Empty spiracles homeobox genes EMX1 and EMX2 regulate WNT pathway activation in sarcomagenesis.

BACKGROUND: Sarcomas are a very heterogeneous group of tumors with intrinsic developmental programs derived from the cell of origin. This implies a functional hierarchy inside tumors governed by sarcoma stem cells. Therefore, genetic and/or epigenetic changes profoundly affect the biology of sarcoma tumor stem cells. EMX genes are proposed to be transcription factors that are involved in the sarcomagenesis process, regardless of the neural or mesodermal embryological sarcoma origin. It has been shown that EMX1 or EMX2 overexpression reduces tumorigenic properties, while reducing the levels of these genes enhances these properties. Furthermore, it has been shown that EMX genes decrease the expression of stem cell regulatory genes and the stem cell phenotype. Taken together, these results indicate that the EMX1 and EMX2 genes negatively regulate these tumor-remodeling populations or sarcoma stem cells, acting as tumor suppressors in sarcoma. METHODS: Bioinformatic analysis, quantitative mRNA and protein expression analysis, cell models of sarcoma by ectopic expression of EMX genes. By cell biology methods we measured tumorigenesis and populations enriched on stem cell phenotypes, either in vitro or in vivo. RESULTS: In this work, we showed that the canonical Wnt pathway is one of the mechanisms that explains the relationships of EMX1/EMX2 and stem cell genes in sarcoma. The Wnt-EMX1/EMX2 relationship was validated in silico with sarcoma patient datasets, in vitro in primary derived sarcoma cell lines, and in vivo. EMX expression was found to negatively regulate the Wnt pathway. In addition, the constitutive activation of the Wnt pathway revers to a more aggressive phenotype with stem cell properties, and stemness gene transcription increased even in the presence of EMX1 and/or EMX2 overexpression, establishing the relationship among the Wnt pathway, stem cell genes and the EMX transcription factors. CONCLUSIONS: Our data showed that Empty Spiracles Homeobox Genes EMX1 and EMX2 represses WNT signalling and activation of WNT pathway bypass EMX-dependent stemness repression and induces sarcomagenesis. These results also suggest the relevance of the Wnt/b-catenin/stemness axis as a therapeutic target in sarcoma.

Cellular senescence or stemness: hypoxia flips the coin.

Cellular senescence is a complex physiological state whose main feature is proliferative arrest. Cellular senescence can be considered the reverse of cell immortalization and continuous tumor growth. However, cellular senescence has many physiological functions beyond being a putative tumor suppressive trait. It remains unknown whether low levels of oxygen or hypoxia, which is a feature of every tissue in the organism, modulate cellular senescence, altering its capacity to suppress the limitation of proliferation. It has been observed that the lifespan of mammalian primary cells is increased under low oxygen conditions. Additionally, hypoxia promotes self-renewal and pluripotency maintenance in adult and embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and cancer stem cells (CSCs). In this study, we discuss the role of hypoxia facilitating senescence bypass during malignant transformation and acquisition of stemness properties, which all contribute to tumor development and cancer disease aggressiveness.

Regulation of sarcomagenesis by the empty spiracles homeobox genes EMX1 and EMX2.

The EMX (Empty Spiracles Homeobox) genes EMX1 and EMX2 are two homeodomain gene members of the EMX family of transcription factors involved in the regulation of various biological processes, such as cell proliferation, migration, and differentiation, during brain development and neural crest migration. They play a role in the specification of positional identity, the proliferation of neural stem cells, and the differentiation of certain neuronal cell phenotypes. In general, they act as transcription factors in early embryogenesis and neuroembryogenesis from metazoans to higher vertebrates. The EMX1 and EMX2's potential as tumor suppressor genes has been suggested in some cancers. Our work showed that EMX1/EMX2 act as tumor suppressors in sarcomas by repressing the activity of stem cell regulatory genes (OCT4, SOX2, KLF4, MYC, NANOG, NES, and PROM1). EMX protein downregulation, therefore, induced the malignance and stemness of cells both in vitro and in vivo. In murine knockout (KO) models lacking Emx genes, 3MC-induced sarcomas were more aggressive and infiltrative, had a greater capacity for tumor self-renewal, and had higher stem cell gene expression and nestin expression than those in wild-type models. These results showing that EMX genes acted as stemness regulators were reproduced in different subtypes of sarcoma. Therefore, it is possible that the EMX genes could have a generalized behavior regulating proliferation of neural crest-derived progenitors. Together, these results indicate that the EMX1 and EMX2 genes negatively regulate these tumor-altering populations or cancer stem cells, acting as tumor suppressors in sarcoma.

Mutation of SPINOPHILIN (PPP1R9B) found in human tumors promotes the tumorigenic and stemness properties of cells.

Rationale: SPINOPHILIN (SPN, PPP1R9B) is an important tumor suppressor involved in the progression and malignancy of different tumors depending on its association with protein phosphatase 1 (PP1) and the ability of the PP1-SPN holoenzyme to dephosphorylate retinoblastoma (pRB). Methods: We performed a mutational analysis of SPN in human tumors, focusing on the region of interaction with PP1 and pRB. We explored the effect of the SPN-A566V mutation in an immortalized non-tumorigenic cell line of epithelial breast tissue, MCF10A, and in two different p53-mutated breast cancer cells lines, T47D and MDA-MB-468. Results: We characterized an oncogenic mutation of SPN found in human tumor samples, SPN-A566V, that affects both the SPN-PP1 interaction and its phosphatase activity. The SPN-A566V mutation does not affect the interaction of the PP1-SPN holoenzyme with pocket proteins pRB, p107 and p130, but it affects its ability to dephosphorylate them during G0/G1 and G1, indicating that the PP1-SPN holoenzyme regulates cell cycle progression. SPN-A566V also promoted stemness, establishing a connection between the cell cycle and stem cell biology via pocket proteins and PP1-SPN regulation. However, only cells with both SPN-A566V and mutant p53 have increased tumorigenic and stemness properties. Conclusions: SPN-A566V, or other equivalent mutations, could be late events that promote tumor progression by increasing the CSC pool and, eventually, the malignant behavior of the tumor.

Breast tumor cells promotes the horizontal propagation of EMT, stemness, and metastasis by transferring the MAP17 protein between subsets of neoplastic cells.

MAP17 (PDZK1IP1) is a small protein regulating inflammation and tumor progression, upregulated in a broad range of carcinomas. MAP17 levels increase during tumor progression in a large percentage of advanced tumors. In the present work, we explored the role of this protein shaping tumor evolution. Here we show that in breast cancer, cells increased MAP17 levels in tumors by demethylation induced multiple changes in gene expression through specific miRNAs downregulation. These miRNA changes are dependent on Notch pathway activation. As a consequence, epithelial mesenchymal transition (EMT) and stemness are induced promoting the metastatic potential of these cells both in vitro and in vivo. Furthermore, MAP17 increased the exosomes in tumor cells, where MAP17 was released as cargo, and this horizontal propagation also increased the EMT in the recipient cells. Importantly, an antibody against MAP17 in the media reduces the EMT and stemness alterations promoted by the conditioned media from MAP17-expressing cells. Therefore, MAP17 expression promotes the horizontal propagation of EMT and metastasis by transferring the MAP17 protein between subsets of neoplastic cells. Thus, MAP17 can be used to describe a new mechanism for cell malignity at distance, without the involvement of genetic or epigenetic modifications. MAP17 can also be taken in consideration as new target for metastatic high-grade breast tumors.

New markers for human ovarian cancer that link platinum resistance to the cancer stem cell phenotype and define new therapeutic combinations and diagnostic tools.

BACKGROUND: Ovarian cancer is the leading cause of gynecologic cancer-related death, due in part to a late diagnosis and a high rate of recurrence. Primary and acquired platinum resistance is related to a low response probability to subsequent lines of treatment and to a poor survival. Therefore, a comprehensive understanding of the mechanisms that drive platinum resistance is urgently needed. METHODS: We used bioinformatics analysis of public databases and RT-qPCR to quantitate the relative gene expression profiles of ovarian tumors. Many of the dysregulated genes were cancer stem cell (CSC) factors, and we analyzed its relation to therapeutic resistance in human primary tumors. We also performed clustering and in vitro analyses of therapy cytotoxicity in tumorspheres. RESULTS: Using bioinformatics analysis, we identified transcriptional targets that are common endpoints of genetic alterations linked to platinum resistance in ovarian tumors. Most of these genes are grouped into 4 main clusters related to the CSC phenotype, including the DNA damage, Notch and C-KIT/MAPK/MEK pathways. The relative expression of these genes, either alone or in combination, is related to prognosis and provide a connection between platinum resistance and the CSC phenotype. However, the expression of the CSC-related markers was heterogeneous in the resistant tumors, most likely because there were different CSC pools. Furthermore, our in vitro results showed that the inhibition of the CSC-related targets lying at the intersection of the DNA damage, Notch and C-KIT/MAPK/MEK pathways sensitize CSC-enriched tumorspheres to platinum therapies, suggesting a new option for the treatment of patients with platinum-resistant ovarian cancer. CONCLUSIONS: The current study presents a new approach to target the physiology of resistant ovarian tumor cells through the identification of core biomarkers. We hypothesize that the identified mutations confer platinum resistance by converging to activate a few pathways and to induce the expression of a few common, measurable and targetable essential genes. These pathways include the DNA damage, Notch and C-KIT/MAPK/MEK pathways. Finally, the combined inhibition of one of these pathways with platinum treatment increases the sensitivity of CSC-enriched tumorspheres to low doses of platinum, suggesting a new treatment for ovarian cancer.

Tumor cell-secreted PLD increases tumor stemness by senescence-mediated communication with microenvironment.

Cancer cells are in continuous communication with the surrounding microenvironment and this communication can affect tumor evolution. In this work, we show that phospholipase D2 (PLD2) was overexpressed in colon tumors and is secreted by cancer cells, inducing senescence in neighboring fibroblasts. This occurs through its lipase domain. Senescence induced by its product, phosphatidic acid, leads to a senescence-associated secretory phenotype (SASP) able to increase the stem properties of cancer cells. This increase in stemness occurs by Wnt pathway activacion. This closes a feedback loop in which senescence acts as a crosspoint for the generation of CSCs mediated by phospholipid metabolism. We also demonstrate the connexion of both phenomena in mouse models in vivo showing that a high PLD2 expression increased stemness and tumorigenesis. Thus, the patients with colon cancer show high levels of PLD2 and SASP factor genes expression correlating with Wnt pathway activation. Therefore, we demonstrate that tumor cell-secreted PLD2 contributes to tumor development by modifying the microenvironment, making it a possible therapeutic target for cancer treatment. This mechanism may also explain the high levels of Wnt pathway activation in colon cancer.

NAMPT Is a Potent Oncogene in Colon Cancer Progression that Modulates Cancer Stem Cell Properties and Resistance to Therapy through Sirt1 and PARP.

Purpose: Colorectal cancer is the second most common cancer in women and the third most common in men worldwide. However, despite current progress, many patients with advanced and metastatic tumors still die from the malignancy. Refractory disease often relies on nicotinamide adenine dinucleotide (NAD)-dependent mechanisms. NAD metabolism and a stable NAD regeneration circuit are required to maintain tissue homeostasis and metabolism. However, high levels of NAD confer therapy resistance to tumors.Experimental Design: Ectopic overexpression of nicotinamide phosphoribosil transferase (NAMPT) and shRNAs in colorectal cancer cell lines, tumorigenic and stemness properties and transcription measurement in culture and in vivo Transcriptional analysis in public databases. Therapeutic approaches.Results: NAMPT, the rate-limiting enzyme responsible for the highest source of physiologic NAD biosynthesis, increases tumorigenic properties and induces cancer stem cell-like properties through pathways that control stem cell signaling, thus enriching the cancer-initiating cell (CIC) population. Furthermore, NAMPT expression correlated with high levels of CIC-like cells in colon tumors directly extracted from patients, and transcription meta-analysis revealed that NAMPT is also a key factor that induces cancer stem pathways in colorectal cancer tumors. This effect is mediated by PARP and SIRT1. In addition, we report a novel NAMPT-driven signature that stratifies prognosis from high to low expression groups. The NAMPT signature contained SIRT1 and PARP1 levels as well as other cancer stem cell-related genes. Finally, NAMPT inhibition increased the sensitivity to apoptosis in both NAMPT-expressing cells and tumorspheres.Conclusions: NAMPT represents a novel therapeutic target in colon cancer progression and relapse, particularly the CIC subset of human colon cancers. Clin Cancer Res; 24(5); 1202-15. ©2017 AACR.

NAMPT overexpression induces cancer stemness and defines a novel tumor signature for glioma prognosis.

Gliomas are the most prevalent primary malignant brain tumors associated with poor prognosis. NAMPT, a rate-limiting enzyme that boosts the nicotinamide adenine dinucleotide (NAD) regeneration in the salvage pathway, is commonly expressed in these tumors. NAD metabolism is required to maintain tissue homeostasis. To maintain metabolism, cancer cells require a stable NAD regeneration circuit. However, high levels of NAD confer resistance to therapy to these tumors, usually treated with Temozolomide (TMZ). We report that NAMPT overexpression in glioma cell lines increases tumorigenic properties controlling stem cell pathways and enriching the cancer-initiating cell (CIC) population. Furthermore, NAMPT expression correlated with high levels of Nanog, CD133 and CIC-like cells in glioblastoma directly extracted from patients. Meta-analysis reveals that NAMPT is also a key factor inducing cancer stem pathways in glioma cells. Furthermore, we report a novel NAMPT-driven signature which stratify prognosis within tumor staging. NAMPT signature also correlates directly with EGFR positive and IDH negative tumors. Finally, NAMPT inhibition increases sensitivity to apoptosis in both NAMPT-expressing cells and tumorspheres. Therefore, NAMPT represents a novel therapeutic target in Glioma progression and relapse.

High casein kinase 1 epsilon levels are correlated with better prognosis in subsets of patients with breast cancer.

Reliable biological markers that predict breast cancer (BC) outcomes after multidisciplinary therapy have not been fully elucidated. We investigated the association between casein kinase 1 epsilon (CK1ε) and the risk of recurrence in patients with BC. Using 168 available tumor samples from patients with BC treated with surgery +/- chemo(radio)therapy, we scored the CK1ε expression as high (≥ 1.5) or low (<1.5) using an immunohistochemical method. Kaplan-Meier analysis was performed to assess the risk of relapse, and Cox proportional hazards analyses were utilized to evaluate the effect of CK1ε expression on this risk. The median age at diagnosis was 60 years (range 35-96). A total of 58% of the patients underwent breast conservation surgery, while 42% underwent mastectomy. Adjuvant chemotherapy and radiation therapy were administered in 101 (60%) and 137 cases (82%), respectively. Relapse was observed in 24 patients (14%). Multivariate analysis found high expression of CK1ε to be associated with a statistically significant higher disease-free survival (DFS) in BC patients with wild-type p53 (Hazard ratio [HR] = 0.33; 95% CI, 0.12-0.91; P = 0.018) or poor histological differentiation ([HR] = 0.34; 95% CI, 0.12-0.94; P = 0.039) or in those without adjuvant chemotherapy ([HR] = 0.11; 95% CI, 0.01-0.97; P = 0.006). Our data indicate that CK1ε expression is associated with DFS in BC patients with wild-type p53 or poor histological differentiation or in those without adjuvant chemotherapy and thus may serve as a predictor of recurrence in these subsets of patients.