Expansion Of Cancer Stem Cells Research Articles

Glucocorticoid receptor signalling activates YAP in breast cancer

The Hippo pathway is an oncosuppressor signalling cascade that plays a major role in the control of cell growth, tissue homoeostasis and organ size. Dysregulation of the Hippo pathway leads to aberrant activation of the transcription co-activator YAP (Yes-associated protein) that contributes to tumorigenesis in several tissues. Here we identify glucocorticoids (GCs) as hormonal activators of YAP. Stimulation of glucocorticoid receptor (GR) leads to increase of YAP protein levels, nuclear accumulation and transcriptional activity in vitro and in vivo. Mechanistically, we find that GCs increase expression and deposition of fibronectin leading to the focal adhesion-Src pathway stimulation, cytoskeleton-dependent YAP activation and expansion of chemoresistant cancer stem cells. GR activation correlates with YAP activity in human breast cancer and predicts bad prognosis in the basal-like subtype. Our results unveil a novel mechanism of YAP activation in cancer and open the possibility to target GR to prevent cancer stem cells self-renewal and chemoresistance.

Shp2 promotes liver cancer stem cell expansion by augmenting β-catenin signaling and predicts chemotherapeutic response of patients.

Shp2 could promote HCC cell dedifferentiation and liver CSC expansion by amplifying β-catenin signaling and may be useful in predicting patient response to chemotherapeutics. (Hepatology 2017;65:1566-1580).

Isolation, Characterization, and Expansion of Cancer Stem Cells.

The ability to isolate, characterize, and expand distinct tumor cell populations from primary tissue or xenografts is vital to identifying molecular mechanisms specific to cancer stem cells. Once cells have been extracted from tissue, there are multiple methods by which they can be sorted and cultured. We will describe the approaches that can be taken from cancer stem cell isolation through expansion, including Magnetic-activated Cell Sorting (MACS), Fluorescence-activated Cell Sorting (FACS), the use of reporter systems, and various cell culture methods.

Leptin-Notch signaling axis is involved in pancreatic cancer progression

Pancreatic cancer (PC) shows a high death rate. PC incidence and prognosis are affected by obesity, a pandemic characterized by high levels of leptin. Notch is upregulated by leptin in breast cancer. Thus, leptin and Notch crosstalk could influence PC progression. Here we investigated in PC cell lines (BxPC-3, MiaPaCa-2, Panc-1, AsPC-1), derived tumorspheres and xenografts whether a functional leptin-Notch axis affects PC progression and expansion of pancreatic cancer stem cells (PCSC). PC cells and tumorspheres were treated with leptin and inhibitors of Notch (gamma-secretase inhibitor, DAPT) and leptin (iron oxide nanoparticle-leptin peptide receptor antagonist 2, IONP-LPrA2). Leptin treatment increased cell cycle progression and proliferation, and the expression of Notch receptors, ligands and targeted molecules (Notch1-4, DLL4, JAG1, Survivin and Hey2), PCSC markers (CD24/CD44/ESA, ALDH, CD133, Oct-4), ABCB1 protein, as well as tumorsphere formation. Leptin-induced effects on PC and tumorspheres were decreased by IONP-LPrA2 and DAPT. PC cells secreted leptin and expressed the leptin receptor, OB-R, which indicates a leptin autocrine/paracrine signaling loop could also affect tumor progression. IONP-LPrA2 treatment delayed the onset of MiaPaCa-2 xenografts, and decreased tumor growth and the expression of proliferation and PCSC markers. Present data suggest that leptin-Notch axis is involved in PC. PC has no targeted therapy and is mainly treated with chemotherapy, whose efficiency could be decreased by leptin and Notch activities. Thus, the leptin-Notch axis could be a novel therapeutic target, particularly for obese PC patients.

Erythrocytosis in hepatocellular carcinoma portends poor prognosis by respiratory dysfunction secondary to mitochondrial DNA mutations.

These findings suggest clinical and therapeutic implications for erythrocytosis in hepatocellular carcinoma. There is an underlying link between mitochondrial function and hypoxia inducible factor alpha signaling, revealing a mechanism of erythrocytosis in a subset of hepatocellular carcinoma patients who may benefit from treatment involving EPO signaling interference. (Hepatology 2017;65:134-151).

Ferritin heavy chain is a negative regulator of ovarian cancer stem cell expansion and epithelial to mesenchymal transition

ObjectivesFerritin is the major intracellular iron storage protein essential for maintaining the cellular redox status. In recent years ferritin heavy chain (FHC) has been shown to be involved also in the control of cancer cell growth. Analysis of public microarray databases in ovarian cancer revealed a correlation between low FHC expression levels and shorter survival. To better understand the role of FHC in cancer, we have silenced the FHC gene in SKOV3 cells.ResultsFHC-KO significantly enhanced cell viability and induced a more aggressive behaviour. FHC-silenced cells showed increased ability to form 3D spheroids and enhanced expression of NANOG, OCT4, ALDH and Vimentin. These features were accompanied by augmented expression of SCD1, a major lipid metabolism enzyme. FHC apparently orchestrates part of these changes by regulating a network of miRNAs.MethodsFHC-silenced and control shScr SKOV3 cells were monitored for changes in proliferation, migration, ability to propagate as 3D spheroids and for the expression of stem cell and epithelial-to-mesenchymal-transition (EMT) markers. The expression of three miRNAs relevant to spheroid formation or EMT was assessed by q-PCR.ConclusionsIn this paper we uncover a new function of FHC in the control of cancer stem cells.

MiR-199a-3p targets stemness-related and mitogenic signaling pathways to suppress the expansion and tumorigenic capabilities of prostate cancer stem cells.

Human cancers exhibit significant cellular heterogeneity featuring tumorigenic cancer stem cells (CSCs) in addition to more differentiated progeny with limited tumor-initiating capabilities. Recent studies suggest that microRNAs (miRNAs) regulate CSCs and tumor development. A previous library screening for differential miRNA expression in CD44+ (and other) prostate CSC vs. non-CSC populations identified miR-199a-3p to be among the most highly under-expressed miRNAs in CSCs. In this study, we characterized the biological functions of miR-199a-3p in CD44+ prostate cancer (PCa) cells and in tumor regeneration. Overexpression of miR-199a-3p in purified CD44+ or bulk PCa cells, including primary PCa, inhibited proliferation and clonal expansion without inducing apoptosis. miR-199a-3p overexpression also diminished tumor-initiating capacities of CD44+ PCa cells as well as tumor regeneration from bulk PCa cells. Importantly, inducible miR-199a-3p expression in pre-established prostate tumors in NOD/SCID mice inhibited tumor growth. Using target prediction program and luciferase assays, we show mechanistically that CD44 is a direct functional target of miR-199a-3p in PCa cells. Moreover, miR-199a-3p also directly or indirectly targeted several additional mitogenic molecules, including c-MYC, cyclin D1 (CCND1) and EGFR. Taken together, our results demonstrate how the aberrant loss of a miRNA-mediated mechanism can lead to the expansion and tumorigenic activity of prostate CSCs, further supporting the development and implementation of miRNA mimics for cancer treatment.

Abstract 2531: Marker free isolation and expansion of cancer stem cells from small cell lung cancer

Abstract Small cell lung cancer (SCLC) accounts for approximately 15% of all lung cancer cases and, contrary to the advances in diagnostics and therapeutic options for non small cell lung cancer, SCLC long term survival has failed to materially improve over the last 3 decades. Early dissemination with hematogenous metastasis, advanced stages at diagnosis, dramatic response to chemotherapy with early and aggressive relapses are the clinical hallmarks. Limited surgical samples, early palliation and limited second line treatment options negating the benefit of repeat biopsies have limited tissue available for research to understand the biology of this relatively rare tumor. Recent data has suggested the presence of rare populations of cells within the primary SCLC tumors which have stem cell-like properties. We hypothesize that novel in vitro culture platforms and methods can be used to isolate and expand cancer stem cells (CSCs) within SCLC tumors and that the expanded CSCs will yield a renewable cell source for research applications and for predictive drug response profiling. Many groups have relied upon cell surface markers such as CD133 and CD24 to identify the CSC population. Our approach is unique in that we have employed a marker-free approach utilizing a combination of chemical and functional isolation. We then use a combination of growth factors, extracellular matrix proteins, and oxygen tension in a 3D perfusion culture system to further isolate, purify and expand isolated cells with the goal of having quantities of functional SCLC stem cells for molecular, proteomic and functional in vitro and in vivo assays. Initial cell sources will include immortalized cell lines and patient derived xenograft lines, with eventual adaptation to primary patient samples. Using numerous techniques, we have qualified these cells based upon marker expression (flow cytometry, RT-PCR), dye exclusion, drug response, and limited dilution assays. Future experiments will confirm stemness with limited dilution tumorigenicity assays in vivo. Initial data suggest poor correlation of canonical CSC marker expression with clonogenic growth in cell lines, supporting the need for unbiased and functional isolation methods. These expanded populations of CSCs may be used to identify novel CSC markers and/or targets, mechanisms of resistance and screen and develop novel therapeutics Our ultimate goals are to be able to use our novel culture platforms and methods to expand CSCs for real time precision medicine applications to identify novel therapies in real time that can eradicate the CSC population and improve outcomes of patients with SCLC. Citation Format: Tessa M. DesRochers, Melissa Millard, Alina Lotstein, Lillia Holmes, Hal E. Crosswell. Marker free isolation and expansion of cancer stem cells from small cell lung cancer. [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 2531.

Abstract 2124: Notch-1 mediated down regulation of PTEN in trastuzumab resistant HER2+ breast cancer

Abstract ErbB-2 (HER2) is over expressed in 15 - 25% of breast cancer. First line therapy for HER2+ breast cancer is a humanized, monoclonal antibody, trastuzumab. Resistance is a major clinical problem. Previous work has suggested that Notch-1 is critical for resistance. PTEN, a breast tumor suppressor and potent inhibitor of the PI3-K/AKT is decreased in HER2+ breast cancer and loss of PTEN predicts for worst outcome. Notch-1 downregulates PTEN expression and we sought to determine the mechanism by which Notch-1 attenuates PTEN and whether this decrease is necessary for trastuzumab resistance. Our hypothesis is that Notch-1 decreases PTEN to maintain trastuzumab resistance. Human HER2+ breast cancer cell lines: sensitive (BT474HS) or resistant (BT474HR and HCC1954) to trastuzumab were used to measure Notch-1 modulation of PTEN expression when treated with trastuzumab. Protein and transcript expression of PTEN were measured by Western blot and RT-PCR, respectively. ChIP assays were preformed to identify whether Notch-1 regulated PTEN expression was mediated by direct recruitment of Notch-1 to the PTEN promoter. Growth and mammosphere forming assays were performed to determine if trastuzumab resistance could be rescued in trastuzumab treated cells with simultaneous Notch-1 and PTEN knockdown, or increased downstream PI3K pathway activity using a constitutive active form of AKT1 (Myr-AKT1). Western blot and RT-PCR results showed that Notch-1 protein levels are increased while PTEN mRNA and protein levels are decreased in trastuzumab resistant cells (BT-474HR) compared to sensitive (BT-474HS) cells. Results revealed that Notch-1 knockdown increased PTEN transcript and protein expression. Notch-1 ChIP assays demonstrated that Notch-1 was enriched on the PTEN promoter and a Notch target gene promoter, HEY1 in resistant cells. Notch1 knockdown significantly decreased proliferation and mammosphere forming efficiency (MFE) of resistant cells regardless of trastuzumab treatment. PTEN knockdown rescued the decrease in proliferation and MFE mediated by Notch-1 knockdown. As PTEN is an attenuator of PI3-K/AKT signaling, we asked whether constitutive activation of AKT1 was sufficient to rescue proliferation upon Notch-1 knockdown. The results showed that myr-AKT1 increased AKT1 signaling and rescued the decrease in proliferation mediated by Notch-1 knockdown. These results demonstrate, to our knowledge, for the first time that Notch-1 is a direct transcriptional repressor of PTEN in HER2+ breast cancer cells. Increased activation of the PI3K pathway through Notch-1 mediated inhibition of PTEN is necessary to maintain proliferation and possibly survival of the cancer stem cell population of trastuzumab resistant cells. Inhibiting the expression and/or activity of Notch-1 in HER2+ breast cancer could prove to be an effective therapy to prevent expansion of cancer stem cells and/or re-sensitize HER2+ breast cancer to ant-HER2 therapy. Citation Format: Andrew T. Baker, Kinnari Pandya, Clodia Osipo. Notch-1 mediated down regulation of PTEN in trastuzumab resistant HER2+ breast cancer. [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 2124.

Abstract 5237: Protection of tocopherols against estrogen-induced breast cancer via mechanisms targeting cancer stem cells

Abstract Estrogens have been implicated to be complete carcinogens through mechanisms involving increased proliferation, oxidative stress and DNA damage. Although the importance of estrogen in breast cancer is well established, the effects of estrogen on breast cancer stem cells are not fully understood. Due to the role of breast cancer stem cells in tumor initiation, maintenance, and progression, targeting cancer stem cells has become important for understanding estrogen-mediated breast carcinogenesis. The vitamin E family, which is consisted of four different forms of tocopherols and tocotrienols, has been shown to exert anti-tumor effects in animal models of various cancers. In the present study, we examined the protective effects of α-, γ-, δ-tocopherol as well as a natural γ-tocopherol rich mixture of tocopherols (γ-TmT) against estrogen-induced expansion of cancer stem-like cells and estrogen-mediated tumor growth. To determine the effects of tocopherols on breast cancer stem cells, the MCF-7 mammosphere cell culture system, which enriches for mammary progenitor cells and putative breast cancer stem cells, was utilized. Treatment of MCF-7 mammosphere with estrogen for six days resulted in an increase in CD44+/CD24- stem-like cell populations as well as the number and size of mammosphere. α-, γ-, δ-Tocopherol, and most significantly γ-TmT, inhibited estrogen-induced mammosphere formation, indicating that tocopherols reverse estrogen-induced expansion of breast cancer stem cells. In MCF-7 xenograft tumor study, MCF-7 cells (5 × 106 cells/mouse) were injected into the mammary fat pad in immunodeficient mice after subcutaneous implantation with 0.72 mg of estrogen pellet. Mice were then administered with diets containing 0.2% α-, γ-, δ-tocopherol and γ-TmT for 5 weeks. Treatment of α-, γ-, δ-tocopherol or γ-TmT reduced the tumor volume by 71% (p<0.05), 55% (p<0.05), 59% (p<0.05) and 42% (p<0.01) as well as tumor weight by 80%, 63% (p<0.05), 61% (p<0.05) and 48% (p<0.05), respectively. Our results suggest that tocopherols significantly repress the expansion of breast cancer stem cell-like population and tumor development. Tocopherols, in particular a naturally occurring tocopherol mixture rich in γ and δ-tocopherols, could thus be effective natural agents for the prevention and treatment of estrogen-induced breast cancer.(This work was supported by NIH R01 AT007036) Citation Format: Min Ji Bak, Soumyasri Das Gupta, Joseph Wahler, Mao-Jung Lee, Yong Lin, Weichung Joe Shih, Chung S Yang, Nanjoo Suh. Protection of tocopherols against estrogen-induced breast cancer via mechanisms targeting 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 5237.

Abstract LB-229: Novel mitochondrial inhibitors hold promise for the eradication of lung cancer stem cells

Abstract Lung cancer therapy failure is attributed not only to the genetic heterogeneity of tumors, but is also promoted by the functional and phenotypic plasticity of different cell populations. The cancer stem cell (CSC) theory postulates that an under-represented population of cells with different growth- and drug-sensitivity properties is responsible for metastatic dissemination and post-therapy relapse. The detailed molecular and phenotypical analysis of CSCs in primary human lung cancers has been limited however, because CSCs have poor proliferative capacity when placed in culture and eventually undergo terminal differentiation. We have developed a novel culturing system that permits the rapid expansion of patient-derived cancer samples and is allowing us to rapidly build a collection of non-small-cell (NSCLC) lung cancers for real-time drug screening as well as for molecular and phenotypical characterization. Compared to other known culturing conditions, this system leads to a stark enrichment of CSCs, defined as such based on the expression of stem cell markers, the ability to differentiate towards multiple lineages and to form tumors at limiting dilutions in immuno-deficient mice. Because CSCs tend to be refractory to targeted or untargeted therapies, we have studied these CSC populations with respect to their metabolic vulnerabilities. CSCs reside in stem cell niches that are deprived of nutrients and of glucose and are characterized by low radical oxygen species (ROS) levels, thus likely relying upon different metabolic requirements relative to other cancer cells which represent the “bulk” of the tumor mass. We found that CSCs populations derived from tumors harboring diverse mutational profiles share, as a common feature, the reliance upon mitochondrial respiration for growth and expansion. We show that this metabolic aberration is sustained, for the most part, by the mitochondrial citrate transporter SLC25A1, whose expression levels are elevated in NSCLC predicting poorest prognostic outcome. SLC25A1 is required for the expansion of CSCs where it promotes the homeostatic energetic output of the mitochondria via oxidative phosphorylation, while overcoming the glycolytic addition, thus essentially reverting the Warburg effect which has been thus far considered a hallmark of tumor cells. We have also developed two chemical inhibitors of SLC25A1 activity and we demonstrate that these inhibitors target lung CSCs by blocking mitochondrial respiratory activity, by inducing mitochondrial dysfunction and by altering the redox environment. These alterations lead to energetic crisis and to cell death. These agents show remarkable anti-tumor activity as single agents in xenograft experiments in mice and they enhance sensitivity to platinum-based chemotherapy. In conclusion we have identified a novel class of agents that specifically targets the mitochondrial metabolism of cancer stem cells. We propose that such activity may permit eradication of the cell population primarily responsible of drug resistance and metastatic dissemination regardless of the mutational status of the tumor of origin. These findings could set the ground for clinical trials employing mitochondrial inhibitors as a rescue-treatment after failure with currently available targeted or untargeted therapies. Citation Format: Harvey Fernandez, Shreyas Grade, Kyu Ah Kim, Mayalin Laforteza, Yuwen Zhang, Chris Albanese, Mikell Paige, Giuseppe Giaccone, Maria L. Avantaggiati. Novel mitochondrial inhibitors hold promise for the eradication of lung 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 LB-229.

Abstract 3350: Verification of mechanism that CSC markers are implicated in poor prognosis for pancreatic ductal adenocarcinoma

Abstract Background: Cancer stem cells (CSCs) refer to a subset of tumor cells that have self-renewal ability and generate plenty of non-CSC cells that comprise a tumor. In addition, CSCs play crucial roles in various processes during tumor progression and metastasis as well. A number of CSC markers candidates have been explored to date, and Aldehyde dehydrogenase 1 (ALDH1), c-Met, and CD44 have been identified as CSC markers in pancreatic ductal adenocarcinoma (PDAC). On the other hand, prostaglandin E2 (PGE2) is one of metabolites in arachidonate cascade and is implicated in the expansion of hematopoietic and tissue stem cell fraction. The aim of this study is to single out the most important CSC marker and elucidate the functional role of PGE2 for CSC expansion in PDAC. Methods: Three CSC markers (ALDH1, CD44, and c-Met) expression was examined by immunohistochemistry in 121 primary surgical specimens of PDAC and analyzed a relationship with clinicopathological factors and clinical outcomes. The clonogenic growth potential of CSC marker-positive PDAC cells was assessed in vitro by growth assays and sphere formation assays. We next investigated the expression of CSC markers and self-renewal related genes in PDAC cell lines with PGE2 or 15-PGDH inhibitor treatment. We further conducted functional experiments using siRNA to identify the critical molecule in PDAC progression. Results: A high level of ALDH1 expression was detected in 63 of the 121 cases, and was significantly associated with large tumor size and poor prognosis in PDAC patiants. On the other hand, CD44 and c-Met expression were not associated with the prognosis. Among CSC markers, the expression of ALDH1 was significantly increased by PGE2 treatment in PDAC cells. By suppressing ALDH1 expression by siRNA, growth and sphere formation potential were inhibited in ALDH1 high-expressing PDAC cells. In contrast, the expression of ALDH1 was remarkably increased by PGE2 or 15-PGDH inhibitor treatment in ALDH1 low-expressing PDAC cells. Finally, we found that Nanog and Oct-4 were down-stream molecules of PGE2-ALDH1 signaling and played crucial roles for PDAC cell expansion. Conclusion: Our results demonstrated that PGE2 positively regulated ALDH1 expression, and the growth and sphere formation potential were promoted by increasing ALDH1 expression, resulting in poor prognosis of PDAC patients. Inhibiting PGE2-ALDH1 signaling could lead to the suppression of tumor growth in PDAC patients. Citation Format: Kota Arima, Takatsugu Ishimoto, Hirohisa Okabe, Yuki Kitano, Risa Inoue, Kensuke Yamamura, Takayoshi Kaida, Takaaki Higashi, Katsunobu Taki, Katsunori Imai, Daisuke Hashimoto, Akira Chikamoto, Toru Beppu, Hideo Baba. Verification of mechanism that CSC markers are implicated in poor prognosis for pancreatic ductal adenocarcinoma. [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 3350.

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.

DDX3 Represses Stemness by Epigenetically Modulating Tumor-suppressive miRNAs in Hepatocellular Carcinoma.

Studies indicate that the presence of cancer stem cells (CSCs) is responsible for poor prognosis of hepatocellular carcinoma (HCC) patients. In this study, the functional role of DDX3 in regulation of hepatic CSCs was investigated. Our results demonstrated that reduced DDX3 expression was not only inversely associated with tumor grade, but also predicted poor prognosis of HCC patients. Knockdown of DDX3 in HCC cell line HepG2 induced stemness gene signature followed by occurrence of self-renewal, chemoreisistance, EMT, migration as well as CSC expansion, and most importantly, DDX3 knockdown promotes tumorigenesis. Moreover, we found positive correlations between DDX3 level and expressions of tumor-suppressive miR-200b, miR-200c, miR-122 and miR-145, but not miR-10b and miR-519a, implying their involvement in DDX3 knockdown-induced CSC phenotypes. In addition, DDX3 reduction promoted up-regulation of DNA methyltransferase 3A (DNMT3A), while neither DNMT3B nor DNMT1 expression was affected. Enriched DNMT3A binding along with hypermethylation on promoters of these tumor-suppressive miRNAs reflected their transcriptional repressions in DDX3-knockdown cells. Furthermore, individual restoration of these tumor-suppressive miRNAs represses DDX3 knockdown-induced CSC phenotypes. In conclusion, our study suggested that DDX3 prevents generation of CSCs through epigenetically regulating a subset of tumor-suppressive miRNAs expressions, which strengthens tumor suppressor role of DDX3 in HCC.

Stemness and chemoresistance in epithelial ovarian carcinoma cells under shear stress.

One of greatest challenges to the successful treatment of cancer is drug resistance. An exciting approach is the eradication of cancer stem cells (CSCs). However, little is known about key signals regulating the formation and expansion of CSCs. Moreover, lack of a reliable predictive preclinical model has been a major obstacle to discover new cancer drugs and predict their clinical activity. Here, in ovarian cancer, a highly chemoresistant tumor that is rapidly fatal, we provide the first evidence demonstrating the causal involvement of mechanical stimulus in the CSC phenotype using a customizable microfluidic platform and three-dimensional spheroids, which most closely mimic tumor behavior. We found that ovarian cancer cells significantly acquired the expression of epithelial-to-mesenchymal transition and CSC markers and a remarkable chemoresistance to clinically relevant doses of frontline chemotherapeutic drugs cisplatin and paclitaxel when grown under fluid shear stress, which corroborates with the physiological attainable levels in the malignant ascites, but not under static condition. Furthermore, we uncovered a new link of microRNA-199a-3p, phosphatidylinositol 3-kinase/Akt, and multidrug transporter activation in shear stress-induced CSC enrichment. Our findings shed new light on the significance of hydrodynamics in cancer progression, emphasizing the need of a flow-informed framework in the development of therapeutics.

WE-FG-BRA-09: Using Graphene Oxide Nano Flakes During Image Guided Radiotherapy to Minimize the Potential of Cancer Recurrence Or Metastasis

Purpose: An increasing number of studies show that cancer stem cells (CSCs) become more invasive (metastatic) and may escape into the blood stream and lymph nodes during radiotherapy (RT), before they have received a lethal dose during RT. Other Studies have shown that Graphene oxide (GO) can selectively inhibit the proliferative expansion of CSCs across multiplicative tumor types. In this study we investigate the feasibility of using GO during radiotherapy (RT) to minimize the escape of CSCs towards preventing cancer metastasis or recurrence. Methods: We hypothesize that sufficient amount of GO nano-flakes (GONFs) released from new design radiotherapy biomaterials (fiducials or spacers) loaded with the GONFs can reach all tumor cells within typical times of 14 or 21days before the beginning of image-guided radiotherapy (IGRT) following implantation. To test this hypothesis, the space-time diffusion of the GONFs was investigated. Knudsen's and Cunningham's numbers were calculated to get the Stokes’ velocities and mobility values, according to these values, diffusion coefficients were calculated. In a previous study it was shown that GONFs concentration of 50 µg/ml were effective. In the diffusion study, 100 µg/ml was chosen as an initial concentration because it has been shown to be relatively non-toxic. Results: The 50 µg/ml concentration in a 2 cm diameter volume of lung tumor could be only achieved using 2 nm and 6 nm GONFs with respective diffusion times of 14 and 21 days. As expected, increased nanoflake size requires longer times to achieve the target 50 µg/ml concentration. Conclusion: The preliminary results indicate the potential of using GONFs delivered via new design radiotherapy biomaterials (e.g. fiducials) to inhibit the proliferative expansion of CSCs. The study avails ongoing in-vivo studies on using GONFs to enhance treatment outcomes for cancer patients.

The retinal determination gene network: from developmental regulator to cancer therapeutic target.

Although originally identified for its function in Drosophila melanogaster eye specification, the Retinal Determination Gene Network (RDGN) is essential for the development of multiple organs in mammals. The RDGN regulates proliferation, differentiation and autocrine signaling, and interacts with other key signaling pathways. Aberrant expression of RDGN members such as DACH, EYA and SIX contributes to tumor initiation and progression; indeed, the levels of RDGN members are clinically prognostic factors in various cancer types. Stimulation or suppression of the activities of these crucial components can block cancer cell proliferation, prevent cancer stem cell expansion and even reverse the EMT process, thereby attenuating malignant phenotypes. Thus, cancer therapeutic interventions targeting RDGN members should be pursued in future studies.

Virtual screening-driven repositioning of etoposide as CD44 antagonist in breast cancer cells.

CD44 is a receptor for hyaluronan (HA) that promotes epithelial-to-mesenchymal transition (EMT), induces cancer stem cell (CSC) expansion, and favors metastasis. Thus, CD44 is a target for the development of antineoplastic agents. In order to repurpose drugs as CD44 antagonists, we performed consensus-docking studies using the HA-binding domain of CD44 and 11,421 molecules. Drugs that performed best in docking were examined in molecular dynamics simulations, identifying etoposide as a potential CD44 antagonist. Ligand competition and cell adhesion assays in MDA-MB-231 cells demonstrated that etoposide decreased cell binding to HA as effectively as a blocking antibody. Etoposide-treated MDA-MB-231 cells developed an epithelial morphology; increased their expression of E-cadherin; and reduced their levels of EMT-associated genes and cell migration. By gene expression analysis, etoposide reverted an EMT signature similarly to CD44 knockdown, whereas other topoisomerase II (TOP2) inhibitors did not. Moreover, etoposide decreased the proportion of CD44+/CD24− cells, lowered chemoresistance, and blocked mammosphere formation. Our data indicate that etoposide blocks CD44 activation, impairing key cellular functions that drive malignancy, thus rendering it a candidate for further translational studies and a potential lead compound in the development of new CD44 antagonists.

Activated Thyroid Hormone Promotes Differentiation and Chemotherapeutic Sensitization of Colorectal Cancer Stem Cells by Regulating Wnt and BMP4 Signaling.

Thyroid hormone is a pleiotropic factor that controls many cellular processes in multiple cell types such as cancer stem cells (CSC). Thyroid hormone concentrations in the blood are stable, but the action of the deiodinases (D2-D3) provides cell-specific regulation of thyroid hormone activity. Deregulation of deiodinase function and thyroid hormone status has been implicated in tumorigenesis. Therefore, we investigated the role of thyroid hormone metabolism and signaling in colorectal CSCs (CR-CSC), where deiodinases control cell division and chemosensitivity. We found that increased intracellular thyroid hormone concentration through D3 depletion induced cell differentiation and sharply mitigated tumor formation. Upregulated BMP4 expression and concomitantly attenuated Wnt signaling accompanied these effects. Furthermore, we demonstrate that BMP4 is a direct thyroid hormone target and is involved in a positive autoregulatory feedback loop that modulates thyroid hormone signaling. Collectively, our findings highlight a cell-autonomous metabolic mechanism by which CR-CSCs exploit thyroid hormone signaling to facilitate their self-renewal potential and suggest that drug-induced cell differentiation may represent a promising therapy for preventing CSC expansion and tumor progression.

Neurotrophin signaling in cancer stem cells.

Cancer stem cells (CSCs), are thought to be at the origin of tumor development and resistance to therapies. Thus, a better understanding of the molecular mechanisms involved in the control of CSC stemness is essential to the design of more effective therapies for cancer patients. Cancer cell stemness and the subsequent expansion of CSCs are regulated by micro-environmental signals including neurotrophins. Over the years, the roles of neurotrophins in tumor development have been well established and regularly reviewed. Especially, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are reported to stimulate tumor cell proliferation, survival, migration and/or invasion, and favors tumor angiogenesis. More recently, neurotrophins have been reported to regulate CSCs. This review briefly presents neurotrophins and their receptors, summarizes their roles in different cancers, and discusses the emerging evidence of neurotrophins-induced enrichment of CSCs as well as the involved signaling pathways.