Embryonic Stemness Research Articles

Abstract 5438: Circulating tumor cells exhibit a tumor stemness defense phenotype in Head and Neck cancer subjects undergoing cisplatin based chemotherapy

Abstract Introduction: We have proposed that tumor has its own defense mechanism similar to an organism having its own defense at the population level. We also proposed that the center of this tumor defense is the hypoxic niche of cancer stem cells (CSCs). Our lab has previously characterized the hypoxic niche of CSCs, and demonstrated that these CSCs reprogram to a highly inflammatory, aggressive and embryonic stemness phenotype during oxidative stress (1). These reprogrammed CSCs, the tumor stemness defense (TSD) phenotype is transient, and exert altruistic defense against bacterial invasion in SCC-25 cell line (2). Further, VEGF/VEGFR1 autocrine signaling was found in cisplatin induced TSD phenotype (4) and Myc/Hif2alpha stemness pathway was found in TSD phenotype of a leukemia stem cell model (3). We hypothesize that the platinum-induced stress may also activate TSD phenotype in the dormant CSC fraction of head and neck squamous cell carcinoma (HNSCC), which may mobilize to the distant tissue via circulation as circulating tumor cells (CTCs). Methods: To test the hypothesis, we isolated EpCAM+/ABCG2+ CTCs (blood, n=14) and EpCAM+/ABCG2+ CSCs (tumor tissue, n=6/14) of HNSCC subjects under cisplatin treatment. CTCs were collected from peripheral blood mononuclear cells (PBMNC) by the immunomagnetic sorting of EpCAM+ cells, and then cells were expanded in a defined serum free media that we used to maintain the hypoxic CSCs (1). We evaluated the EpCAM+/ABCG2+ CTCs and EpCAM+/ABCG2+ CSCs for TSD phenotype by performing qPCR gene expression, Boyden chamber assay of invasion, clonogenic assay and serial transplantation assay in NOD/SCID mice. Data was compared with the EpCAM+/ABCG2- cell population. We also developed a pre-clinical model of cisplatin-induced reprogramming of HNSCC derived CSCs to understand the molecular mechanisms. Results: In 8/14 patient derived EpCAM+/ABCG2+ CTCs and EpCAM+/ABCG2+ CSCs showed TSD phenotype associated gene expression, high migration/invasion, self sufficiency (ability to form spheroid when grown in serum free media without growth factors), enhanced secretion of protumorigenic growth factors; VEGF, SDF-1, PIGF, and HMGB1. These CTCs and CSCs have shown tumor formation with significant CSC frequency, when injected to NOD/SCID mice. Finally, using a SCC-25 cell line derived pre-clinical model, we demonstrated that cisplatin therapy reprogram CSCs to TSD phenotype. Conclusion: Our work indicates that CTCs with TSD phenotype may indicate the interaction between cisplatin and CSC niche defense. Using the pre-clinical model, the TSD phenotype based CSC niche defense mechanism may be further explored to develop markers for therapy response, metastasis or recurrence in HNSCC.

Generation of Polyploid Giant Cancer Cells (PGCCs) from Hey Ovarian Cancer Cells and Analysis of Embryonic Properties.

Polyploid giant cancer cells (PGCCs) play a fundamental role in tumor initiation, dormancy, drug resistance, and metastasis, although the detailed biology of PGCCs remains poorly understood. The lack of literature on establishing a reproducible in vitro system for generating PGCCs is the leading technological obstacle to studying the biology of PGCCs. Here we provide a detailed protocol for generating stable PGCCs from Hey cancer cells and studying the PGCCs' embryonic stemness. This protocol includes (1) generating PGCCs of high purity in 2D culture by exposing Hey cells to paclitaxel, monitoring the cell cycle and amitotic budding of daughter cells from PGCCs, and collecting and studying the daughter cells; (2) inducing PGCCs to form spheroids expressing embryonic stemness markers and observing the spheroids' cleavage and blastocyst-like structure; and (3) inducing redifferentiation of PGCCs into different lineages of differentiatedcells.

SALL4 is a CRL3REN/KCTD11 substrate that drives Sonic Hedgehog-dependent medulloblastoma

The Sonic Hedgehog (SHH) pathway is crucial regulator of embryonic development and stemness. Its alteration leads to medulloblastoma (MB), the most common malignant pediatric brain tumor. The SHH-MB subgroup is the best genetically characterized, however the molecular mechanisms responsible for its pathogenesis are not fully understood and therapeutic benefits are still limited. Here, we show that the pro-oncogenic stemness regulator Spalt-like transcriptional factor 4 (SALL4) is re-expressed in mouse SHH-MB models, and its high levels correlate with worse overall survival in SHH-MB patients. Proteomic analysis revealed that SALL4 interacts with REN/KCTD11 (here REN), a substrate receptor subunit of the Cullin3-RING ubiquitin ligase complex (CRL3REN) and a tumor suppressor lost in ~30% of human SHH-MBs. We demonstrate that CRL3REN induces polyubiquitylation and degradation of wild type SALL4, but not of a SALL4 mutant lacking zinc finger cluster 1 domain (ΔZFC1). Interestingly, SALL4 binds GLI1 and cooperates with HDAC1 to potentiate GLI1 deacetylation and transcriptional activity. Notably, inhibition of SALL4 suppresses SHH-MB growth both in murine and patient-derived xenograft models. Our findings identify SALL4 as a CRL3REN substrate and a promising therapeutic target in SHH-dependent cancers.

Rat Plantar Fascia Stem/Progenitor Cells Showed Lower Expression of Ligament Markers and Higher Pro-Inflammatory Cytokines after Intensive Mechanical Loading or Interleukin-1β Treatment In Vitro.

The pathogenesis of plantar fasciitis is unclear, which hampers the development of an effective treatment. The altered fate of plantar fascia stem/progenitor cells (PFSCs) under overuse-induced inflammation might contribute to the pathogenesis. This study aimed to isolate rat PFSCs and compared their stem cell-related properties with bone marrow stromal cells (BMSCs). The effects of inflammation and intensive mechanical loading on PFSCs' functions were also examined. We showed that plantar fascia-derived cells (PFCs) expressed common MSC surface markers and embryonic stemness markers. They expressed lower Nanog but higher Oct4 and Sox2, proliferated faster and formed more colonies compared to BMSCs. Although PFCs showed higher chondrogenic differentiation potential, they showed low osteogenic and adipogenic differentiation potential upon induction compared to BMSCs. The expression of ligament markers was higher in PFCs than in BMSCs. The isolated PFCs were hence PFSCs. Both IL-1β and intensive mechanical loading suppressed the mRNA expression of ligament markers but increased the expression of inflammatory cytokines and matrix-degrading enzymes in PFSCs. In summary, rat PFSCs were successfully isolated. They had poor multi-lineage differentiation potential compared to BMSCs. Inflammation after overuse altered the fate and inflammatory status of PFSCs, which might lead to poor ligament differentiation of PFSCs and extracellular matrix degeneration. Rat PFSCs can be used as an in vitro model for studying the effects of intensive mechanical loading-induced inflammation on matrix degeneration and erroneous stem/progenitor cell differentiation in plantar fasciitis.

Suppression of NANOG Expression Reduces Drug Resistance of Cancer Stem Cells in Glioblastoma.

Glioblastoma (GBM) is an aggressive and incurable primary brain tumor that harbors therapy-resistant cancer stem cells (CSCs). Due to the limited effectiveness of conventional chemotherapies and radiation treatments against CSCs, there is a critical need for the development of innovative therapeutic approaches. Our previous research revealed the significant expression of embryonic stemness genes, NANOG and OCT4, in CSCs, suggesting their role in enhancing cancer-specific stemness and drug resistance. In our current study, we employed RNA interference (RNAi) to suppress the expression of these genes and observed an increased susceptibility of CSCs to the anticancer drug, temozolomide (TMZ). Suppression of NANOG expression induced cell cycle arrest in CSCs, specifically in the G0 phase, and it concomitantly decreased the expression of PDK1. Since PDK1 activates the PI3K/AKT pathway to promote cell proliferation and survival, our findings suggest that NANOG contributes to chemotherapy resistance in CSCs through PI3K/AKT pathway activation. Therefore, the combination of TMZ treatment with RNAi targeting NANOG holds promise as a therapeutic strategy for GBM.

Suppression of P-cadherin expression as a key regulatory element for embryonic stem cell stemness.

In embryonic stem (ES) cell colonies, a small subpopulation that changes cell shape and loses pluripotency often appears in two-dimensional (2D) cultures, even in the presence of a stemness factor. We have previously shown that membrane translocation of the syntaxin4, t-SNARE protein contributes to this phenomenon. Here, we show that ES cells in three-dimensional (3D) aggregates do not succumb to extruded syntaxin4 owing to suppressed expression of P-cadherin protein. While extracellular expression of syntaxin4 led to the striking upregulation of P-cadherin mRNA in both 2D and 3D-ES cells, morphological changes and appreciable expression of P-cadherin protein were detected only in 2D-ES cells. Importantly, the introduction of an expression cassette for P-cadherin practically reproduced the effects induced by extracellular syntaxin4, where the transgene product was clearly detected in 2D-, but not 3D-ES cells. An expression construct for P-cadherin-Venus harboring an in-frame insertion of the P2A sequence at the joint region gave fluorescent signals only in the cytoplasm of 2D-ES cells, demonstrating translational regulation of P-cadherin. These results provide the mechanistic insight into the uncontrollable differentiation in 2D-ES cells and shed light on the validity of the "embryoid body protocol commonly used for ES cell handling" for directional differentiation.Key words: differentiation, embryoid body, ES cells, P-cadherin, syntaxin4.

Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus.

SummaryBackgroundHuman cytomegalovirus (HCMV) infection has been actively implicated in complex neoplastic processes. Beyond oncomodulation, the molecular mechanisms that might underlie HCMV-induced oncogenesis are being extensively studied. Polycomb repressive complex 2 (PRC2) proteins, in particular enhancer of zeste homolog 2 (EZH2) are associated with cancer progression. Nevertheless, little is known about EZH2 activation in the context of HCMV infection and breast oncogenesis.MethodsHerein, we identified EZH2 as a downstream target for HCMV-induced Myc upregulation upon acute and chronic infection with high-risk strains using a human mammary epithelial model.FindingsWe detected polyploidy and CMV-transformed HMECs (CTH) cells harboring HCMV and dynamically undergoing the giant cells cycle. Acquisition of embryonic stemness markers positively correlated with EZH2 and Myc expression. EZH2 inhibitors curtail sustained CTH cells’ malignant phenotype. Besides harboring polyploid giant cancer cells (PGCCs), tumorigenic breast biopsies were characterized by an enhanced EZH2 and Myc expression, with a strong positive correlation between EZH2 and Myc expression, and between PGCC count and EZH2/Myc expression in the presence of HCMV. Further, we isolated two HCMV strains from EZH2HighMycHigh basal-like tumors which replicate in MRC5 cells and transform HMECs toward CTH cells after acute infection.InterpretationOur data establish a potential link between HCMV-induced Myc activation, the subsequent EZH2 upregulation, and polyploidy induction. These data support the proposed tumorigenesis properties of EZH2/Myc, and allow the isolation of two oncogenic HCMV strains from EZH2HighMycHigh basal breast tumors while identifying EZH2 as a potential therapeutic target in the management of breast cancer, particularly upon HCMV infection.FundingThis work was supported by grants from the University of Franche-Comté (UFC) (CR3300), the Région Franche-Comté (2021-Y-08292 and 2021-Y-08290) and the Ligue contre le Cancer (CR3304) to Georges Herbein. Zeina Nehme is a recipient of a doctoral scholarship from the municipality of Habbouch. Sandy Haidar Ahmad is recipient of a doctoral scholarship from Lebanese municipality. Ranim El Baba is a recipient of a doctoral scholarship from Hariri foundation for sustainable human development.

IL-6 promotes drug resistance through formation of polyploid giant cancer cells and stromal fibroblast reprogramming

To understand the role of polyploid giant cancer cells (PGCCs) in drug resistance and disease relapse, we examined the mRNA expression profile of PGCCs following treatment with paclitaxel in ovarian cancer cells. An acute activation of IL-6 dominated senescence-associated secretory phenotype lasted 2–3 weeks and declined during the termination phase of polyploidy. IL-6 activates embryonic stemness during the initiation of PGCCs and can reprogram normal fibroblasts into cancer-associated fibroblasts (CAFs) via increased collagen synthesis, activation of VEGF expression, and enrichment of CAFs and the GPR77 + /CD10 + fibroblast subpopulation. Blocking the IL-6 feedback loop with tocilizumab or apigenin prevented PGCC formation, attenuated embryonic stemness and the CAF phenotype, and inhibited tumor growth in a patient-derived xenograft high-grade serous ovarian carcinoma model. Thus, IL-6 derived by PGCCs is capable of reprogramming both cancer and stromal cells and contributes to the evolution and remodeling of cancer. Targeting IL-6 in PGCCs may represent a novel approach to combating drug resistance.

High Phosphoglycerate Dehydrogenase Expression Induces Stemness and Aggressiveness in Thyroid Cancer.

Background: We examined the changes in glucose metabolites of papillary thyroid cancer (PTC) and identified phosphoglycerate dehydrogenase (PHGDH) as a potential target. The role of PHGDH in the proliferation and tumorigenesis of thyroid cancer cells and its clinical significance were analyzed. Methods: Glucose metabolites of various thyroid tissues were analyzed via targeted metabolomics analysis. In vitro experiments using shPHGDHs, inhibitor (NCT503), or PHGDH overexpression in thyroid cell lines (BCPAP, 8505C, and Nthy-Ori) were performed. In vivo experiments were performed by using shPHGDH. Human tissue samples and The Cancer Genome Atlas (TCGA) data were used to validate the experimental findings. Results:PHGDH knockdown in BCPAP and 8505c cell lines significantly inhibited cell viability, colony formation, and tumor spheroid formation compared with the control. In addition, treatment with NCT503 showed similar results. PHGDH inhibition by both knockdown and treatment with NCT503 significantly inhibited the expression of embryonic cancer stemness markers (Oct4, Sox2, KLF4, and Nanog). PHGDH overexpression in Nthy-Ori cells significantly increased cell viability and colony formation. The stemness markers were significantly increased after PHGDH overexpression. PHGDH knockdown significantly inhibited tumor growth in an in vivo mouse xenograft study using 8505c cells. The protein expression of Oct4 in tumors was significantly reduced after PHGDH knockdown. The associations between PHGDH expression and stemness markers were confirmed in the TCGA data and human thyroid tissue samples. Positive PHGDH protein expression was associated with metastases of PTC. Conclusions:PHGDH expression is induced in thyroid cancer and is associated with stemness and aggressiveness of PTC.

An FGFR/AKT/SOX2 Signaling Axis Controls Pancreatic Cancer Stemness.

Cancer stemness is associated with high malignancy and low differentiation, as well as therapeutic resistance of tumors including pancreatic ductal adenocarcinoma (PDAC). Fibroblast growth factors (FGFs) exert pleiotropic effects on a variety of cellular processes and functions including embryonic stem cell pluripotency and cancer cell stemness via the activation of four tyrosine kinase FGF receptors (FGFRs). FGF ligands have been a major component of the cocktail of growth factors contained in the cancer stemness-inducing (CSI) and organoid culture medium. Although FGF/FGFR signaling has been hypothesized to maintain cancer stemness, its function in this process is still unclear. We report that inhibition of FGF/FGFR signaling impairs sphere-forming ability of PDAC in vitro, and knocking down FGFR1 and FGFR2 decreased their tumorigenesis abilities in vivo. Mechanistically, we demonstrated that SOX2 is down-regulated upon loss of FGFR signaling. The overexpression of SOX2 in SOX2-negative cells, which normally do not display stemness capabilities, is sufficient to induce spheroid formation. Additionally, we found that AKT phosphorylation was reduced upon FGFR signaling inhibition. The inhibition of AKT using specific pharmacological inhibitors in the context of CSI medium leads to the loss of spheroid formation associated with loss of SOX2 nuclear expression and increased degradation. We demonstrate that an FGFR/AKT/SOX2 axis controls cancer stemness in PDAC and therefore may represent an important therapeutic target in the fight against this very aggressive form of cancer.

The NANOG Transcription Factor Induces Type 2 Deiodinase Expression and Regulates the Intracellular Activation of Thyroid Hormone in Keratinocyte Carcinomas.

Type 2 deiodinase (D2), the principal activator of thyroid hormone (TH) signaling in target tissues, is expressed in cutaneous squamous cell carcinomas (SCCs) during late tumorigenesis, and its repression attenuates the invasiveness and metastatic spread of SCC. Although D2 plays multiple roles in cancer progression, nothing is known about the mechanisms regulating D2 in cancer. To address this issue, we investigated putative upstream regulators of D2 in keratinocyte carcinomas. We found that the expression of D2 in SCC cells is positively regulated by the NANOG transcription factor, whose expression, besides being causally linked to embryonic stemness, is associated with many human cancers. We also found that NANOG binds to the D2 promoter and enhances D2 transcription. Notably, blockage of D2 activity reduced NANOG-induced cell migration as well as the expression of key genes involved in epithelial–mesenchymal transition in SCC cells. In conclusion, our study reveals a link among endogenous endocrine regulators of cancer, thyroid hormone and its activating enzyme, and the NANOG regulator of cancer biology. These findings could provide the basis for the development of TH inhibitors as context-dependent anti-tumor agents.

Abstract 4297: MYC HIF-2 alpha embryonic stemness genes mediate a transcriptional network to maintain asymmetric self-renewal in oral cancer

Abstract Cancer Stem Cells (CSCs) explains therapeutic failure across several tumor types. Despite being evolutionarily successful, the extreme ability to self-renew is asymmetric and limited to CSCs in heterogeneous tumor population. Thus, understanding molecular mechanisms maintaining CSCs asymmetric self-renewal would be therapeutically critical.Here, we identified putative molecular transcriptional network that may regulate CSCs asymmetric self-renewal. We developed 4-NQO (4-Nitroquinoline 1-oxide) induced mouse model of oral carcinogenesis that exhibited similar phenotype as human oral cancer. ABCG2+ CSCs enriched from 4-NQO derived mouse tumors and human oral cancer (n=7) patients indicated high expression of embryonic stemness genes such as Nanog, Sox-2, and Oct-4 in addition to MYC and HIF-2a. Higher engraftment potential was observed when ABCG2+ cells were injected to NOD/SCID mice, while HIF-2a silencing markedly increased mice survival during engraftments. ChIP assay revealed preferential binding of MYC to HIF-2a promoter regions in ABCG2+ vs ABCG2- cells. Nanog and Oct4 facilitated MYC binding to HIF-2a while gene silencing of Nanog and Sox2 led to loss of MYC and HIF-2a expression, indicating the possible regulation by MYC and HIF-2a. Further, we utilized clinical data from The Cancer Genome Atlas (TCGA) and demonstrated significant correlation between HIF-2a/MYC and embryonic stemness genes on Structural Equation Modeling Analysis (SEMA) platform, thereby confirming our in vitro/in vivofindings. Thus,our results may provide evidence of a biologically robust stemness transcriptional network regulating the asymmetric self-renewal of ABCG2+ CSCs. Note: This abstract was not presented at the meeting. Citation Format: Bidisha Pal, Wael Tasabehji, Sandhya Sorra, Joyeeta Talukdar, Bikul Das. MYC HIF-2 alpha embryonic stemness genes mediate a transcriptional network to maintain asymmetric self-renewal in oral cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4297.

Inhibition of transcription factor T-cell factor 3 (TCF3) using the oligodeoxynucleotide strategy increases embryonic stem cell stemness: possible application in regenerative medicine.

The transcription factor T-cell factor 3 (TCF3), one component of the Wnt pathway, is known as a cell-intrinsic inhibitor of many pluripotency genes in embryonic stem cells (ESCs) that influences the balance between pluripotency and differentiation. In this study, the effects of inhibition of TCF3 transcription factor on the stemness of mouse ESCs (mESCs) were investigated using the decoy oligodeoxynucleotides (ODNs) strategy. The TCF3 decoy and its scramble ODNs were designed and synthesized. The interaction specificity of the TCF3 decoy with the TCF3 transcription factor was evaluated by the electrophoretic mobility shift assay. Subcellular localization was carried out using fluorescence and confocal microscopy. Self-renewal and pluripotency of mESCs were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), cell cycle and apoptosis, alkaline phosphatase (ALP), embryoid body (EB) formation, and real-time assays. All experiments were performed in triplicate. The results showed that knockdown of TCF3 by decoy ODNs transfection in mESCs led to an increase in the cell proliferation, ALP enzyme activity, and master regulatory stemness genes and a decrease in the number and diameter of EBs. These results supported TCF3 as a potential target to maintain the pluripotency and self-renewal capacity of mESCs. Knockdown of the TCF3 transcription factor using decoy ODNs can be a promising method to maintain the stemness of stem cells in regenerative medicine and cell therapy researches.

Differential sequences of exosomal NANOG DNA as a potential diagnostic cancer marker.

NANOG has been demonstrated to play an essential role in the maintenance of embryonic stem cells, and its pseudogene, NANOGP8, is suggested to promote the cancer stem cell phenotype. As the roles of these genes are intimately involved with glioblastoma multiforme progression and exosomes are critical in intercellular communication, we conducted a detailed analysis of the association of the NANOG gene family with exosomes to identify diagnostic markers for cancer. Exosomes were precipitated from conditioned culture media from various cell lines, and NANOG gene fragments were directly amplified without DNA isolation using multiple primer sets. The use of the enzymes AlwNI and SmaI with restriction fragment length polymorphism analysis functioned to distinguish NANOGP8 from other NANOG family members. Collectively, results suggest that the NANOG DNA associated with exosomes is not full length and that mixed populations of the NANOG gene family exist. Furthermore, sequence analysis of exosomal DNA amplified with a NANOGP8 specific primer set frequently showed an insertion of a 22 bp sequence into the 3’ UTR. The occurrence rate of this insertion was significantly higher in exosomal DNA clones from cancer cells as compared to normal cells. We have detected mixed populations of NANOG DNA associated with exosomes and have identified preferential modulations in the sequences from cancer samples. Our findings, coupled with the properties of exosomes, may allow for the detection of traditionally inaccessible cancers (i.e. GBM) through minimally invasive techniques. Further analysis of exosomal DNA sequences of NANOG and other embryonic stemness genes (OCT3/4, SOX2, etc.) may establish a robust collection of exosome based diagnostic markers, and further elucidate the mechanisms of cancer formation, progression, and metastasis.

Abstract P3-03-17: Can polyploid tumor cells possessing stem cell features be induced in resistant breast carcinomas?

Abstract Cancer stem cells are believed to be responsible for radio- and chemoresistance of malignant tumors. In vitro studies demonstrate that ionizing radiation is capable of reprogramming cancer cells from non-stem state into stem state [1, 2]. Moreover, the embryonic stemness cassette was found to be expressed in tumor cells (including breast cancer cells) after they were polyploidized as a result of genotoxic stress [2, 3], thus prompting us to suppose that the polyploid cells and their descendants released by depolyploidization can possess stem cell characteristics. The aim of the current work is to test whether polyploid cells having stem cell features can be also induced in vivo, namely in locally advanced breast carcinomas as a result of neoadjuvant chemotherapy (NAC), assuming that this process is not autonomous, but rather stipulated by the tumor microenvironment. The study population consisted of 30 breast cancer patients of age ranged from 31 to 75 y. o. diagnosed in the Latvian Oncology Center of the Riga East University Hospital between 2013 and 2015. The tissue specimens were collected after the patients' informed consent was obtained in accordance with the Ethics regulations. The clinico-pathologic information about these patients, including Ki-67 index and the status of ER, PR and HER2 receptors, was obtained from the aforementioned clinics. The majority of patients (n = 28) had locally advanced breast cancer, predominantly Stage III disease. Both diagnostic biopsy and operation material, such as primary tumors surgically removed after NAC using standard doses of paclitaxel and doxorubicin, were subjected to DNA content analysis with image cytometry. Ploidy-related parameters, such as DNA index and the percentage of cells exceeding 4.5c (presumably proliferating and polyploid cells), were determined. Immunofluorescence staining was applied to evaluate expression of such markers/factors as proliferation (Ki-67), stemness (SOX2 and NANOG) and invasiveness (CD44). At the time of diagnosis, 14 patients had primary tumors possessing near-triploid clones, and these cases in comparison with 16 other cases comprised of near-euploid clones had 4.5-fold increase of percentages of cells exceeding the ploidy of 4.5c (p < 0.05) and 1.3-fold increase of percentages of cells positive for Ki-67 (p > 0.05). Of 10 cases diagnosed as “triple-negative”, 6 were near-triploid. Among those cases that showed the resistance to NAC (grades 1 and 2 by Miller-Payne histopathologic scoring), 67% were near-triploid. Polyploidization, which in some resistant cases is gained by NAC, was likely to be attributed to near-triploid clones. Notably, polyploid cells were positive for Ki-67, SOX2, NANOG, and CD44. Thus, these non-quiescent polyploid cells can possess the invasiveness and self-renewal features that were also seen in descendants after depolyploidization. Perhaps, reversible polyploidy plays a definite role in gaining the resistance of tumor cells to chemo- and radiotherapy in vivo. 1. Ghisolfi L. et al. PLoS ONE 2012; 7: e43628. 2. Lagadec C. et al. Stem Cells 2012; 30: 833-44. 3. Salmina K. et al. Exp Cell Res 2010; 316: 2099-112. Citation Format: Gerashchenko BI, Salmina K, Eglitis J, Erenpreisa J. Can polyploid tumor cells possessing stem cell features be induced in resistant breast carcinomas? [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-17.

KLF4 is regulated by RAS/RAF/MEK/ERK signaling through E2F1 and promotes melanoma cell growth

Melanoma is the most lethal form of skin cancer and treatment of metastatic melanoma remains challenging. BRAF/MEK inhibitors show only temporary benefit due the occurrence of resistance and immunotherapy is effective only in a subset of patients. To improve patient survival, there is a need to better understand molecular mechanisms that drive melanoma growth and operate downstream of the mitogen activated protein kinase (MAPK) signaling. The Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor that plays a critical role in embryonic development, stemness and cancer, where it can act either as oncogene or tumor suppressor. KLF4 is highly expressed in post-mitotic epidermal cells, but its role in melanoma remains unknown. Here, we address the function of KLF4 in melanoma and its interaction with the MAPK signaling pathway. We find that KLF4 is highly expressed in a subset of human melanomas. Ectopic expression of KLF4 enhances melanoma cell growth by decreasing apoptosis. Conversely, knock-down of KLF4 reduces melanoma cell proliferation and induces cell death. In addition, depletion of KLF4 reduces melanoma xenograft growth in vivo. We find that the RAS/RAF/MEK/ERK signaling positively modulates KLF4 expression through the transcription factor E2F1, which directly binds to KLF4 promoter. Overall, our data demonstrate the pro-tumorigenic role of KLF4 in melanoma and uncover a novel ERK1/2-E2F1-KLF4 axis. These findings identify KLF4 as a possible new molecular target for designing novel therapeutic treatments to control melanoma growth.

Abstract B03: Pancreatic cancer stem cells: can it be restrained?

Abstract Over the past decades world-wide, research towards not only understanding the biology, but also attempting to decrease the mortality rate in pancreatic carcinoma has become the forefront in cancers of solid tumor. Recent evidences suggest that pancreatic carcinomas harbour a distinct subpopulation of putative cancer stem cells defined by their self-renewal capacity, differentiation ability, tumorigenicity and ability to drive towards metastasis. Therefore, there is not only a need to identify this population, and also design newer approaches to target these chemo-resistant cell populations, especially in pancreatic cancer which till date shows 5-year survival rate to be below 5%. Our study identified the potential of CD44hiCD24lo MiaPaCa2/PanC1 cells to possess lower capability to generate spheroids whereas CD44hiCD24hi MiaPaCa2/PanC1 showed higher spheroid forming capacity. Spheroids obtained from both cell-lines showed similar metastatic cancer gene expression pattern. However, a distinctive CXCR4 expression levels were observed between CD44hiCD24lo PanC1-spheroids and CD44hiCD24hi PanC1-spheroids. It was also observed that EMT, CXCR4 and Muc1 were highly expressed in MiaPaCa2-spheroids. Hence, suggesting only a sub-population of tumor cells behave as “cancer initiating cells/stem-like cells” which are associated with cell-cell adhesion and cell matrix interaction. In order to determine the role of cancer stem-like cells (spheroids/CSCs) to migrate and invade to metastasize particularly in pancreatic carcinoma, experiments were designed to evaluate their self-renewal capability and epithelial to mesenchymal transition (EMT) and their association with developmental genes. An exponential spheroid formation was observed along with stem cell transcriptional genes (Oct4 and Nanog). Interestingly, MiaPaCa2-spheroids exhibited higher expression levels of Sox-2, MMP-9 and Vimentin in comparison to PanC1-spheroids suggesting that MiaPaCa2-spheroids may be more aggressive or has higher invasive potential. It appears that pancreatic CSCs undergoing EMT probably express higher levels of Sox-9, Ngn-3, Pdx-1 and c-Myc genes which attributes to its embryonic stemness. The results from the study clearly project the role of CSCs in pancreatic carcinoma and thus further studies to delineate the mechanism which might lead to suppression of pancreatic CSCs is crucial. In this direction, a pilot study has been initiated to explore the potential of Mesenchymal Stem Cells to target the pancreatic CSCs. Citation Format: Neha Chopra, Sangeeta Choudhury, Saima Wajid, Kriti Jain, Rizwana Mirza.{Authors}. Pancreatic cancer stem cells: can it be restrained? [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B03.

Disentangling the aneuploidy and senescence paradoxes: a study of triploid breast cancers non-responsive to neoadjuvant therapy.

Aneuploid cells should have a reduced proliferation rate due to difficulty in proceeding through mitosis. However, contrary to this, high aneuploidy is associated with aggressive tumour growth and poor survival prognosis, in particular in triploid breast cancer. A further paradox revolves around the observation that, while cell senescence should inhibit proliferation, the senescence marker p16INK4a correlates with poor treatment outcome in patients with a very aggressive triple-negative breast carcinoma (TNBC). In this study, we aim to pour light on the possible relationship of these conundrums with polyploidy of tumour cells. We performed detailed analysis of DNA histogram profiles in diagnostic core biopsies of 30 cases of operable breast cancer and found that near triploidy in TNBC and other forms correlated with weak or no response to neoadjuvant chemotherapy (NAC) as scored by Miller-Payne index. Polyploid cells in operation samples from tumours that were non-responsive to NAC treatment were Ki67 and CD44 positive. In addition, polyploid cells were positive for markers of embryonic stemness (OCT4, SOX2, NANOG) and senescence (p16INK4a). The relationship patterns between p16INK4a and NANOG were heterogeneous, with predominantly mutually exclusive expression but also synergistic and intermediate variants in the same samples. We conclude that the aneuploidy and senescence paradoxes can be explained by the mutual platform of polyploidy, conferring genomic and epigenetic instability as a survival advantage. Such cells are able to bypass aneuploidy restrictions of conventional mitosis and overcome the barrier of senescence by a shift to self-renewal, resulting in progression of cancer.

Valproic acid and 5-azacytidine promote an increase of stemness phenotype in human osteosarcomas

Osteosarcoma (OS) is the most common pediatric tumour of bone in the world. It leads to local invasion and early metastasis to lung. The prognosis is poor with the 5-year survival rate of 65% for patients with primary tumour and 20% for patients with metastasis. OS contain a small cell population with stem cell like properties, referred to as cancer stem cells characterized by CD133 expression [1,2]. Recent studies have implicated epigenetic alterations, including DNA methylation and histone modifications, as prominent factors that contribute to the phenotype of cancer progenitor or stem cells. Here, we examined the epigenetic effects of the HDAC inhibitor valproic acid (VPA) and demethylating agent, 5’azacytidine (DAC), on the stemness phenotype in osteosarcoma cell lines. Saos-2 and MG63 cell lines were treated with 0,5 mM VPA and 3µM DAC for 48 hours alone, and in combination. CD133 expression as well as stemness markers expression including OCT4, Sox2 and Nanog was analyzed by flow cytometry and real-time PCR. Vimentin and osteocalcin levels were also tested. Sarcospheres formation rate was assessed as spheres number/seed single cells number. Specific histone modifications including H3-trymethyl-k9, H3-acetyl-k9, H3-trymethil-k27 and global methylation were analysed by flow cytometry and immunofluorescence. Moreover, soft agar and invasion assays were performed. Our findings indicated that DAC or VPA and their combination induced an increase of stemness characteristics of OS cells in terms of high expression of CD133, OCT4, Sox2 and Nanog and high sarcospheres-forming efficiency. Interestingly, combined treatment with DAC and VPA induced an increase of CD133 expression in a synergistic manner in all cell lines. Vimentin resulted up-regulated after treatment, whereas, the level of osteocalcin remained similar before and after treatment. Furthermore, soft agar assay revealed major colony-forming efficiency in treated cells compared to untreated cells, whereas invasion potential decreased after drug treatment. Interestingly, histone modification analyses correlated with those typical of embryonic stemness with an increase of H3-acetyl-k9 and decrease of H3-trymethyl-k9, and H3-trymethil-k27 after drug treatment as well as global methylation decreased in treated cells. In conclusion, DAC and VPA induced an increase of stemness associated to an decrease of global methylation, increase of acetylation and decrease of H3-trymethyl-k9, and H3-trymethil-k27. This study was supported by MIUR-EU funds (PON 01_02834 and PRIN 2011/13)

Abstract P2-06-05: Expression of the pluripotency transcription factor SOX2 in primary breast cancers (BCs): Correlation with clinicopathological features (CPfs) and recurrence

Abstract BACKGROUND SOX2 is one of the pluripotency transcription factors expressed by stem cells, which plays a central role in controlling the expression of genes implicated in embryonic development and stemness manteinance. Key regulators of embryonic stem cell (ESC) identity, such as NANOG, SOX2, OCT4 and GDF3 resulted overexpressed in a variety of solid tumors with a possible role in cancer progression and prognosis. SOX2 expression has mainly been reported in basal-like BC subtype, suggesting a role in conferring a less differentiated phenotype. In our analysis we evaluated a heterogenous group of BC tissues to determine whether the expression of ESC-regulating genes correlated with CPfs and recurrence. METHODS 140 primary invasive BC specimens were collected from 137 female patients who underwent surgery. mRNA expression for SOX2, OCT4, NANOG, GDF3 genes was assessed by RT-PCR. Immunoistochemistry (IHC) was performed for SOX2 with mouse monoclonal antibody (1:50, Y17, Santa Cruz Biotechnology, USA). Correlations with molecular subtypes, menopausal status, grading, ER, PR, ki67 (≤ 20% and > 20%), HER2, T-size and node status were evaluated by Fisher's exact test and χ2 test. Association of ESC-genes, CPfs and DFS was estimated by univariate and multivariate Cox-regression analysis (p≤ 0.05). Survival analysis (DFS and OS) were calculated by Kaplan-Meier curves and compared by log-rank test. RESULTS In 117 samples assessable by RT-PCR the genes resulted expressed as follows: NANOG=52 (44.5%), SOX2=11 (9.4%), GDF3=9 (7.2%), OCT-4=0. Correlation of mRNA gene expression with CPfs was statistically significant between NANOG and grade 2, GDF3 and node-negative status, SOX2 and higher ki67 (p=0.019, p=0.029, p= 0.035, respectively). Six out of 11 SOX2+ tumors were HER2+ (data not statistically significant); in the remaining 5 samples the fluorescence in situ hybridization was performed but no HER2 amplification was detected. At univariate analysis of DFS, SOX2 expression (HR=2,36; p=0.002), ki67 (HR= 2,19; p=0,028), T-size >1 (HR=2,06; p=2.011), node-status (HR=2,21; p= 0.014); ER/PR(HR=0,58/HR=0,59, p=0.065/p=0.068) resulted statistically significant. At multivariate analysis, SOX2 (HR=2,99; 95% CI 1,41-6,30; p=0.004), node-status (HR=2,44; 95%CI 1,25-4,76; p=0.009) and T-size >1 (HR=1,77; 95% CI=0,99-3,13; p=0.051) were independently associated with increased risk of recurrence. An earlier recurrence was observed in SOX2+ patients (median 34.9 months; 95% CI: 7.5-62.2) than SOX2- patients (median: 60.3 months; 95% CI: 32.6-88.1) (p=0.017); OS resulted shorter in SOX2+ (68.2 months 95%CI: 63.7-151.4 vs 145.3 months 95% CI: 80.5-210.2) albeit not statistically significant (p=0.104). IHC analysis showed a positive score for SOX2 protein expression in all of 11 samples with SOX2 mRNA amplification; SOX2+ protein was not detected in 20 samples randomly selected among the tissues not expressing SOX2 mRNA. CONCLUSIONS Our analysis confirm that ESC-regulating genes correlate with specific CPfs (grading, node-status and ki67). Notably, SOX2 resulted to be an independent prognostic factor, as it was associated with a risk of recurrence increased by 3 times, irrespective of other CPfs. Citation Format: Barbara Pistilli, Giovanni Benedetti, Mauro Finicelli, Tiziana Squillaro, Andrea Marcellusi, Tommasina Biscotti, Alfredo Santinelli, Paola Mariani, Paolo Decembrini, Giancarlo Ciccioli, Luciano Latini, Antonio Giordano, Umberto Galderisi. Expression of the pluripotency transcription factor SOX2 in primary breast cancers (BCs): Correlation with clinicopathological features (CPfs) and recurrence [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-06-05.