Upregulation of the canonical signaling pathway of interferon-gamma is associated with glioblastoma progression.

BACKGROUND: Glioblastoma is a brain malignant tumor grade IV, highly invasive. Alterations in several signaling pathways are involved in glioblastoma development. In this work, we evaluated the IFN-γ canonical signaling pathway in glioblastoma cells and its effect on cell viability and migration. METHODS: The levels of STAT1/pSTAT1, IRF1, and PD-L1 in LN-18 glioblastoma cells were analyzed using western blotting. Cell viability was evaluated by calcein-AM/propidium iodide assays, and a wound healing assay was used to study the migration of glioblastoma cells treated with IFN-γ. Our aim was to determine the expression of IFN-γ signaling elements in cell lines and tissue from glioblastoma samples and examine the relationship between these elements and the survival of glioblastoma patients. The following platforms were utilized for analysis: the CCLE (Cancer Cell Line Encyclopedia), UALCAN (University of Alabama at Birmingham Cancer data analysis Portal), GEPIA (Gene Expression Profiling Interactive Analysis), and GENT2 (Gene Expression patterns across Normal and Tumor tissues). RESULTS: Our results evidenced that IFN-γ signaling increases non-phosphorylated and phosphorylated STAT1 levels and promotes the upregulation of IRF1 and PD-L1 in glioblastoma cells. The activation of IFN-γ signaling increased cell migration without affecting the viability of glioblastoma cells. Furthermore, in silico analysis showed that the elements of IFN-γ signaling pathways (IFNGR1/IFNGR2/STAT1/IRF1) are upregulated in human glioblastoma samples. The upregulation of IFN-γ signaling was associated with shorter survival in glioblastoma patients. CONCLUSION: IFN-γ signaling pathway is upregulated in glioblastoma, displaying pro-tumor activity. Thus, IFN-γ signaling elements may be potential biomarkers and targets for treating glioblastoma.

Canonical Wnt Pathway Is Involved in Chemoresistance and Cell Cycle Arrest Induction in Colon Cancer Cell Line Spheroids.

The presence of cancer stem cells (CSCs) has been associated with the induction of drug resistance and disease recurrence after therapy. 5-Fluorouracil (5FU) is widely used as the first-line treatment of colorectal cancer (CRC). However, its effectiveness may be limited by the induction of drug resistance in tumor cells. The Wnt pathway plays a key role in the development and CRC progression, but it is not clearly established how it is involved in CSCs resistance to treatment. This work aimed to investigate the role played by the canonical Wnt/ß-catenin pathway in CSCs resistance to 5FU treatment. Using tumor spheroids as a model of CSCs enrichment of CRC cell lines with different Wnt/ß-catenin contexts, we found that 5FU induces in all CRC spheroids tested cell death, DNA damage, and quiescence, but in different proportions for each one: RKO spheroids were very sensitive to 5FU, while SW480 were less susceptible, and the SW620 spheroids, the metastatic derivative of SW480 cells, displayed the highest resistance to death, high clonogenic capacity, and the highest ability for regrowth after 5FU treatment. Activating the canonical Wnt pathway with Wnt3a in RKO spheroids decreased the 5FU-induced cell death. But the Wnt/ß-catenin pathway inhibition with Adavivint alone or in combination with 5FU in spheroids with aberrant activation of this pathway produced a severe cytostatic effect compromising their clonogenic capacity and diminishing the stem cell markers expression. Remarkably, this combined treatment also induced the survival of a small cell subpopulation that could exit the arrest, recover SOX2 levels, and re-grow after treatment.

Lysophosphatidylinositol Promotes Chemotaxis and Cytokine Synthesis in Mast Cells with Differential Participation of GPR55 and CB2 Receptors.

Mast cells (MCs) are the main participants in the control of immune reactions associated with inflammation, allergies, defense against pathogens, and tumor growth. Bioactive lipids are lipophilic compounds able to modulate MC activation. Here, we explored some of the effects of the bioactive lipid lysophosphatidylinositol (LPI) on MCs. Utilizing murine bone marrow-derived mast cells (BMMCs), we found that LPI did not cause degranulation, but slightly increased FcεRI-dependent ß-hexosaminidase release. However, LPI induced strong chemotaxis together with changes in LIM kinase (LIMK) and cofilin phosphorylation. LPI also promoted modifications to actin cytoskeleton dynamics that were detected by an increase in cell size and interruptions in the continuity of the cortical actin ring. The chemotaxis and cortical actin ring changes were dependent on GPR55 receptor activation, since the specific agonist O1602 mimicked the effects of LPI and the selective antagonist ML193 prevented them. The LPI and O1602-dependent stimulation of BMMC also led to VEGF, TNF, IL-1α, and IL-1ß mRNA accumulation, but, in contrast with chemotaxis-related processes, the effects on cytokine transcription were dependent on GPR55 and cannabinoid (CB) 2 receptors, since they were sensitive to ML193 and to the specific CB2 receptor antagonist AM630. Remarkably, GPR55-dependent BMMC chemotaxis was observed towards conditioned media from distinct mouse and human cancer cells. Our data suggest that LPI induces the chemotaxis of MCs and leads to cytokine production in MC in vitro with the differential participation of GPR55 and CB2 receptors. These effects could play a significant role in the recruitment of MCs to tumors and the production of MC-derived pro-angiogenic factors in the tumor microenvironment.

Hepatic C9 cells switch their behaviour in short or long exposure to soft substrates.

BACKGROUND INFORMATION: There have been several studies to understand the influence of stiffness of the culture substrates for different types of adherent cells. It is generally accepted that cell proliferation, spreading and focal adhesions increase with higher matrix stiffness. However, what remains unclear is whether this kind of cell behaviour may be reverted by culturing on soft substrates those cell lines that were originally selected or primed on stiff surfaces. RESULTS: Here, we studied the influence of substrate softness on proliferation, adhesion and morphology of the highly proliferative hepatic C9 cell line. We cultured C9 cells on soft and stiff polydimethylsiloxane (PDMS) substrates prepared with two different elastic moduli in the range of 10 and 200 kPa, respectively. Lower cell proliferation was observed on substrates with lower stiffness without affecting cell viability. The proliferation rate of C9 cell line along with extracellular growth-regulated kinase (ERK) phosphorylation was decreased on soft PDMS. Despite this cell line has been described as a hepatic epithelial cell, our characterisation demonstrated that the origin of C9 cells is uncertain, although clearly epithelial, with the expression of markers of several hepatic cells. Surprisingly, consecutive passages of C9 cells on soft PDMS did not alter this mesenchymal phenotype, vimentin expression did not decrease when culturing cells in soft substrates, even though the ERK phosphorylation levels eventually were increased after several passages on soft PDMS, triggering again an increase of cell proliferation. CONCLUSIONS AND SIGNIFICANCE: This study shows that the exposure of C9 cells to soft substrates promoted a decrease of cell proliferation rate, as reported for other types of cells on PDMS, whereas a much longer term exposure caused cells to adapt to softness after trained for several passages, reactivating proliferation. During this phenomenon, the morphology and phenotype of trained cells was modified accompanying the increase of cell proliferation rate contrary to the effect observed in short periods of cell culture. In contrast to previous reports, cell death was not observed during these experiments, discarding a cell selection mechanism and suggesting soft cell adaptation may be limited in time in C9 cells.

Fyn kinase mediates cortical actin ring depolymerization required for mast cell migration in response to TGF-ß in mice.

Transforming growth factor-ß (TGF-ß) is a potent mast cell (MC) chemoattractant able to modulate local inflammatory reactions. The molecular mechanism leading to TGF-ß-directed MC migration is not fully described. Here we analyzed the role of the Src family protein kinase Fyn on the main TGF-ß-induced cytoskeletal changes leading to MC migration. Utilizing bone marrow-derived mast cells (BMMCs) from WT and Fyn-deficient mice we found that BMMC migration to TGF-ß was impaired in the absence of the kinase. TGF-ß caused depolymerization of the cortical actin ring and changes on the phosphorylation of cofilin, LIMK and CAMKII only in WT cells. Defective cofilin activation and phosphorylation of regulatory proteins was detected in Fyn-deficient BMMCs and this finding correlated with a lower activity of the catalytic subunit of the phosphatase PP2A. Diminished TGF-ß-induced chemotaxis of Fyn-deficient cells was also observed in an in vivo model of MC migration (bleomycin-induced scleroderma). Our results show that Fyn kinase is an important positive effector of TGF-ß-induced chemotaxis through the control of PP2A activity and this is relevant to pathological processes that are related to TGF-ß-dependent mast cell migration.

Protein Tyrosine Kinase Fyn Regulates TLR4-Elicited Responses on Mast Cells Controlling the Function of a PP2A-PKCα/ß Signaling Node Leading to TNF Secretion.

Mast cells produce proinflammatory cytokines in response to TLR4 ligands, but the signaling pathways involved are not fully described. In this study, the participation of the Src family kinase Fyn in the production of TNF after stimulation with LPS was evaluated using bone marrow-derived mast cells from wild-type and Fyn-deficient mice. Fyn(-/-) cells showed higher LPS-induced secretion of preformed and de novo-synthesized TNF. In both cell types, TNF colocalized with vesicle-associated membrane protein (VAMP)3-positive compartments. Addition of LPS provoked coalescence of VAMP3 and its interaction with synaptosomal-associated protein 23; those events were increased in the absence of Fyn. Higher TNF mRNA levels were also observed in Fyn-deficient cells as a result of increased transcription and greater mRNA stability after LPS treatment. Fyn(-/-) cells also showed higher LPS-induced activation of TAK-1 and ERK1/2, whereas IκB kinase and IκB were phosphorylated, even in basal conditions. Increased responsiveness in Fyn(-/-) cells was associated with a lower activity of protein phosphatase 2A (PP2A) and augmented activity of protein kinase C (PKC)α/ß, which was dissociated from PP2A and increased its association with the adapter protein neuroblast differentiation-associated protein (AHNAK, desmoyokin). LPS-induced PKCα/ß activity was associated with VAMP3 coalescence in WT and Fyn-deficient cells. Reconstitution of MC-deficient Wsh mice with Fyn(-/-) MCs produced greater LPS-dependent production of TNF in the peritoneal cavity. Our data show that Fyn kinase is activated after TLR4 triggering and exerts an important negative control on LPS-dependent TNF production in MCs controlling the inactivation of PP2Ac and activation of PKCα/ß necessary for the secretion of TNF by VAMP3(+) carriers.

Novel regulation of Ski protein stability and endosomal sorting by actin cytoskeleton dynamics in hepatocytes.

TGF-ß-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-ß signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-ß and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-ß/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-ß and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration.

Actin-cytoskeleton polymerization differentially controls the stability of Ski and SnoN co-repressors in normal but not in transformed hepatocytes.

BACKGROUND: Ski and SnoN proteins function as transcriptional co-repressors in the TGF-ß pathway. They regulate cell proliferation and differentiation, and their aberrant expression results in altered TGF-ß signalling, malignant transformation, and alterations in cell proliferation. METHODS: We carried out a comparative characterization of the endogenous Ski and SnoN protein regulation by TGF-ß, cell adhesion disruption and actin-cytoskeleton rearrangements between normal and transformed hepatocytes; we also analyzed Ski and SnoN protein stability, subcellular localization, and how their protein levels impact the TGF-ß/Smad-driven gene transcription. RESULTS: Ski and SnoN protein levels are lower in normal hepatocytes than in hepatoma cells. They exhibit a very short half-life and a nuclear/cytoplasmic distribution in normal hepatocytes opposed to a high stability and restricted nuclear localization in hepatoma cells. Interestingly, while normal cells exhibit a transient TGF-ß-induced gene expression, the hepatoma cells are characterized by a strong and sustained TGF-ß-induced gene expression. A novel finding is that Ski and SnoN stability is differentially regulated by cell adhesion and cytoskeleton rearrangements in the normal hepatocytes. The inhibition of protein turnover down-regulated both Ski and SnoN co-repressors impacting the kinetic of expression of TGF-ß-target genes. CONCLUSION: Normal regulatory mechanisms controlling Ski and SnoN stability, subcellular localization and expression are altered in hepatocarcinoma cells. GENERAL SIGNIFICANCE: This work provides evidence that Ski and SnoN protein regulation is far more complex in normal than in transformed cells, since many of the normal regulatory mechanisms are lost in transformed cells.

Downregulation of SnoN oncoprotein induced by antibiotics anisomycin and puromycin positively regulates transforming growth factor-ß signals.

BACKGROUND: SnoN and Ski proteins function as Smad transcriptional corepressors and are implicated in the regulation of diverse cellular processes such as proliferation, differentiation and transformation. Transforming growth factor-ß (TGF-ß) signaling causes SnoN and Ski protein degradation via proteasome with the participation of phosphorylated R-Smad proteins. Intriguingly, the antibiotics anisomycin (ANS) and puromycin (PURO) are also able to downregulate Ski and SnoN proteins via proteasome. METHODS: We explored the effects of ANS and PURO on SnoN protein downregulation when the activity of TGF-ß signaling was inhibited by using different pharmacological and non-pharmacological approaches, either by using specific TßRI inhibitors, overexpressing the inhibitory Smad7 protein, or knocking-down TßRI receptor or Smad2 by specific shRNAs. The outcome of SnoN and Ski downregulation induced by ANS or PURO on TGF-ß signaling was also studied. RESULTS: SnoN protein downregulation induced by ANS and PURO did not involve the induction of R-Smad phosphorylation but it was abrogated after TGF-ß signaling inhibition; this effect occurred in a cell type-specific manner and independently of protein synthesis inhibition or any other ribotoxic effect. Intriguingly, antibiotics seem to require components of the TGF-ß/Smad pathway to downregulate SnoN. In addition, SnoN protein downregulation induced by antibiotics favored gene transcription induced by TGF-ß signaling. CONCLUSIONS: ANS and PURO require TGF-ß/Smad pathway to induce SnoN and Ski protein downregulation independently of inducing R-Smad2 phosphorylation, which facilitates TGF-ß signaling. GENERAL SIGNIFICANCE: Antibiotic analogs lacking ribotoxic effects are useful as pharmacological tools to study TGF-ß signaling by controlling Ski and SnoN protein levels.

Deregulation of interferon-gamma receptor 1 expression and its implications for lung adenocarcinoma progression.

Interferon-gamma (IFN-γ) plays a dual role in cancer; it is both a pro- and an antitumorigenic cytokine, depending on the type of cancer. The deregulation of the IFN-γ canonic pathway is associated with several disorders, including vulnerability to viral infections, inflammation, and cancer progression. In particular, the interplay between lung adenocarcinoma (LUAD) and viral infections appears to exist in association with the deregulation of IFN-γ signaling. In this mini-review, we investigated the status of the IFN-γ signaling pathway and the expression level of its components in LUAD. Interestingly, a reduction in IFNGR1 expression seems to be associated with LUAD progression, affecting defenses against viruses such as severe acute respiratory syndrome coronavirus 2. In addition, alterations in the expression of IFNGR1 may inhibit the antiproliferative action of IFN-γ signaling in LUAD.

Transforming growth factor-ß/SMAD Target gene SKIL is negatively regulated by the transcriptional cofactor complex SNON-SMAD4.

The human SKI-like (SKIL) gene encodes the SMAD transcriptional corepressor SNON that antagonizes TGF-ß signaling. SNON protein levels are tightly regulated by the TGF-ß pathway: whereas a short stimulation with TGF-ß decreases SNON levels by its degradation via the proteasome, longer TGF-ß treatment increases SNON levels by inducing SKIL gene expression. Here, we investigated the molecular mechanisms involved in the self-regulation of SKIL gene expression by SNON. Bioinformatics analysis showed that the human SKIL gene proximal promoter contains a TGF-ß response element (TRE) bearing four groups of SMAD-binding elements that are also conserved in mouse. Two regions of 408 and 648 bp of the human SKIL gene (∼2.4 kb upstream of the ATG initiation codon) containing the core promoter, transcription start site, and the TRE were cloned for functional analysis. Binding of SMAD and SNON proteins to the TRE region of the SKIL gene promoter after TGF-ß treatment was demonstrated by ChIP and sequential ChIP assays. Interestingly, the SNON-SMAD4 complex negatively regulated basal SKIL gene expression through binding the promoter and recruiting histone deacetylases. In response to TGF-ß signal, SNON is removed from the SKIL gene promoter, and then the activated SMAD complexes bind the promoter to induce SKIL gene expression. Subsequently, the up-regulated SNON protein in complex with SMAD4 represses its own expression as part of the negative feedback loop regulating the TGF-ß pathway. Accordingly, when the SNON-SMAD4 complex is absent as in some cancer cells lacking SMAD4 the regulation of some TGF-ß target genes is modified.

Calcium-sensing receptor inhibits TGF-ß-signaling by decreasing Smad2 phosphorylation.

Calcium-sensing receptor (CaSR) contributes to maintain homeostatic levels of extracellular calcium. In addition, CaSR controls other cellular activities such as proliferation and migration, particularly in cells not related to extracellular calcium homeostasis, potentially by cross-talking with parallel signaling pathways. Here we report that CaSR attenuates transforming growth factor-ß (TGF-ß)-signaling in hepatic C9 cells and in transfected HEK293 cells. Wild type CaSR interferes with TGF-ß-dependent Smad2 phosphorylation and induces its proteasomal degradation, resulting in a decrease of TGF-ß-dependent transcriptional activity, whereas an inactivating CaSR mutant does not transduce an inhibitory effect of extracellular calcium on TGF-ß signaling. Attenuation of TGF-ß signaling in response to extracellular calcium is linked to Rab11-dependent CaSR-trafficking with the intervention of CaSR carboxyl-terminal tail. Our data suggest that CaSR might regulate TGF-ß-dependent cellular responses mediated by TGF-ß signaling inhibition.

TGF-ß/SMAD canonical pathway induces the expression of transcriptional cofactor TAZ in liver cancer cells.

The TGF-ß and Hippo pathways are critical for liver size control, regeneration, and cancer progression. The transcriptional cofactor TAZ, also named WWTR1, is a downstream effector of Hippo pathway and plays a key role in the maintenance of liver physiological functions. However, the up-regulation of TAZ expression has been associated with liver cancer progression. Recent evidence shows crosstalk of TGF-ß and Hippo pathways, since TGF-ß modulates TAZ expression through different mechanisms in a cellular context-dependent manner but supposedly independent of SMADs. Here, we evaluate the molecular interplay between TGF-ß pathway and TAZ expression and observe that TGF-ß induces TAZ expression through SMAD canonical pathway in liver cancer HepG2 cells. Therefore, TAZ cofactor is a primary target of TGF-ß/SMAD-signaling, one of the pathways altered in liver cancer.

Casiopeina II-gly and bromo-pyruvate inhibition of tumor hexokinase, glycolysis, and oxidative phosphorylation.

The copper-based drug Casiopeina II-gly (CasII-gly) shows potent antineoplastic effect and diminishes mitochondrial metabolism on several human and rodent malignant tumors. To elucidate whether CasII-gly also affects glycolysis, (a) the flux through the complete pathway and the initial segment and (b) the activities of several glycolytic enzymes of AS-30D hepatocarcinoma cells were determined. CasII-gly (IC50 = 0.74-6.7 µM) was more effective to inhibit 24-72 h growth of several human carcinomas than 3-bromopyruvate (3BrPyr) (IC50 = 45-100 µM) with no apparent effect on normal human-proliferating lymphocytes and HUVECs. In short-term 60-min experiments, CasII-gly increased tumor cell lactate production and glycogen breakdown. CasII-gly was 1.3-21 times more potent than 3BrPyr and cisplatin to inhibit tumor HK. As CasII-gly inhibited the soluble and mitochondrial HK activities and the flux through the HK-TPI glycolytic segment, whereas PFK-1, GAPDH, PGK, PYK activities and HPI-TPI segment flux were not affected, the data suggested glycogenolysis activation induced by HK inhibition. Accordingly, glycogen-depleted as well as oligomycin-treated cancer cells became more sensitive to CasII-gly. The inhibition time-course of HK by CasII-gly was slower than that of OxPhos in AS-30D cells, indicating that glycolytic toxicity was secondary to mitochondria, the primary CasII-gly target. In long-term 24-h experiments with HeLa cells, 5 µM CasII-gly inhibited OxPhos (80%), glycolysis (40%), and HK (42%). The present data indicated that CasII-gly is an effective multisite anticancer drug simultaneously targeting mitochondria and glycolysis.

Decoding the Therapeutic Implications of the ERα Stability and Subcellular Distribution in Breast Cancer.

Approximately 70% of all breast cancer cases are estrogen receptor-alpha positive (ERα+) and any ERα signaling pathways deregulation is critical for the progression of malignant mammary neoplasia. ERα acts as a transcription factor that promotes the expression of estrogen target genes associated with pro-tumor activity in breast cancer cells. Furthermore, ERα is also part of extranuclear signaling pathways related to endocrine resistance. The regulation of ERα subcellular distribution and protein stability is critical to regulate its functions and, consequently, influence the response to endocrine therapies and progression of this pathology. This minireview highlights studies that have deciphered the molecular mechanisms implicated in controlling ERα stability and nucleo-cytoplasmic transport. These mechanisms offer information about novel biomarkers, therapeutic targets, and promising strategies for breast cancer treatment.

Mast Cell-Tumor Interactions: Molecular Mechanisms of Recruitment, Intratumoral Communication and Potential Therapeutic Targets for Tumor Growth.

Mast cells (MCs) are tissue-resident immune cells that are important players in diseases associated with chronic inflammation such as cancer. Since MCs can infiltrate solid tumors and promote or limit tumor growth, a possible polarization of MCs to pro-tumoral or anti-tumoral phenotypes has been proposed and remains as a challenging research field. Here, we review the recent evidence regarding the complex relationship between MCs and tumor cells. In particular, we consider: (1) the multifaceted role of MCs on tumor growth suggested by histological analysis of tumor biopsies and studies performed in MC-deficient animal models; (2) the signaling pathways triggered by tumor-derived chemotactic mediators and bioactive lipids that promote MC migration and modulate their function inside tumors; (3) the possible phenotypic changes on MCs triggered by prevalent conditions in the tumor microenvironment (TME) such as hypoxia; (4) the signaling pathways that specifically lead to the production of angiogenic factors, mainly VEGF; and (5) the possible role of MCs on tumor fibrosis and metastasis. Finally, we discuss the novel literature on the molecular mechanisms potentially related to phenotypic changes that MCs undergo into the TME and some therapeutic strategies targeting MC activation to limit tumor growth.

Cholesterol: recapitulation of its active role during liver regeneration.

Liver regeneration is a compensatory hyperplasia produced by several stimuli that promotes proliferation in order to provide recovery of the liver mass and architecture. This process involves complex signalling cascades that receive feedback from autocrine and paracrine pathways, recognized by parenchymal as well as non-parenchymal cells. Nowadays the dynamic role of lipids in biological processes is widely recognized; however, a systematic analysis of their importance during liver regeneration is still missing. Therefore, in this review we address the role of lipids including the bioactive ones such as sphingolipids, but with special emphasis on cholesterol. Cholesterol is not only considered as a structural component but also as a relevant lipid involved in the control of the intermediate metabolism of different liver cell types such as hepatocytes, hepatic stellate cells and Kupffer cells. Cholesterol plays a significant role at the level of specific membrane domains, as well as modulating the expression of sterol-dependent proteins. Moreover, several enzymes related to the catabolism of cholesterol and whose activity is down regulated are related to the protection of liver tissue from toxicity during the process of regeneration. This review puts in perspective the necessity to study and understand the basic mechanisms involving lipids during the process of liver regeneration. On the other hand, the knowledge acquired in this area in the past years, can be considered invaluable in order to provide further insights into processes such as general organogenesis and several liver-related pathologies, including steatosis and fibrosis.

Identification of genes modulated by interferon gamma in breast cancer cells.

Interferon gamma (IFNγ) plays a context-dependent dual tumor-suppressor and pro-tumorigenic roles in cancer. IFNγ induces morphological changes in breast cancer (BC) cells with or without estrogen receptor alpha (ERα) expression. However, IFNγ-regulated genes in BC cells remain unexplored. Here, we performed a cDNA microarray analysis of MCF-7 (ERα+) and MDA-MB-231 (HER2-/PR-/ERα-) cells with and without IFNγ treatment. We identified specific IFNγ-modulated genes in each cell type, and a small group of genes regulated by IFNγ common in both cell types. IFNγ treatment for an extended time mainly repressed gene expression shared by both cell types. Nonetheless, some of these IFNγ-repressed genes were seemingly deregulated in human mammary tumor samples, along with decreased IFNGR1 (an IFNγ receptor) expression. Thus, IFNγ signaling-elicited anti-tumor activities may be mediated by the downregulation of main IFNγ target genes in BC; however, it may be deregulated by the tumor microenvironment in a tumor stage-dependent manner.

Quantitative Expression of Key Cancer Markers in the AS-30D Hepatocarcinoma Model.

Hepatocellular carcinoma is one of the cancers with the highest mortality rate worldwide. HCC is often diagnosed when the disease is already in an advanced stage, making the discovery and implementation of biomarkers for the disease a critical aim in cancer research. In this study, we aim to quantify the transcript levels of key signaling molecules relevant to different pathways known to participate in tumorigenesis, with special emphasis on those related to cancer hallmarks and epithelial-mesenchymal transition, using as a model the murine transplantable hepatocarcinoma AS-30D. Using qPCR to quantify the mRNA levels of genes involved in tumorigenesis, we found elevated levels for Tgfb1 and Spp1, two master regulators of EMT. A mesenchymal signature profile for AS-30D cells is also supported by the overexpression of genes encoding for molecules known to be associated to aggressiveness and metastatic phenotypes such as Foxm1, C-met, and Inppl1. This study supports the use of the AS-30D cells as an efficient and cost-effective model to study gene expression changes in HCC, especially those associated with the EMT process.

Fabrication of Adhesive Substrate for Incorporating Hydrogels to Investigate the Influence of Stiffness on Cancer Cell Behavior.

Stiffness control of cell culture platforms provides researchers in cell biology with the ability to study different experimental models in conditions of mimicking physiological or pathological microenvironments. Nevertheless, the signal transduction pathways and drug sensibility of cancer cells have been poorly characterized widely using biomimetic platforms because the limited experience of cancer cell biology groups about handling substrates with specific mechanical properties. The protein cross-linking and stiffening control are crucial checkpoints that could strongly affect cell adhesion and spreading, misrepresenting the data acquired, and also generating inaccurate cellular models. Here, we introduce a simple method to adhere to polyacrylamide (PAA) hydrogels on glass coverslips without any special treatment for mechanics studies in cancer cell biology. By using a commercial photosensitive glue, Loctite 3525, it is possible to polymerize PAA hydrogels directly on glass surfaces. Furthermore, we describe a cross-linking reaction method to attach proteins to PAA as an alternative method to Sulfo-SANPAH cross-linking, which is sometimes difficult to implement and reproduce. In this chapter, we describe a reliable procedure to fabricate ECM protein-cross-linked PAA hydrogels for mechanotransduction studies on cancer cells.