RESUMEN

BACKGROUND: p63 is a transcription factor with intrinsic pioneer factor activity and pleiotropic functions. Transforming growth factor ß (TGFß) signaling via activation and cooperative action of canonical, SMAD, and non-canonical, MAP-kinase (MAPK) pathways, elicits both anti- and pro-tumorigenic properties, including cell stemness and invasiveness. TGFß activates the ΔNp63 transcriptional program in cancer cells; however, the link between TGFß and p63 in unmasking the epigenetic landscape during tumor progression allowing chromatin accessibility and gene transcription, is not yet reported. METHODS: Small molecule inhibitors, including protein kinase inhibitors and RNA-silencing, provided loss of function analyses. Sphere formation assays in cancer cells, chromatin immunoprecipitation and mRNA expression assays were utilized in order to gain mechanistic evidence. Mass spectrometry analysis coupled to co-immunoprecipitation assays revealed novel p63 interactors and their involvement in p63-dependent transcription. RESULTS: The sphere-forming capacity of breast cancer cells was enhanced upon TGFß stimulation and significantly decreased upon ΔNp63 depletion. Activation of TGFß signaling via p38 MAPK signaling induced ΔNp63 phosphorylation at Ser 66/68 resulting in stabilized ΔNp63 protein with enhanced DNA binding properties. TGFß stimulation altered the ratio of H3K27ac and H3K27me3 histone modification marks, pointing towards higher H3K27ac and increased p300 acetyltransferase recruitment to chromatin. By silencing the expression of ΔNp63, the TGFß effect on chromatin remodeling was abrogated. Inhibition of H3K27me3, revealed the important role of TGFß as the upstream signal for guiding ΔNp63 to the TGFß/SMAD gene loci, as well as the indispensable role of ΔNp63 in recruiting histone modifying enzymes, such as p300, to these genomic regions, regulating chromatin accessibility and gene transcription. Mechanistically, TGFß through SMAD activation induced dissociation of ΔNp63 from NURD or NCOR/SMRT histone deacetylation complexes, while promoted the assembly of ΔNp63-p300 complexes, affecting the levels of histone acetylation and the outcome of ΔNp63-dependent transcription. CONCLUSIONS: ΔNp63, phosphorylated and recruited by TGFß to the TGFß/SMAD/ΔNp63 gene loci, promotes chromatin accessibility and transcription of target genes related to stemness and cell invasion.

Asunto(s)

Asunto(s)

RESUMEN

Intracellular signaling by the pleiotropic cytokine transforming growth factor-ß (TGF-ß) is inhibited by Smad7 in a feedback control mechanism. The activity of Smad7 is tightly regulated by multiple post-translational modifications. Using resin-assisted capture and metabolic labeling methods, we show here that Smad7 is S-palmitoylated in mammary epithelial cell models that are widely studied because of their strong responses to TGF-ß and their biological relevance to mammary development and tumor progression. S-palmitoylation of Smad7 is mediated by zDHHC17, a member of a family of 23 S-acyltransferase enzymes. Moreover, we identified four cysteine residues (Cys202, Cys225, Cys415, and Cys417) in Smad7 as palmitoylation acceptor sites. S-palmitoylation of Smad7 on Cys415 and Cys417 promoted the translocation of Smad7 from the nucleus to the cytoplasm, enhanced the stability of the Smad7 protein, and enforced its inhibitory effect on TGF-ß-induced Smad transcriptional response. Thus, our findings reveal a new post-translational modification of Smad7, and highlight an important role of S-palmitoylation to enhance inhibition of TGF-ß/Smad signaling by Smad7.

Asunto(s)

RESUMEN

BACKGROUND: Long non-coding RNAs (lncRNAs) regulate cellular processes by interacting with RNAs or proteins. Transforming growth factor ß (TGFß) signaling via Smad proteins regulates gene networks that control diverse biological processes, including cancer cell migration. LncRNAs have emerged as TGFß targets, yet, their mechanism of action and biological role in cancer remain poorly understood. METHODS: Whole-genome transcriptomics identified lncRNA genes regulated by TGFß. Protein kinase inhibitors and RNA-silencing, in combination with cDNA cloning, provided loss- and gain-of-function analyses. Cancer cell-based assays coupled to RNA-immunoprecipitation, chromatin isolation by RNA purification and protein screening sought mechanistic evidence. Functional validation of TGFß-regulated lncRNAs was based on new transcriptomics and by combining RNAscope with immunohistochemical analysis in tumor tissue. RESULTS: Transcriptomics of TGFß signaling responses revealed down-regulation of the predominantly cytoplasmic long intergenic non-protein coding RNA 707 (LINC00707). Expression of LINC00707 required Smad and mitogen-activated protein kinase inputs. By limiting the binding of Krüppel-like factor 6 to the LINC00707 promoter, TGFß led to LINC00707 repression. Functionally, LINC00707 suppressed cancer cell invasion, as well as key fibrogenic and pro-mesenchymal responses to TGFß, as also attested by RNA-sequencing analysis. LINC00707 also suppressed Smad-dependent signaling. Mechanistically, LINC00707 interacted with and retained Smad proteins in the cytoplasm. Upon TGFß stimulation, LINC00707 dissociated from the Smad complex, which allowed Smad accumulation in the nucleus. In vivo, LINC00707 expression was negatively correlated with Smad2 activation in tumor tissues. CONCLUSIONS: LINC00707 interacts with Smad proteins and limits the output of TGFß signaling, which decreases LINC00707 expression, thus favoring cancer cell invasion. Video Abstract.

Asunto(s)

RESUMEN

Activation of platelet-derived growth factor (PDGF) receptors α and ß (PDGFRα and PDGFRß) at the cell surface by binding of PDGF isoforms leads to internalization of receptors, which affects the amplitude and kinetics of signaling. Ubiquitination of PDGF receptors in response to ligand stimulation is mediated by the Casitas b-lineage lymphoma (Cbl) family of ubiquitin ligases, promoting internalization and serving as a sorting signal for vesicular trafficking of receptors. We report here that another E3 ligase, i.e., tripartite motif-containing protein 21 (TRIM21), contributes to the ubiquitination of PDGFRß in human primary fibroblasts AG1523 and the osteosarcoma cell line U2OS and regulates basal levels of PDGFRß. We found that siRNA-mediated depletion of TRIM21 led to decreased ubiquitination of PDGFRß in response to PDGF-BB stimulation, while internalization from the cell surface and the rate of ligand-induced degradation of the receptor were not affected. Moreover, induction of TRIM21 decreased the levels of PDGFRß in serum-starved cells, and even more in growing cells, in the absence of PDGF stimulation. Consistently, siRNA knockdown of TRIM21 caused accumulation of the total amount of PDGFRß, both in the cytoplasm and on the cell surface, without affecting mRNA levels of the receptor. We conclude that TRIM21 acts post-translationally and maintains basal levels of PDGFRß, thus suggesting that ubiquitination of PDGFRß by TRIM21 may direct a portion of receptor for degradation in growing cells in a ligand-independent manner.

Asunto(s)

RESUMEN

BACKGROUND: The platelet-derived growth factor (PDGF) family of ligands exerts their cellular effects by binding to α- and ß-tyrosine kinase receptors (PDGFRα and PDGFRß, respectively). SUMOylation is an important posttranslational modification (PTM) which regulates protein stability, localization, activation and protein interactions. A mass spectrometry screen has demonstrated SUMOylation of PDGFRα. However, the functional role of SUMOylation of PDGFRα has remained unknown. RESULTS: In the present study, we validated that PDGFRα is SUMOylated on lysine residue 917 as was previously reported using a mass spectrometry approach. Mutation of lysine residue 917 to arginine (K917R) in PDGFRα substantially decreased SUMOylation, indicating that this amino acid residue is a major SUMOylation site. Whereas no difference in the stability of wild-type and mutant receptor was observed, the K917R mutant PDGFRα was less ubiquitinated than wild-type PDGFRα. The internalization and trafficking of the receptor to early and late endosomes were not affected by the mutation, neither was the localization of the PDGFRα to Golgi. However, the K917R mutant PDGFRα showed delayed activation of PLC-γ and enhanced activation of STAT3. Functional assays showed that the mutation of K917 of PDGFRα decreased cell proliferation in response to PDGF-BB stimulation. CONCLUSIONS: SUMOylation of PDGFRα decreases ubiquitination of the receptor and affects ligand-induced signaling and cell proliferation.

Asunto(s)

RESUMEN

In the present study, we show that the inhibitor of the apoptosis-stimulating protein of p53 (iASPP) physically interacts with the hyaluronan receptor CD44 in normal and transformed cells. We noticed that the CD44 standard isoform (CD44s), but not the variant isoform (CD44v), bound to iASPP via the ankyrin-binding domain in CD44s. The formation of iASPP-CD44s complexes was promoted by hyaluronan stimulation in fibroblasts but not in epithelial cells. The cellular level of p53 affected the amount of the iASPP-CD44 complex. iASPP was required for hyaluronan-induced CD44-dependent migration and adhesion of fibroblasts. Of note, CD44 altered the sub-cellular localization of the iASPP-p53 complex; thus, ablation of CD44 promoted translocation of iASPP from the nucleus to the cytoplasm, resulting in increased formation of a cytoplasmic iASPP-p53 complex in fibroblasts. Overexpression of iASPP decreased, but CD44 increased the level of intracellular reactive oxygen species (ROS). Knock-down of CD44s, in the presence of p53, led to increased cell growth and cell density of fibroblasts by suppression of p27 and p53. Our observations suggest that the balance of iASPP-CD44 and iASPP-p53 complexes affect the survival and migration of fibroblasts.

RESUMEN

The liver kinase B1 (LKB1) controls cellular metabolism and cell polarity across species. We previously established a mechanism for negative regulation of transforming growth factor ß (TGFß) signaling by LKB1. The impact of this mechanism in the context of epithelial polarity and morphogenesis remains unknown. After demonstrating that human mammary tissue expresses robust LKB1 protein levels, whereas invasive breast cancer exhibits significantly reduced LKB1 levels, we focused on mammary morphogenesis studies in three dimensional (3D) acinar organoids. CRISPR/Cas9-introduced loss-of-function mutations of STK11 (LKB1) led to profound defects in the formation of 3D organoids, resulting in amorphous outgrowth and loss of rotation of young organoids embedded in matrigel. This defect was associated with an enhanced signaling by TGFß, including TGFß auto-induction and induction of transcription factors that mediate epithelial-mesenchymal transition (EMT). Protein marker analysis confirmed a more efficient EMT response to TGFß signaling in LKB1 knockout cells. Accordingly, chemical inhibition of the TGFß type I receptor kinase largely restored the morphogenetic defect of LKB1 knockout cells. Similarly, chemical inhibition of the bone morphogenetic protein pathway or the TANK-binding kinase 1, or genetic silencing of the EMT factor SNAI1, partially restored the LKB1 knockout defect. Thus, LKB1 sustains mammary epithelial morphogenesis by limiting pathways that promote EMT. The observed downregulation of LKB1 expression in breast cancer is therefore predicted to associate with enhanced EMT induced by SNAI1 and TGFß family members.

Asunto(s)

RESUMEN

Breast cancer is a common cancer in women. Breast cancer cells synthesize large amounts of hyaluronan to assist their proliferation, survival, migration and invasion. Accumulation of hyaluronan and overexpression of its receptor CD44 and hyaluronidase TMEM2 in breast tumors correlate with tumor progression and reduced overall survival of patients. Currently, the only known small molecule inhibitor of hyaluronan synthesis is 4-methyl-umbelliferone (4-MU). Due to the importance of hyaluronan for breast cancer progression, our aim was to identify new, potent and chemically distinct inhibitors of its synthesis. Here, we report a new small molecule inhibitor of hyaluronan synthesis, the thymidine analog 5'-Deoxy-5'-(1,3-Diphenyl-2-Imidazolidinyl)-Thymidine (DDIT). This compound is more potent than 4-MU and displays significant anti-tumorigenic properties. Specifically, DDIT inhibits breast cancer cell proliferation, migration, invasion and cancer stem cell self-renewal by suppressing HAS-synthesized hyaluronan. DDIT appears as a promising lead compound for the development of inhibitors of hyaluronan synthesis with potential usefulness in breast cancer treatment.

RESUMEN

Transforming growth factor-ß (TGF-ß) signaling has important roles during embryonic development and in tissue homeostasis. TGF-ß ligands exert cellular effects by binding to type I (TßRI) and type II (TßRII) receptors and inducing both SMAD-dependent and SMAD-independent intracellular signaling pathways, the latter of which includes the activation of the tyrosine kinase Src. We investigated the mechanism by which TGF-ß stimulation activates Src in human and mouse cells. Before TGF-ß stimulation, inactive Src was complexed with TßRII. Upon TGF-ß1 stimulation, TßRII associated with and phosphorylated TßRI at Tyr182. Binding of Src to TßRI involved the interaction of the Src SH2 domain with phosphorylated Tyr182 and the interaction of the Src SH3 domain with a proline-rich region in TßRI and led to the activation of Src kinase activity and Src autophosphorylation. TGF-ß1-induced Src activation required the kinase activities of TßRII and Src but not that of TßRI. Activated Src also phosphorylated TßRI on several tyrosine residues, which may stabilize the binding of Src to the receptor. Src activation was required for the ability of TGF-ß to induce fibronectin production and migration in human breast carcinoma cells and to induce α-smooth muscle actin and actin reorganization in mouse fibroblasts. Thus, TGF-ß induces Src activation by stimulating a direct interaction with TßRI that depends on tyrosine phosphorylation of TßRI by TßRII.

Asunto(s)

RESUMEN

The transcription factor SNAI1 mediates epithelial-mesenchymal transition, fibroblast activation and controls inter-tissue migration. High SNAI1 expression characterizes metastatic triple-negative breast carcinomas, and its knockout by CRISPR/Cas9 uncovered an epithelio-mesenchymal phenotype accompanied by reduced signaling by the cytokine TGFß. The SNAI1 knockout cells exhibited plasticity in differentiation, drifting towards the luminal phenotype, gained stemness potential and could differentiate into acinar mammospheres in 3D culture. Loss of SNAI1 de-repressed the transcription factor FOXA1, a pioneering factor of mammary luminal progenitors. FOXA1 induced a specific gene program, including the androgen receptor (AR). Inhibiting AR via a specific antagonist regenerated the basal phenotype and blocked acinar differentiation. Thus, loss of SNAI1 in the context of triple-negative breast carcinoma cells promotes an intermediary luminal progenitor phenotype that gains differentiation plasticity based on the dual transcriptional action of FOXA1 and AR. This function of SNAI1 provides means to separate cell invasiveness from progenitor cell de-differentiation as independent cellular programs.

Asunto(s)

RESUMEN

Glioblastoma multiforme (GBM) is a lethal brain tumor, characterized by enhanced proliferation and invasion, as well as increased vascularization and chemoresistance. The expression of the hyaluronan receptor CD44 has been shown to correlate with GBM progression and poor prognosis. Here, we sought to elucidate the molecular mechanisms by which CD44 promotes GBM progression by knocking out (KO) CD44, employing CRISPR/Cas9 gene editing in U251MG cells. CD44-depleted cells exhibited an impaired proliferation rate, as shown by the decreased cell numbers, decreased Ki67-positive cell nuclei, diminished phosphorylation of CREB, and increased levels of the cell cycle inhibitor p16 compared to control cells. Furthermore, the CD44 KO cells showed decreased stemness and increased senescence, which was manifested upon serum deprivation. In stem cell-like enriched spheres, RNA-sequencing analysis of U251MG cells revealed a CD44 dependence for gene signatures related to hypoxia, the glycolytic pathway, and G2 to M phase transition. Partially similar results were obtained when cells were treated with the γ-secretase inhibitor DAPT, which inhibits CD44 cleavage and therefore inhibits the release of the intracellular domain (ICD) of CD44, suggesting that certain transcriptional responses are dependent on CD44-ICD. Interestingly, the expression of molecules involved in hyaluronan synthesis, degradation, and interacting matrix proteins, as well as of platelet-derived growth factor (PDGF) isoforms and PDGF receptors, were also deregulated in CD44 KO cells. These results were confirmed by the knockdown of CD44 in another GBM cell line, U2990. Notably, downregulation of hyaluronan synthase 2 (HAS2) impaired the hypoxia-related genes and decreased the CD44 protein levels, suggesting a CD44/hyaluronan feedback circuit contributing to GBM progression.

RESUMEN

BACKGROUND: Transforming growth factor ß (TGFß) is overexpressed in several advanced cancer types and promotes tumor progression. We have reported that the intracellular domain (ICD) of TGFß receptor (TßR) I is cleaved by proteolytic enzymes in cancer cells, and then translocated to the nucleus in a manner dependent on the endosomal adaptor proteins APPL1/2, driving an invasiveness program. How cancer cells evade TGFß-induced growth inhibition is unclear. METHODS: We performed microarray analysis to search for genes regulated by APPL1/2 proteins in castration-resistant prostate cancer (CRPC) cells. We investigated the role of TßRI and TRAF6 in mitosis in cancer cell lines cultured in 10% FBS in the absence of exogenous TGFß. The molecular mechanism of the ubiquitination of AURKB by TRAF6 in mitosis and the formation of AURKB-TßRI complex in cancer cell lines and tissue microarrays was also studied. FINDINGS: During mitosis and cytokinesis, AURKB-TßRI complexes formed in midbodies in CRPC and KELLY neuroblastoma cells. TRAF6 induced polyubiquitination of AURKB on K85 and K87, protruding on the surface of AURKB to facilitate its activation. AURKB-TßRI complexes in patient's tumor tissue sections correlated with the malignancy of prostate cancer. INTERPRETATION: The AURKB-TßRI complex may become a prognostic biomarker for patients with risk of developing aggressive PC. FUNDING: Swedish Medical Research Council (2019-01598, ML; 2015-02757 and 2020-01291, CHH), the Swedish Cancer Society (20 0964, ML), a regional agreement between Umeå University and Region Västerbotten (ALF; RV-939377, -967041, -970057, ML). The European Research Council (787472, CHH). KAW 2019.0345, and the Kempe Foundation SMK-1866; ML. National Microscopy Infrastructure (NMI VR-RFI 2016-00968).

Asunto(s)

RESUMEN

Complexity in mechanisms that drive cancer development and progression is exemplified by the transforming growth factor ß (TGF-ß) signaling pathway, which suppresses early-stage hyperplasia, yet assists aggressive tumors to achieve metastasis. Of note, several molecules, including mRNAs, non-coding RNAs, and proteins known to be associated with the TGF-ß pathway have been reported as constituents in the cargo of extracellular vesicles (EVs). EVs are secreted vesicles delimited by a lipid bilayer and play critical functions in intercellular communication, including regulation of the tumor microenvironment and cancer development. Thus, this review aims at summarizing the impact of EVs on TGF-ß signaling by focusing on mechanisms by which EV cargo can influence tumorigenesis, metastatic spread, immune evasion and response to anti-cancer treatment. Moreover, we emphasize the potential of TGF-ß-related molecules present in circulating EVs as useful biomarkers of prognosis, diagnosis, and prediction of response to treatment in cancer patients.

RESUMEN

Transforming growth factor ß (TGFß) induces epithelial-mesenchymal transition (EMT), which correlates with stemness and invasiveness. Mesenchymal-epithelial transition (MET) is induced by TGFß withdrawal and correlates with metastatic colonization. Whether TGFß promotes stemness and invasiveness simultaneously via EMT remains unclear. We established a breast cancer cell model expressing red fluorescent protein (RFP) under the E-cadherin promoter. In 2D cultures, TGFß induced EMT, generating RFPlow cells with a mesenchymal transcriptome, and regained RFP, with an epithelial transcriptome, after MET induced by TGFß withdrawal. RFPlow cells generated robust mammospheres, with epithelio-mesenchymal cell surface features. Mammospheres that were forced to adhere generated migratory cells, devoid of RFP, a phenotype which was inhibited by a TGFß receptor kinase inhibitor. Further stimulation of RFPlow mammospheres with TGFß suppressed the generation of motile cells, but enhanced mammosphere growth. Accordingly, mammary fat-pad-transplanted mammospheres, in the absence of exogenous TGFß treatment, established lung metastases with evident MET (RFPhigh cells). In contrast, TGFß-treated mammospheres revealed high tumour-initiating capacity, but limited metastatic potential. Thus, the biological context of partial EMT and MET allows TGFß to differentiate between pro-stemness and pro-invasive phenotypes.

Asunto(s)

RESUMEN

Interaction of platelet-derived growth factor (PDGF) isoforms with their receptors results in activation and internalization of receptors, with a concomitant activation of downstream signalling pathways. Ubiquitination of PDGFRs serves as a mark to direct the internalization and sorting of the receptors. By overexpressing a panel of deubiquitinating enzymes (DUBs), we found that USP17 and USP4 efficiently deubiquitinate PDGF receptor ß (PDGFRß) and are able to remove both Lys63 and Lys48-linked polyubiquitin chains from the receptor. Deubiquitination of PDGFRß did not affect its stability, but regulated the timing of its trafficking, whereby USP17 prolonged the presence of the receptor at the cell surface, while USP4 affected the speed of trafficking towards early endosomes. Induction of each of the DUBs in BJhTERT fibroblasts and U2OS osteosarcoma cells led to prolonged and/or shifted activation of STAT3 in response to PDGF-BB stimulation, which in turn led to increased transcriptional activity of STAT3. Induction of USP17 promoted acute upregulation of the mRNA expression of STAT3-inducible genes STAT3, CSF1, junB and c-myc, while causing long-term changes in the expression of myc and CDKN1A. Deletion of USP17 was lethal to fibroblasts, while deletion of USP4 led to a decreased proliferative response to stimulation by PDGF-BB. Thus, USP17- and USP4-mediated changes in ubiquitination of PDFGRß lead to dysregulated signalling and transcription downstream of STAT3, resulting in defects in the control of cell proliferation.

Asunto(s)

RESUMEN

The role of liver kinase B1 (LKB1) in glioblastoma (GBM) development remains poorly understood. LKB1 may regulate GBM cell metabolism and has been suggested to promote glioma invasiveness. After analyzing LKB1 expression in GBM patient mRNA databases and in tumor tissue via multiparametric immunohistochemistry, we observed that LKB1 was localized and enriched in GBM tumor cells that co-expressed SOX2 and NESTIN stemness markers. Thus, LKB1-specific immunohistochemistry can potentially reveal subpopulations of stem-like cells, advancing GBM patient molecular pathology. We further analyzed the functions of LKB1 in patient-derived GBM cultures under defined serum-free conditions. Silencing of endogenous LKB1 impaired 3D-gliomasphere frequency and promoted GBM cell invasion in vitro and in the zebrafish collagenous tail after extravasation of circulating GBM cells. Moreover, loss of LKB1 function revealed mitochondrial dysfunction resulting in decreased ATP levels. Treatment with the clinically used drug metformin impaired 3D-gliomasphere formation and enhanced cytotoxicity induced by temozolomide, the primary chemotherapeutic drug against GBM. The IC50 of temozolomide in the GBM cultures was significantly decreased in the presence of metformin. This combinatorial effect was further enhanced after LKB1 silencing, which at least partially, was due to increased apoptosis. The expression of genes involved in the maintenance of tumor stemness, such as growth factors and their receptors, including members of the platelet-derived growth factor (PDGF) family, was suppressed after LKB1 silencing. The defect in gliomasphere growth caused by LKB1 silencing was bypassed after supplementing the cells with exogenous PFDGF-BB. Our data support the parallel roles of LKB1 in maintaining mitochondrial homeostasis, 3D-gliomasphere survival, and hindering migration in GBM. Thus, the natural loss of, or pharmacological interference with LKB1 function, may be associated with benefits in patient survival but could result in tumor spread.

Asunto(s)

RESUMEN

Glioma-initiating cells (GICs), a major source of glioblastoma recurrence, are characterized by the expression of neural stem cell markers and the ability to grow by forming nonadherent spheres under serum-free conditions. Bone morphogenetic proteins (BMPs), members of the transforming growth factor-ß family, induce differentiation of GICs and suppress their tumorigenicity. However, the mechanisms underlying the BMP-induced loss of GIC stemness have not been fully elucidated. Here, we show that paired related homeobox 1 (PRRX1) induced by BMPs decreases the CD133-positive GIC population and inhibits tumorigenic activity of GICs in vivo. Of the two splice isoforms of PRRX1, the longer isoform, pmx-1b, but not the shorter isoform, pmx-1a, induces GIC differentiation. Upon BMP stimulation, pmx-1b interacts with the DNA methyltransferase DNMT3A and induces promoter methylation of the PROM1 gene encoding CD133. Silencing DNMT3A maintains PROM1 expression and increases the CD133-positive GIC population. Thus, pmx-1b promotes loss of stem cell-like properties of GICs through region-specific epigenetic regulation of CD133 expression by recruiting DNMT3A, which is associated with decreased tumorigenicity of GICs.

Asunto(s)

RESUMEN

Glioblastoma (GBM) is a brain malignancy characterized by invasiveness to the surrounding brain tissue and by stem-like cells, which propagate the tumor and may also regulate invasiveness. During brain development, polarity proteins, such as Par3, regulate asymmetric cell division of neuro-glial progenitors and neurite motility. We, therefore, studied the role of the Par3 protein (encoded by PARD3) in GBM. GBM patient transcriptomic data and patient-derived culture analysis indicated diverse levels of expression of PARD3 across and independent from subtypes. Multiplex immunolocalization in GBM tumors identified Par3 protein enrichment in SOX2-, CD133-, and NESTIN-positive (stem-like) cells. Analysis of GBM cultures of the three subtypes (proneural, classical, mesenchymal), revealed decreased gliomasphere forming capacity and enhanced invasiveness upon silencing Par3. GBM cultures with suppressed Par3 showed low expression of stemness (SOX2 and NESTIN) but higher expression of differentiation (GFAP) genes. Moreover, Par3 silencing reduced the expression of a set of genes encoding mitochondrial enzymes that generate ATP. Accordingly, silencing Par3 reduced ATP production and concomitantly increased reactive oxygen species. The latter was required for the enhanced migration observed upon silencing of Par3 as anti-oxidants blocked the enhanced migration. These findings support the notion that Par3 exerts homeostatic redox control, which could limit the tumor cell-derived pool of oxygen radicals, and thereby the tumorigenicity of GBM.

Asunto(s)

RESUMEN

We previously reported that bone morphogenetic protein (BMP) signaling promotes tumorigenesis in gynecologic cancer cells. BMP2 enhances proliferation of ovarian and endometrial cancer cells via c-KIT induction, and triggers epithelial-mesenchymal transition (EMT) by SNAIL and/or SLUG induction, leading to increased cell migration. However, the downstream effectors of BMP signaling in gynecological cancer cells have not been clearly elucidated. In this study, we performed RNA-sequencing of Ishikawa endometrial and SKOV3 ovarian cancer cells after BMP2 stimulation, and identified TNFRSF12A, encoding fibroblast growth factor-inducible 14 (FN14) as a common BMP2-induced gene. FN14 knockdown suppressed BMP2-induced cell proliferation and migration, confirmed by MTS and scratch assays, respectively. In addition, FN14 silencing augmented chemosensitivity of SKOV3 cells. As a downstream effector of BMP signaling, FN14 modulated both c-KIT and SNAIL expression, which are important for growth and migration of ovarian and endometrial cancer cells. These results support the notion that the tumor promoting effects of BMP signaling in gynecological cancers are partially attributed to FN14 induction.

Asunto(s)