Using a Dual CRISPR/Cas9 Approach to Gain Insight into the Role of LRP1B in Glioblastoma.

LRP1B remains one of the most altered genes in cancer, although its relevance in cancer biology is still unclear. Recent advances in gene editing techniques, particularly CRISPR/Cas9 systems, offer new opportunities to evaluate the function of large genes, such as LRP1B. Using a dual sgRNA CRISPR/Cas9 gene editing approach, this study aimed to assess the impact of disrupting LRP1B in glioblastoma cell biology. Four sgRNAs were designed for the dual targeting of two LRP1B exons (1 and 85). The U87 glioblastoma (GB) cell line was transfected with CRISPR/Cas9 PX459 vectors. To assess LRP1B-gene-induced alterations and expression, PCR, Sanger DNA sequencing, and qRT-PCR were carried out. Three clones (clones B9, E6, and H7) were further evaluated. All clones presented altered cellular morphology, increased cellular and nuclear size, and changes in ploidy. Two clones (E6 and H7) showed a significant decrease in cell growth, both in vitro and in the in vivo CAM assay. Proteomic analysis of the clones' secretome identified differentially expressed proteins that had not been previously associated with LRP1B alterations. This study demonstrates that the dual sgRNA CRISPR/Cas9 strategy can effectively edit LRP1B in GB cells, providing new insights into the impact of LRP1B deletions in GBM biology.

The Chick CAM as an In Vivo System to Study Stem Cell Activity.

The chick embryo chorioallantoic membrane (CAM), an extensively vascularized extraembryonic membrane, has been widely used to study several aspects of tumor development including tumor-induced angiogenesis, tumor cell proliferation, and metastasis. Based on the tumor cell/CAM system, we focused here on the identification and quantification of cancer stem cells. We validated the CAM model as a suitable model to evaluate stem cell activity in a given mixed cell population.

Neuroprotection in early stages of Alzheimer's disease is promoted by transthyretin angiogenic properties.

BACKGROUND: While still controversial, it has been demonstrated that vascular defects can precede the onset of other AD hallmarks features, making it an important therapeutic target. Given that the protein transthyretin (TTR) has been established as neuroprotective in AD, here we investigated the influence of TTR in the vasculature. METHODS: We evaluated the thickness of the basement membrane and the length of brain microvessels, by immunohistochemistry, in AßPPswe/PS1A246E (AD) transgenic mice and non-transgenic mice (NT) bearing one (TTR+/-) or two (TTR+/+) copies of the TTR gene. The angiogenic potential of TTR was evaluated in vitro using the tube formation assay, and in vivo using the chick chorioallantoic membrane (CAM) assay. RESULTS: AD transgenic mice with TTR genetic reduction, AD/TTR+/-, exhibited a thicker BM in brain microvessels and decreased vessel length than animals with normal TTR levels, AD/TTR+/+. Further in vivo investigation, using the CAM assay, revealed that TTR is a pro-angiogenic molecule, and the neovessels formed are functional. Also, TTR increased the expression of key angiogenic molecules such as proteins interleukins 6 and 8, angiopoietin 2, and vascular endothelial growth factor, by endothelial cells, in vitro, under tube formation conditions. We showed that while TTR reduction also leads to a thicker BM in NT mice, this effect is more pronounced in AD mice than in NT animals, strengthening the idea that TTR is a neuroprotective protein. We also studied the effect of TTR tetrameric stabilization on BM thickness, showing that AD mice treated with the TTR tetrameric stabilizer iododiflunisal (IDIF) displayed a significant reduction of BM thickness and increased vessel length, when compared to non-treated littermates. CONCLUSION: Our in vivo results demonstrate the involvement of TTR in angiogenesis, particularly as a modulator of vascular alterations occurring in AD. Since TTR is decreased early in AD, its tetrameric stabilization can represent a therapeutic avenue for the early treatment of AD through the maintenance of the vascular structure.

Increased LAMP2A levels correlate with a shorter disease-free survival of HER2 negative breast cancer patients and increased breast cancer cell viability.

Chaperone Mediated Autophagy (CMA) is a selective autophagy pathway deregulated in many cancers. In this study, we were aiming at understanding the importance of CMA in breast cancer. To this end, we examined the expression of the CMA markers HSP8 and LAMP2A in different breast cancer cell lines and found a wide range of LAMP2A expression levels across the cell lines analyzed. Next, we applied a specific immunohistochemical staining protocol to a tissue microarray derived from a cohort of 365 breast cancer patients. Therefore, we were able to find a correlation of high LAMP2A but not HSPA8 (HSC70) with worse disease free survival in patients with HER2 negative tumors (p = 0.026) which was independent prognostic parameter from pT category, pN category and grading in a multivariate model (HR = 1.889; 95% CI = 1.039-3.421; p = 0.037). In line, low LAMP2A levels decrease proliferation of the breast cancer cell lines T47D and MCF-7 in vitro. Our data suggest that LAMP2A supports a more severe breast cancer cell phenotype.

The Chick Chorioallantoic Membrane Model: A New In Vivo Tool to Evaluate Breast Cancer Stem Cell Activity.

The high plasticity of cancer stem-like cells (CSCs) allows them to differentiate and proliferate, specifically when xenotransplanted subcutaneously into immunocompromised mice. CSCs are highly tumorigenic, even when inoculated in small numbers. Thus, in vivo limiting dilution assays (LDA) in mice are the current gold standard method to evaluate CSC enrichment and activity. The chick embryo chorioallantoic membrane (CAM) is a low cost, naturally immune-incompetent and reproducible model widely used to evaluate the spontaneous growth of human tumor cells. Here, we established a CAM-LDA assay able to rapidly reproduce tumor specificities-in particular, the ability of the small population of CSCs to form tumors. We used a panel of organotropic metastatic breast cancer cells, which show an enrichment in a stem cell gene signature, enhanced CD44+/CD24-/low cell surface expression and increased mammosphere-forming efficiency (MFE). The size of CAM-xenografted tumors correlate with the number of inoculated cancer cells, following mice xenograft growth pattern. CAM and mice tumors are histologically comparable, displaying both breast CSC markers CD44 and CD49f. Therefore, we propose a new tool for studying CSC prevalence and function-the chick CAM-LDA-a model with easy handling, accessibility, rapid growth and the absence of ethical and regulatory constraints.

Conjugation of the T1 sequence from CCN1 to fibrin hydrogels for therapeutic vascularization.

Hydrogel matrices with angiogenic properties are much desirable for therapeutic vascularization strategies, namely to provide vascular supply to ischemic areas, transplanted cells, or bioengineered tissues. Here we report the pro-angiogenic effect of fibrin (Fb) functionalization with the T1 sequence from the angiogenic inducer CCN1, forseeing its use in the injured brain and spinal cord. Fibrin functionalization with 40 µM of T1 peptide effectively improved cellular sprouting of human brain microvascular endothelial cells (hCMEC/D3) in the absence of vascular endothelial growth factor (VEGF), without impacting the viscoelastic properties of Fb, cell viability, or proliferation. The pro-angiogenic effect of immobilized T1 was potentiated in the presence of VEGF and partially mediated through α6ß1 integrin. The tethering of T1 also enhanced sprouting of human cord blood-derived outgrowth endothelial cells (OEC). Still, to elicit such effect, a higher input T1 concentration was required (60 µM), in line with the lower protein levels of α6 and ß1 integrin subunits found in OEC comparing to hCMEC/D3, prior to embedment in Fb gel. Finally, the ability of T1-functionalized Fb in inducing cappilary invasion in vivo was assessed using the CAM assay, which evidenced a significant increase in the number of newly formed vessels at sites of implantation of T1-functionalized Fb, in the absence of soluble angiogenic factors. Overall these results demonstrate the potential of T1 peptide-presenting gels for use in therapeutic vascularization approaches. Considering T1 neurite-extension promoting capability and pro-angiogenic properties, T1-functionalized Fb hydrogels are particularly promising for application in the injured central nervous system.

Low Doses of Ionizing Radiation Enhance the Angiogenic Potential of Adipocyte Conditioned Medium.

At low doses, ionizing radiation activates endothelial cells and promotes angiogenesis. However, it is still unknown if other cells may contribute to this process. In this study, the effect of low-dose ionizing radiation (LDIR) in modulating the pro-angiogenic potential of adipocytes was investigated. Adipocytes are known to secrete multiple angiogenic factors and adipokines that induce angiogenesis. In this work, a confluent monolayer of 3T3-L1 pre-adipocytes was exposed to low doses (0.1 and 0.3 Gy) and to higher doses (0.5, 0.8 and 1.0 Gy), as control. Our data show that the adipocyte-conditioned media (A-CM) from mature adipocytes differentiated from low-dose irradiated pre-adipocytes presented a higher angiogenic potential, compared to mature adipocytes differentiated from sham-irradiated control preadipocytes. The vascular endothelial growth factor (VEGF)-A levels were significantly increased in A-CM from the 0.1 Gy (P < 0.05) and 0.3 Gy (P < 0.01) experimental conditions and a significant increase was found in response to 0.3 Gy dose of radiation for VEGF-C, angiopoietin-2 (ANG-2) and hepatocyte growth factor (HGF). Moreover, 0.3 Gy dose of radiation significantly increased the expression of matrix metalloproteinase (MMP)-2 active forms. In vitro, the A-CM from the 0.1 and 0.3 Gy doses experimental conditions significantly accelerated endothelial cell migration after an in vitro wound healing assay. Importantly, in vivo, the A-CM corresponding to the 0.3 Gy experimental condition significantly induced the growth of more blood vessels towards the inoculation area in the chick embryo chorioallantoic membrane (CAM). In conclusion, this work reveals a new mechanism by which low-dose radiation might promote angiogenesis, enhancing the angiogenic potential of A-CM.

The Chick Chorioallantoic Membrane (CAM) Assay as a Three-dimensional Model to Study Autophagy in Cancer Cells.

The chick chorioallantoic membrane (CAM) is an extra-embryonic organ and thus well accessible for seeding and harvesting 3D cell cultures. Samples from CAM assays are suitable for protein and gene expression analysis as well as for immuno-histochemical studies. Here we present the CAM assay as a possible model to study autophagy in different types of cancer using immunohistochemistry. Compared with other 3D and xenograft models, the CAM assay displays several advantages such as lower costs, shorter experimental times, physiological environment and reproducibility. Macroautophagy hereafter simply referred to as "autophagy" is a conserved cellular catabolic process that degrades and recycles cellular components. Under basal conditions, autophagy contributes to the maintenance of cellular homeostasis whereas under cellular stress, such as starvation or hypoxia, autophagy is activated as a survival mechanism. Dysregulation of autophagy has been described in many diseases. In cancer, autophagy has been suggested to play a dual role. Whereas autophagy has been reported to play a tumor suppressive role in early stages, it seems to be rather tumor supportive in later stages. Here we provide a method to study autophagy in 3D microtumors of cancer cells grown on the CAM.

Codon misreading tRNAs promote tumor growth in mice.

Deregulation of tRNAs, aminoacyl-tRNA synthetases and tRNA modifying enzymes are common in cancer, raising the hypothesis that protein synthesis efficiency and accuracy (mistranslation) are compromised in tumors. We show here that human colon tumors and xenograft tumors produced in mice by two epithelial cancer cell lines mistranslate 2- to 4-fold more frequently than normal tissue. To clarify if protein mistranslation plays a role in tumor biology, we expressed mutant Ser-tRNAs that misincorporate Ser-at-Ala (frequent error) and Ser-at-Leu (infrequent error) in NIH3T3 cells and investigated how they responded to the proteome instability generated by the amino acid misincorporations. There was high tolerance to both misreading tRNAs, but the Ser-to-Ala misreading tRNA was a more potent inducer of cell transformation, stimulated angiogenesis and produced faster growing tumors in mice than the Ser-to-Leu misincorporating tRNA. Upregulation of the Akt pathway and the UPR were also observed. Most surprisingly, the relative expression of both misreading tRNAs increased during tumor growth, suggesting that protein mistranslation is advantageous in cancer contexts. These data highlight new features of protein synthesis deregulation in tumor biology.

Inhibitory Effects of Antagonists of Growth Hormone-Releasing Hormone (GHRH) in Thyroid Cancer.

Growth hormone-releasing hormone (GHRH) is a peptide hormone secreted by the hypothalamus that regulates the synthesis and secretion of growth hormone (GH) in the pituitary. The extra-hypothalamic GHRH and its cognate receptors (GHRHR and splice variants) play a mitogenic role by stimulating cell proliferation and preventing apoptotic cell death. It is well established that GHRH antagonists inhibit the growth, tumorigenicity, and metastasis of various human malignancies. In this work, we studied the effect of two new GHRH antagonists, MIA602 and MIA690, on thyroid cancer. We studied the effect of MIA602 and MIA690 on thyroid cancer in vitro, using human thyroid cancer cell lines, and in vivo, using chicken embryo chorioallantoic membrane (CAM) assays. We found that mRNA for GHRH and GHRH receptor is expressed in thyroid cell lines and in samples of thyroid tumors. Immunohistochemistry confirmed the expression of GHRHR protein in specimens of thyroid tumor. We observed that GHRH antagonists inhibited the growth and increased apoptosis of thyroid cancer cells. In vivo, the antagonists inhibited growth and angiogenesis of engrafted thyroid tumors. Our results suggest that GHRH expression may play a role in growth of thyroid cancer and that GHRH antagonists can be a therapeutic option for thyroid cancer patients.

Osteopontin-a splice variant is overexpressed in papillary thyroid carcinoma and modulates invasive behavior.

Osteopontin (OPN) is a matricellular protein overexpressed in cancer cells and modulates tumorigenesis and metastasis, including in thyroid cancer (TC). The contribution of each OPN splice variant (OPN-SV), named OPNa, OPNb and OPNc, in TC is currently unknown. This study evaluates the expression of total OPN (tOPN) and OPN-SV in TC tissues and cell lines, their correlation with clinicopathological, molecular features and their functional roles. We showed that tOPN and OPNa are overexpressed in classic papillary thyroid carcinoma (cPTC) in relation to adjacent thyroid, adenoma and follicular variant of papillary thyroid carcinoma (fvPTC) tissues. In cPTC, OPNa overexpression is associated with larger tumor size, vascular invasion, extrathyroid extension and BRAFV600E mutation. We found that TC cell lines overexpressing OPNa exhibited increased proliferation, migration, motility and in vivo invasion. Conditioned medium secreted from cells overexpressing OPNa induce MMP2 and MMP9 metalloproteinases activity. In summary, we described the expression pattern of OPN-SV in cPTC samples and the key role of OPNa expression on activating TC tumor progression features. Our findings highlight OPNa variant as TC biomarker, besides being a putative target for cPTC therapeutic approaches.

miR-195 in human primary mesenchymal stromal/stem cells regulates proliferation, osteogenesis and paracrine effect on angiogenesis.

Mesenchymal Stromal/Stem Cells (MSC) are currently being explored in diverse clinical applications, including regenerative therapies. Their contribution to regeneration of bone fractures is dependent on their capacity to proliferate, undergo osteogenesis and induce angiogenesis. This study aimed to uncover microRNAs capable of concomitantly regulate these mechanisms. Following microRNA array results, we identified miR-195 and miR-497 as downregulated in human primary MSC under osteogenic differentiation. Overexpression of miR-195 or miR-497 in human primary MSC leads to a decrease in osteogenic differentiation and proliferation rate. Conversely, inhibition of miR-195 increased alkaline phosphatase expression and activity and cells proliferation. Then, miR-195 was used to study MSC capacity to recruit blood vessels in vivo. We provide evidence that the paracrine effect of MSC on angiogenesis is diminishedwhen cells over-express miR-195. VEGF may partially mediate this effect, as its expression and secreted protein levels are reduced by miR-195, while increased by anti-miR-195, in human MSC. Luciferase reporter assays revealed a direct interaction between miR-195 and VEGF 3´-UTR in bone cancer cells. In conclusion, our results suggest that miR-195 regulates important mechanisms for bone regeneration, specifically MSC osteogenic differentiation, proliferation and control of angiogenesis; therefore, it is a potential target for clinical bone regenerative therapies.

Interleukin-1B signalling leads to increased survival of gastric carcinoma cells through a CREB-C/EBPß-associated mechanism.

BACKGROUND: Polymorphisms in inflammation-related genes have been associated with a risk of gastric carcinoma (GC). However, the biological mechanisms underlying these associations are still elusive. Our objective was to determine whether chronic inflammation-associated IL1Β signalling, as seen in the context of Helicobacter pylori infection, could be linked to gastric carcinogenesis by modulating the behaviour of gastric epithelial cells. METHODS: The effect of IL1B was assessed by studying the expression and activation status of the IL1Β-activated transcription factors C/EBPß and CREB in GC cell lines. Interaction between CREB and C/EBPß was explored through interference RNA, chromatin immunoprecipitation and chemical inhibition. CREB and C/EBPß expression was analysed in 66 samples of primary GC and in normal gastric mucosa. GC cell growth was analysed in vitro by BrdU incorporation and in vivo employing a chicken embryo chorioallantoic membrane model. RESULTS: We found that IL1B regulates the expression/activation status of both C/EBPß and CREB in GC cells through an ERK1/2-dependent mechanism. Our results show that CREB is a direct transactivator of CEBPB, acting as an upstream effector in this regulatory mechanism. Furthermore, we found CREB to be overexpressed in 94 % of GC samples and significantly associated with C/EBPß expression (P < 0.05). Finally, we demonstrated both in vitro and in vivo that CREB can mediate IL1B-induced GC cell proliferation. CONCLUSIONS: Our results support the hypothesis that the effect of chronic inflammation on gastric carcinogenesis, as seen in the context of genetically susceptible individuals infected with Helicobacter pylori, includes the modulation of signalling pathways that regulate survival mechanisms in epithelial cells. IL1B is able to increase the expression/activation status of CREB and its target gene C/EBPß, which are mandatory for GC cell survival. Our results may help inform new strategies for the prevention and treatment of GC, including the control of chronic inflammation.

Expression of ST3GAL4 leads to SLe(x) expression and induces c-Met activation and an invasive phenotype in gastric carcinoma cells.

Sialyl-Lewis X (SLe(x)) is a sialylated glycan antigen expressed on the cell surface during malignant cell transformation and is associated with cancer progression and poor prognosis. The increased expression of sialylated glycans is associated with alterations in the expression of sialyltransferases (STs). In this study we determined the capacity of ST3GAL3 and ST3GAL4 sialyltransferases to synthesize the SLe(x) antigen in MKN45 gastric carcinoma cells and evaluated the effect of SLe(x) overexpression in cancer cell behavior both in vitro and in vivo using the chicken chorioallantoic membrane (CAM) model. The activation of tyrosine kinase receptors and their downstream molecular targets was also addressed. Our results showed that the expression of ST3GAL4 in MKN45 gastric cancer cells leads to the synthesis of SLe(x) antigens and to an increased invasive phenotype both in vitro and in the in vivo CAM model. Analysis of phosphorylation of tyrosine kinase receptors showed a specific increase in c-Met activation. The characterization of downstream molecular targets of c-Met activation, involved in the invasive phenotype, revealed increased phosphorylation of FAK and Src proteins and activation of Cdc42, Rac1 and RhoA GTPases. Inhibition of c-Met and Src activation abolished the observed increased cell invasive phenotype. In conclusion, the expression of ST3GAL4 leads to SLe(x) antigen expression in gastric cancer cells which in turn induces an increased invasive phenotype through the activation of c-Met, in association with Src, FAK and Cdc42, Rac1 and RhoA GTPases activation.

Transcription initiation arising from E-cadherin/CDH1 intron2: a novel protein isoform that increases gastric cancer cell invasion and angiogenesis.

Disruption of E-cadherin (CDH1 gene) expression, subcellular localization or function arises during initiation and progression of almost 90% of all epithelial carcinomas. Nevertheless, the mechanisms through which this occurs are largely unknown. Previous studies showed the importance of CDH1 intron 2 sequences for proper gene and protein expression, supporting these as E-cadherin cis-modulators. Through RACE and RT-PCR, we searched for transcription events arising from CDH1 intron 2 and discovered several new transcripts. One, named CDH1a, with high expression in spleen and absent from normal stomach, was demonstrated to be translated into a novel isoform, differing from canonical E-cadherin in its N-terminal, as determined by mass spectrometry. Quantitative and functional assays showed that when overexpressed in an E-cadherin negative context, CDH1a replaced canonical protein interactions and functions. However, when co-expressed with canonical E-cadherin, CDH1a increased cell invasion and angiogenesis. Further, interferon-induced gene IFITM1 and IFI27 levels were increased upon CDH1a overexpression. Effects on invasion and IFITM1 and IFI27 expression were reverted upon CDH1a-specific knockdown. Importantly, CDH1a was de novo expressed in gastric cancer cell lines. This study presents a new mechanism by which E-cadherin functions are impaired by cis-regulatory mechanisms possibly with the involvement of inflammatory machinery. If confirmed in other cancer models, our data enclose potential for designing targeted therapies to rescue E-cadherin function.