D Promotes Tumor Growth and Invasion in Endometrial Cancer

The platelet-derived growth factor-D (PDGF-D) was demonstrated to be able to promote tumor growth and invasion in human malignancies. However, little is known about its roles in endometrial cancer. In the present study, we investigated the expression and functions of PDGF-D in human endometrial cancer. Alterations of PDGF-D mRNA and protein were determined by real time PCR, western blot and immunohistochemical staining. Up-regulation of PDGF-D was achieved by stably transfecting the pcDNA3-PDGF-D plasmids into ECC-1 cells; and knockdown of PDGF-D was achieved by transient transfection with siRNA-PDGF-D into Ishikawa cells. The MTT assay, colony formation assay and Transwell assay were used to detect the effects of PDGF-D on cellular proliferation and invasion. The xenograft assay was used to investigate the functions of PDGF-D in vivo. Compared to normal endometrium, more than 50% cancer samples showed over-expression of PDGF-D (p < 0.001), and high level of PDGF-D was correlated with late stage (p = 0.003), deep myometrium invasion (p < 0.001) and lympha vascular space invasion (p = 0.006). In vitro, over-expressing PDGF-D in ECC-1 cells significantly accelerated tumor growth and promoted cellular invasion by increasing the level of MMP2 and MMP9; while silencing PDGF-D in Ishikawa cells impaired cell proliferation and inhibited the invasion, through suppressing the expression of MMP2 and MMP9. Moreover, we also demonstrated that over-expressed PDGF-D could induce EMT and knockdown of PDGF-D blocked the EMT transition. Consistently, in xenografts assay, PDGF-D over-expression significantly promoted tumor growth and tumor weights. We demonstrated that PDGF-D was commonly over-expressed in endometrial cancer, which was associated with late stage deep myometrium invasion and lympha vascular space invasion. Both in vitro and in vivo experiments showed PDGF-D could promote tumor growth and invasion through up-regulating MMP2/9 and inducing EMT. Thus, we propose targeting PDGF-D to be a potent strategy for endometrial cancer treatment.

Impact of deviated balance of regulatory and cytotoxic T cells on release and reseeding of circulating tumor cells in hepatocellular carcinoma.

e22133 Background: Our previous data showed that a preoperative CTC ≥2 was significantly related with relapse of hepatocellular carcinoma (HCC) after surgery. However, how CTCs managed to evade immune surveillance, invade into circulation and reseed in the original organ still remains largely unclear. In this study, we illustrated the pro-tumor effect of Treg infiltration in the context of CTC releasing and reseeding. Methods: Using CellSearch system, 115 HCC patients were tested for CTCs prior to resection. Foxp3+, CD8+ and Granzyme B-positive (GrB+) tumor -infiltrating lymphocytes were evaluated by immunohistochemistry (IHC) in tissue microarrays. For IHC markers, the cutoff for stratification subsets was the median value. The pro-invasiveness properties of Tregs were analyzed in both in vitro and vivo assays. Results: Twenty-two patients with high Tregs and low activated CTLs in tumoral tissues had increased number of CTCs compared to 22 patients with low Tregs and high activated CTLs (6.68 vs. 0.91, P&lt;0.001). In 77 patients with detectable CTC, high Tregs and low activated CTLs in peritumoral tissues was significantly associated with an increased incidence of HCC recurrence (80.5% vs. 19.4%, P&lt;0.001), and was an independent prognostic factor for HCC early recurrence (P=0.007). Further in vitro study showed Tregs-derived TGF-β promoted HCC cell migration and invasion; down-regulated E-cadherin and up-regulated N-cadherin, vimentin, fibronectin and snail. The data in human tumor sample also showed that the TGF-β level had positive relationships with Treg count, N-cadherin level and CTC count; and had inverse relationship with E-cadherin (all P values &lt;0.05). In mice, depletion of Tregs by neutralizing antibody abolished intrahepatic colonization and lung metastasis. Conclusions: Local immune contexture in HCC orchestrates with biological behavior of CTCs. High intratumoral Tregs may promote the release of CTCs through inducing tumor cells EMT transition via TGF-β secretion. High peritumoral Tregs created an immune-subversive microenvironment by suppressing CTL to favor colonization of CTCs, which led to recurrence after HCC resection.

Abstract LB-318: Reshaping the cancer proteome: therapeutic targeting of signaling and resistance in cancer by antisense manipulation of RNA processing.

Abstract Activation of the receptor tyrosine kinase (RTK) signaling pathways represents a key aspect of tumorigenesis in a broad range of human cancers. Thus, targeting of pathogenic RTKs, mainly with small molecule inhibitors and antibodies, has become an attractive therapeutic approach for cancer therapy. However, resistance by multiple mechanisms, including mutational re-activation of RTK signaling, activation of alternative RTK pathways, EMT transition and others, inevitably occurs over time. We recently identified a number of novel secreted soluble decoy RTKs isoforms (sdRTKs) generated by a U1-snRNP-dependent alternative intronic polyadenylation mechanism. We therefore developed a novel antisense-based method to effectively activate the expression of these sdRTKs, both in vitro and in vivo. This leads to the generation of endogenous dominant negative variants of RTKs, which can specifically affect the signaling of pathogenic RTKs and therefore provides a novel cancer therapy approach. Employing specific antisense compounds designed to activate intronic polyA sites, we are able to induce natural truncated EGFR variants that encode its extracellular ligand-binding domain but lack the intracellular signaling domain. These compounds thus can inhibit EGFR signaling by a three fold-mechanism: 1) removal of FL receptor, 2) sequestration of ligands and 3) non-productive dimerization with residual receptors. They are therefore designed to affect signaling both of targeted cells and of bystanders. Here we will show that, using this approach, we can target EGFR signaling both in vitro and in tumors. Importantly, with this method we can bypass the emergence of resistance typically seen with targeted drug treatments. Further development of this methodology will therefore provide the basis for an exciting novel approach to targeting and inhibiting oncogenic EGFR signaling and other pathways in a variety of cancers. Citation Format: Luca Cartegni, lee Spraggon. Reshaping the cancer proteome: therapeutic targeting of signaling and resistance in cancer by antisense manipulation of RNA processing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-318. doi:10.1158/1538-7445.AM2013-LB-318

Abstract 4023: Comparison of transcriptional signatures in US African American and Kenyan TNBC samples identifies differential expression in key oncogenic pathways.

Abstract Strong epidemiological data supports breast cancer (BC) as the second leading cause of cancer death among US African American (AA) women, with a 20% greater mortality rate than that in Caucasians (Cauc). Collection of similar BC incidence and mortality data for Eastern Africa is limited, however, it is known that BC presents as advanced-stage disease, comprised mainly of poorly differentiated cancers that are less likely to be hormone responsive. Our combined investigative team continues to analyze a cohort of indigenous Kenyan BC patients, in order to begin to understand biological similarities and differences between BC in native African and AA patients. Previous related gene expression studies conducted in a Triple Negative Breast Cancer (TNBC) stage-matched multi-ethnic US cohort suggested differential expression patterns across ethnicities. We are extending this focus to similar studies involving native African samples. Archived BC pathology samples (FFPE) were either obtained from University of Miami or from a native African tumor bank (Kenya). Forty-seven BC samples were contributed from Kenya, and re-analyzed (in Miami) for ER/PR/ Her2Neu status, resulting in confirmation of 29 Kenyan TNBC cases. 10 um sections were cut from each tumor FFPE block, and following RNA isolation and cDNA preparation, hybridized to the Almac breast-enriched gene expression array (Breast Cancer DSA Research Tool). After quality control assessment of array data, a total of 60,856 gene/probes were analyzed by RMA; normalized, log transformed to the median, and compared on GeneSpring® analytical software. Data sets were separated by further analysis based on node status [(N0-Kenyan-7, AA-10); (Mixed Node-Kenyan-16, AA-7)]. Unpaired student's T-test was performed on all subsets and the resulting p-values were corrected for Multiple Testing using Benjamin-Hochberg. Differentially expressed genes/probes were extracted for each subset using p-value &amp;lt; 0.01 and fold change of &amp;gt; 1.5. These probe/gene lists were further analyzed using GeneGo pathway analysis and enriched pathways were identified. Comparisons of transcriptional differences between Kenyan and AA TNBC, regardless of node status, suggest expression alterations in several key pathways, including EMT Transition, Cytoskeleton Remodeling, Immune Response and Epigenetic Control. These preliminary results are being validated by QPCR and other methodologies. In conclusion, we have demonstrated that high quality RNA can be extracted from archived FFPE tumor samples, including samples from East Africa. This observation raises the possibility of future genomic analyses using archived samples. We are intrigued by our study observations, which suggest that inherent gene expression differences exist between Kenyan and AA TNBC samples in previously-recognized pathways important in onocogenesis. Citation Format: Lisa L. Baumbach-Reardon, Julie Getz, Mary Ellen Ahearn, Carmen Gomez, Peter Bird, John Carpten, Mark Pegram. Comparison of transcriptional signatures in US African American and Kenyan TNBC samples identifies differential expression in key oncogenic pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4023. doi:10.1158/1538-7445.AM2013-4023

Abstract 3311: Role of nicastrin in the maintenance of breast cancer stem cells and invasion

Abstract Background: Cancer stem-cells may be involved in development and metastasis of tumours, including breast cancer. Initial identification of breast cancer stem cells (BCSCs) was based on a combination of CD44+, CD24- and Lin- markers. Later, several markers such as aldehyde dehydrogenase 1 (ALDH1) were defined. The notch signaling pathway has been implicated in BCSC survival. Gamma-secretase (GS) is an enzymatic multi-protein complex composed of presenilin 1 (PS1), nicastrin (NCT), anterior pharynx-defective phenotype 1 (APH-1), and the PS enhancer 2 (PEN-2) proteins, which activates Notch. We have previously shown that high levels of NCT observed in 47.5% of BC patients, confers worse overall survival in the ERα negative patients. Here, we have explored targeting NCT, the vital component of GS assembly and function, to determine its role in the maintenance of the BCSC pool. Methods: Stable NCT knock-down HCC1806 breast cancer cells (HCC1806 ShNCT) and control cells (HCC1806 ShLUC) were produced by lentiviral infections and puromicin selection. Stable NCT overexpressing MCF10A cells (MCF10ANCT) and control cells (MCF10ACTRL) were produced by retroviral infections and puromicin selection. The sulphorhodamine B and three-dimentional (3D) assay in Matrigel were used to measure the proliferation rate of these cells. Invasion assay was perfomed using Matrigel-coated Boyden chamber. Stem cell content was evaluated by the percentage of CD44+/CD24- and ALDEFLUORhigh cells and mammospheres formation assay. In vivo expreriments were performed by subcutaneous injection of HCC1806 ShLUC and ShNCT into nude mice. Results: Stable knockdown of NCT in HCC1806 breast cancer cells achieved the following effects: disruption of GS formation and GS/Notch activity, reduction of the pool of BCSC as defined by CD44+/CD24- and ALDH1+ phenotype and inhibition of BCSC cell invasion. Moreover, NCT silencing reduced breast cancer xenograft formation in vivo. Intriguingly, MCF10ANCT presented significant morphological alterations when cultured in a 3D context. Compared to control cells, the epithelial-mesenchymal (EMT) and invasion cell markers, vimentin, Twist and Snail are significantly up-regulated in MCF10ANCT cells. In addition, the over-expression of NCT promoted the generation of a BCSC phenotype characterized by the CD44+/CD24- phenotype and increased ALDH1 activity (up to 3 fold). Finally, using DAPT and Wortmannin as pharmacological inhibitors of Notch and AKT signalling pathways respectively, we found a reversion of the malignant phenotype of MCF10ANCT cells in terms of invasion and stem cell content. Conclusions: These results suggest that therapies directed against NCT may not only reduce tumor size but also the number and the invasive capacity of BCSC. Moreover, we have demonstrated that, even in normal breast epithelial cell line MCF10A, high expression of NCT can induce a Notch/pAKT-dependent EMT transition program. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3311. doi:1538-7445.AM2012-3311

The role of the mesenchymal stem cells on breast cancer: friends or foes?

Mesenchymal stem cells (MSCs) have been the subject of an increased interest. Because of their ability to give rise to bone, cartilage, fat and muscle, their role in regenerative medicine has been extensively studied and the fact that they can be recruited at sites of inflammation and tissue repair has prompted their potential use as tissue regenerative cells. Contextually there has been a growing interest in the role of MSCs in cancer progression. The nature of the relationship between MSCs and tumor cells appears dual, with effects pro- as well as anti-tumorigenic. This paradox depends on the source and the degree of differentiation of MSCs and the tumor model used. Moreover, with the large range of cytokines and growth factors they produce, MSCs exert regulatory function on apoptosis, angiogenesis and an immunomodulatory role. Here we evaluate the interaction between MSCs derived from the periprosthetic capsule of mastectomyzed women and the breast cancer cell line MCF-7. Capsular tissue around breast implants is a normal inflammatory reaction versus a foreign body and it is rich of MSCs. To asses how MSCs interact with tumor cells, MCF-7 cells were incubated with medium previously conditioned by MSCs or directly co-coltured with MSCs; subsequently, we evaluated the proliferation and the expression of genes implicated in different pathways (angiogenesis, proliferation, anti-apoptosis, EMT transition). Our results showed that MCF-7 cells cultured together MSCs or using their conditioned medium have a more elevated proliferation rate but tumour cells seem less aggressive, like attested by a reduction of the expression of selected genes. The understanding of the mechanisms that control the interaction between MSCs and tumor cells is still at an early stage but recent literature confirm that MSCs and their progeny are not innocent bystanders in the tumor microenvironment. The study of these interactions is a critical area of future investigation that is needed to better define their role in cancer progression and their potential as therapeutic agents or targets.

Cancer treatments transform residual cancer cell phenotype

BackgroundPhysiologic wound repair and tissue regeneration are associated with distinct cellular behaviors triggered by tissue damage. Normally quiescent stem cells proliferate to regenerate damaged tissue, while relatively immobile epithelial cells can transform into a motile, tissue invasive phenotype through a partial epithelial-mesenchymal transition. These distinct cellular behaviors may have particular relevance to how cancer cells can be predicted to behave after treatments damaging a tumor.Presentation of the hypothesisSurgery, chemotherapy, and radiation therapy trigger highly conserved wound healing pathways that: (1) facilitate the phenotypic transformation of surviving cancer cells into a highly mobile, metastatic phenotype through an EMT or epithelial-mesenchymal transition and (2) induce residual cancer stem cell proliferation.Testing the hypothesisTissue damage caused by cancer treatments will trigger the release of distinct cytokines with established roles in physiologic wound healing, EMT induction, and stem cell activation. They will be released rapidly after treatment and detectable in the patient's blood. Careful histologic evaluation of cancerous tissue before and after treatment will reveal cellular changes suggestive of EMT induction (down regulation of cytokeratin expression) and cancer stem cell enrichment (stem cell markers upregulated).Implications of the hypothesisCancer cells surviving treatment will be more capable of metastasis and resistant to conventional therapies than the pre-treatment population of cancer cells. These changes will develop rapidly after treatment and, in distinct contrast to selection pressures fostering such changes, be triggered by highly conserved wound repair signals released after tissue damage. This pattern of tissue (tumor) repair may be amenable to treatment intervention at the time it is upregulated.