Positive correlation between the nuclear expression of GPER and pGLI3 in prostate cancer tissues from patients with different Gleason scores.

Prostate cancer (PCa) is the most prevalent cause of death in the male population worldwide. The G Protein-Coupled Estrogen Receptor (GPER) has been gaining relevance in the development of PCa. Hedgehog (Hh) pathway activation is associated with aggressiveness, metastasis, and relapse in PCa patients. To date, no studies have evaluated the crosstalk between the GPER and the Hh pathway along different group grades in PCa. We conducted an analysis of paraffin-embedded tissues derived from patients with different prognostic grade of PCa using immunohistochemistry. Expression and correlation between GPER and glioma associated oncogene homologue (GLI) transcriptional factors in the parenchyma and stroma of PCa tumors were evaluated. Our results indicate that GPER is highly expressed in the nucleus and increases with higher grade groups. Additionally, GPER's expression correlates with pGLI3 nuclear expression across different grade groups in PCa tissues; however, whether the receptor induces the activation of GLI transcriptional factors, or the latter modulate the expression of GPER is yet to be discovered, as well as the functional consequence of this correlation.

Arsenic Trioxide Triggers Apoptosis of Metastatic Oral Squamous Cells Carcinoma with Concomitant Downregulation of GLI1 in Hedgehog Signaling.

Given the lack of advances in Oral Squamous Cell Carcinoma (OSCC) therapy in recent years, pharmacological strategies to block OSCC-related signaling pathways have gained prominence. The present study aimed to evaluate the therapeutic potential of Arsenic Trioxide (ATO) concerning its antitumoral effects and the inhibition of the Hedgehog (HH) pathway in OSCC. Initially, ATO cytotoxicity was assessed in a panel of cell lines. Cell viability, cell cycle, death patterns, and cell morphology were analyzed, as well as the effect of ATO on the expression of HH pathway components. After the cytotoxic assay, HSC3 cells were chosen for all in vitro assays. ATO increased apoptotic cell death and nuclear fragmentation in the sub-G1 cell cycle phase and promoted changes in cell morphology. In addition, the reduced expression of GLI1 indicated that ATO inhibits HH activity. The present study provides evidence of ATO as an effective cytotoxic drug for oral cancer treatment in vitro.

LncRNA SOX2-OT regulates AKT/ERK and SOX2/GLI-1 expression, hinders therapy, and worsens clinical prognosis in malignant lung diseases.

The involvement of LncRNA SOX2-overlapping transcript (SOX2-OT), SOX2, and GLI-1 transcription factors in cancer has been well documented. Nonetheless, it is still unknown whether co-expressed SOX2-OT/SOX2 or SOX2-OT/SOX2/GLI-1 axes are epigenetically/transcriptionally involved in terms of resistance to oncology therapy and in poorer clinical outcomes for patients with lung cancer. We evaluated the role of SOX2-OT/SOX2 and SOX2-OT/SOX2/GLI-1 axes using RT-qPCR, western blot, immunofluorescence analyses, gene silencing, cellular cytotoxic, and ChIP-qPCR assays on human cell lines, solid lung malignant tumors, and normal lung tissue. We detected that the SOX2-OT/SOX2/GLI-1 axis promotes resistance to tyrosine kinase inhibitor (TKI)-erlotinib and cisplatin-based therapy. Evidence from this study show that SOX2-OT modulates the expression/activation of EGFR-pathway members AKT/ERK. Further, both SOX2-OT and GLI-1 genes are epigenetically regulated at their promoter sequences, in an LncRNA SOX2-OT-dependent manner, mainly through modifying the enrichment of the activation histone mark H3K4me3/H3K27Ac, versus the repressive histone mark H3K9me3/H3K27me3. In addition, we identified that inhibition of SOX2-OT and reduced expression of SOX2/GLI-1 sensitizes lung cancer cells to EGFR/TKI-erlotinib or cisplatin-based treatment. Finally, we show that high co-expression of SOX2-OT/SOX2 transcripts and SOX2/GLI-1 proteins appears to correlate with a poor clinical prognosis and lung malignant phenotype. Collectively, these results present evidence that LncRNA SOX2-OT modulates an orchestrated resistance mechanism, promoting poor prognosis and human lung malignancy through genetic, epigenetic, and post-translational mechanisms.

Hedgehog pathway activation in oral squamous cell carcinoma: cancer-associated fibroblasts exhibit nuclear GLI-1 localization.

The purpose of this study was to evaluate the expression of Hedgehog (HH) signaling molecules (SHH and GLI-1) by cancer-associated fibroblasts (CAF) in oral squamous cell carcinoma (OSCC). Immunohistochemistry was used to detect molecular HH signaling and CAF-related protein expression, including α-SMA and S100A4, in 70 samples of human OSCC. The colocalization of α-SMA and S100A4 with SHH was also evaluated by double-staining. In vitro study was performed using primary normal oral fibroblast (NOF) and CAF through immunofluorescence and Western Blot for CAF-proteins, SHH, and GLI-1. Forty-five cases (64.28%) were positive for α-SMA exclusively in tumor stroma, and S100A4 was identified in the cytoplasm of CAFs in 94.28% (n = 66) of the cases. With respect to stromal cells, 64 (91.43%) OSCC cases were positive for SHH, and 31 were positive for GLI-1 (44.29%); positive correlations were found between SHH and α-SMA (p < 0.0001, φ = 0.51), as well as between SHH and S100A4 (p = 0.087, φ = 0.94). Protein expression of SHH and GLI-1 was observed in primary CAFs and NOFs. Although SHH was found to be localized in the cellular cytoplasm of both cell types, GLI-1 was present only in the nuclei of CAF. Our results indicate that CAFs are not only potential sources of HH ligands in tumor stroma, but may also respond to HH signaling through nuclear GLI-1 activation. We further observed that elevated SHH expression by OSCC cells was associated with higher CAF density, reinforcing the chemoattractant role played by these molecules.

Lupeol inhibits LPS-induced neuroinflammation in cerebellar cultures and induces neuroprotection associated to the modulation of astrocyte response and expression of neurotrophic and inflammatory factors.

In the central nervous system (CNS), neuroinflammation, especially that modulated by the cell response of astrocytes and microglia, is associated with damage to neurons in neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease and, Multiple Sclerosis. Lupeol is a dietary triterpene that has demonstrated biological activities as antioxidant. This study investigated the anti-inflammatory and neuroprotective effects of lupeol in an in vitro model of neuroinflammation in primary cerebellar cultures. Cultures were obtained from 6-day-old Wistar rats, subjected to inflammatory damage with lipopolysaccharide (LPS, 1 µg/mL) and treated with lupeol (0.1 µM). We observed, after a 48-hour treatment, through Fluorjade-B staining and immunocytochemistry (ICQ) for ßIII-tubulin, that lupeol induced neuroprotection in cultures submitted to inflammatory damage. On the other hand, through ICQ for GFAP, it was possible to observe that lupeol modulated the astrocyte morphology for Bergmann glia-like phenotype and, especially for velate astrocyte-like phenotype, both phenotypes associated with the neuroprotective profile. Moreover, RT-qPCR analysis showed that lupeol induced the down-regulation of the mRNA expression for proinflammatory markers TNF, iNOS and NLRP3, as well as the production of nitric oxide (method of Greiss), which were up-regulated by LPS, and also induced up-regulation of the mRNA expression for arginase and IL-6 mRNA. In addition, lupeol induced up-regulation of mRNA expression for neurotrophins GDNF and NGF and also for the sonic hedgehog-Gli pathway. Together, these results lead to the conclusion that lupeol inhibits neuroinflammation in cerebellar cultures and induces neuroprotection associated with the modulation of astrocyte response and expression of neurotrophic and inflammatory factors.

LepR+ cells dispute hegemony with Gli1+ cells in bone marrow fibrosis.

Bone marrow fibrosis is a reactive process, and a central pathological feature of primary myelofibrosis. Revealing the origin of fibroblastic cells in the bone marrow is crucial, as these cells are considered an ideal, and essential target for anti-fibrotic therapy. In 2 recent studies, Decker et al. (2017) and Schneider et al. (2017), by using state-of-the-art techniques including in vivo lineage-tracing, provide evidence that leptin receptor (LepR)-expressing and Gli1-expressing cells are responsible for fibrotic tissue deposition in the bone marrow. However, what is the relationship between these 2 bone marrow cell populations, and what are their relative contributions to bone marrow fibrosis remain unclear. From a drug development perspective, these works bring new cellular targets for bone marrow fibrosis.

Identification of novel non-coding RNA-based negative feedback regulating the expression of the oncogenic transcription factor GLI1.

Non-coding RNAs are a complex class of nucleic acids, with growing evidence supporting regulatory roles in gene expression. Here we identify a non-coding RNA located head-to-head with the gene encoding the Glioma-associated oncogene 1 (GLI1), a transcriptional effector of multiple cancer-associated signaling pathways. The expression of this three-exon GLI1 antisense (GLI1AS) RNA in cancer cells was concordant with GLI1 levels. siRNAs knockdown of GLI1AS up-regulated GLI1 and increased cellular proliferation and tumor growth in a xenograft model system. Conversely, GLI1AS overexpression decreased the levels of GLI1, its target genes PTCH1 and PTCH2, and cellular proliferation. Additionally, we demonstrate that GLI1 knockdown reduced GLI1AS, while GLI1 overexpression increased GLI1AS, supporting the role of GLI1AS as a target gene of the GLI1 transcription factor. Activation of TGFß and Hedgehog signaling, two known regulators of GLI1 expression, conferred a concordant up-regulation of GLI1 and GLI1AS in cancer cells. Finally, analysis of the mechanism underlying the interplay between GLI1 and GLI1AS indicates that the non-coding RNA elicits a local alteration of chromatin structure by increasing the silencing mark H3K27me3 and decreasing the recruitment of RNA polymerase II to this locus. Taken together, the data demonstrate the existence of a novel non-coding RNA-based negative feedback loop controlling GLI1 levels, thus expanding the repertoire of mechanisms regulating the expression of this oncogenic transcription factor.