Probes for INS

Abstract

AIM:
Long non-coding RNAs (lncRNAs) are potential biomarkers for breast cancer risk stratification. LncRNA expression has been investigated primarily by RNA sequencing, quantitative reverse transcription PCR or microarray techniques. In this study, six breast cancer-implicated lncRNAs were investigated by chromogenic in situ hybridisation (CISH).

METHODS:
Invasive breast carcinoma (IBC), ductal carcinoma in situ (DCIS) and normal adjacent (NA) breast tissues from 52 patients were screened by CISH. Staining was graded by modified Allred scoring.

RESULTS:
HOTAIR, H19 and KCNQ1OT1 had significantly higher expression levels in IBC and DCIS than NA (p<0.05), and HOTAIR and H19 were expressed more strongly in IBC than in DCIS tissues (p<0.05). HOTAIR and KCNQ101T were expressed in tumour cells; H19 and MEG3 were expressed in stromal microenvironment cells; MALAT1 was expressed in all cells strongly and ZFAS1 was negative or weakly expressed in all specimens.

CONCLUSION:
These data corroborate the involvement of three lncRNAs (HOTAIR, H19 and KCNQ1OT1) in breast tumourigenesis and support lncRNA CISH as a potential clinical assay. Importantly, CISH allows identification of the tissue compartment expressing lncRNA.

Renal cell carcinoma (RCC) is one of the most lethal urologic cancers. About one-third of RCC patients already have distal metastasis at the time of diagnosis. There is growing evidence that Hox antisense intergenic RNA (HOTAIR) plays essential roles in metastasis in several types of cancers. However, the precise mechanism by which HOTAIR enhances malignancy remains unclear, especially in RCC. Here, we demonstrated that HOTAIR enhances RCC-cell migration by regulating the insulin growth factor-binding protein 2 (IGFBP2) expression. HOTAIR expression in tumors was significantly correlated with nuclear grade, lymph-node metastasis, and lung metastasis. High HOTAIR expression was associated with a poor prognosis in both our dataset and The Cancer Genome Atlas dataset. Migratory capacity was enhanced in RCC cell lines in a HOTAIR-dependent manner. HOTAIR overexpression accelerated tumorigenicity and lung metastasis in immunodeficient mice. Microarray analysis revealed that IGFBP2 expression was upregulated in HOTAIR-overexpressing cells compared with control cells. The enhanced migration activity of HOTAIR-overexpressing cells was attenuated by IGFBP2 knockdown. IGFBP2 and HOTAIR were co-expressed in clinical RCC samples. Our findings suggest that the HOTAIR-IGFBP2 axis plays critical roles in RCC metastasis and may serve as a novel therapeutic target for advanced RCC.

Cilia are highly conserved organelles, which serve critical roles in development and physiology. Motile cilia are expressed in a limited range of tissues, where they principally regulate local extracellular fluid dynamics. In contrast, primary cilia are expressed by many vertebrate cell types during interphase, and are intimately involved in the cell cycle and signal transduction. Notably, primary cilia are essential for vertebrate hedgehog pathway activity. Improved detection of motile cilia may assist in the diagnosis of some pathologic entities such as Rathke's cleft cysts, whereas characterizing primary cilia in neoplastic tissues may implicate cilia-dependent signaling pathways as critical for tumorigenesis. We show that immunohistochemistry for the nuclear transcription factor FOXJ1, a master regulator of motile ciliogenesis, robustly labels the motile ciliated epithelium of Rathke's cleft cysts. FOXJ1 expression discriminates Rathke's cleft cysts from entities in the sellar/suprasellar region with overlapping histologic features such as craniopharyngiomas. Co-immunohistochemistry for FOXJ1 and markers that highlight motile cilia such as acetylated tubulin (TUBA4A) and the small GTPase ARL13B further enhance the ability to identify diagnostic epithelial cells. In addition to highlighting motile cilia, ARL13B immunohistochemistry also robustly highlights primary cilia in formalin-fixed paraffin-embedded sections. Primary cilia are present throughout the neoplastic epithelium of adamantinomatous craniopharyngioma, but are limited to basally oriented cells near the fibrovascular stroma in papillary craniopharyngioma. Consistent with this differing pattern of primary ciliation, adamantinomatous craniopharyngiomas express significantly higher levels of SHH, and downstream targets such as PTCH1 and GLI2, compared with papillary craniopharyngiomas. In conclusion, motile ciliated epithelium can be readily identified using immunohistochemistry for FOXJ1, TUBA4A, and ARL13B, facilitating the diagnosis of Rathke's cleft cysts. Primary cilia can be identified by ARL13B immunohistochemistry in routine pathology specimens. The widespread presence of primary cilia in adamantinomatous craniopharyngioma implicates cilia-dependent hedgehog signaling in the pathogenesis of adamantinomatous craniopharyngioma.

Triple-negative breast cancers (TNBCs) represent a heterogeneous disease characterized by several molecular subtypes with different prognoses and responses to therapy. For a correct clinical management of TNBC patients the knowledge of the gene regulation mechanisms related to tumor progression and drug response has become fundamental.

LncRNAs regulate gene expression through various processes, including chromatin modification, transcription and post-transcription and they are emerging as important cancer biomarkers being involved in tumor pathogenesis, metastatic progression and drug resistance.

In this study we aimed to analyze the expression of the lncRNA HOTAIR, mainly involved in breast cancer disease, in a large case series of TNBC patients. We used ISH methods by a RNA probe to better define its staining in tumor tissues and its relation with clinical-pathological parameters and outcomes of patients.

Our results show that high HOTAIR expression in tumor tissues is strongly correlated with lymph nodes metastasis (LNM) (p=0.039), as reported also for other tumor types, and has a direct strong association with Androgen Receptor (AR) expression (p= 0.019).

These data confirm the prognostic role of HOTAIR in TNBC, and, its involvement in the regulation of AR pathway, suggests the possibility to establish new therapeutic strategies for AR+TNBC patients.

Obesity increases the risk of hepatocellular carcinoma (HCC), but precise identification and characterization of druggable oncogenic pathways that contribute to the progression of NAFLD to HCC, and hence to the increased incidence and aggressiveness of HCC in obese individuals is lacking. In this regard, we demonstrate that the Indian Hedgehog (Ihh) signaling pathway is upregulated in the fatty livers of mice consuming a high fat diet, and furthermore sustained in HCC tumors specifically within the context of a NAFLD microenvironment. Using a diet-induced mouse model of HCC wherein only obese mice develop HCC, targeted ablation of hepatocyte-secreted Ihh results in a decreased tumor burden and lower grade tumors. Ihh activation regulates the transdifferentiation of ciliated stellate cells and proliferation of Epcam+ ductal cells to promote fibrosis. Mechanistically, increased expression of hitherto uncharacterized effectors of Hh pathway, namely Myc and Tgf-β2 is critical to the observed physiology. This pro-tumorigenic response is driven by increased expression of Wnt5a to effect a poorly-differentiated and invasive tumor phenotype. Wnt5a secreted from activated stellate cells act on Ror2-expressing hepatocytes. We further demonstrate that Wnt5a expression is also elevated in poorly-differentiated HCC cells, suggesting that these ligands are also able to function in an autocrine positive feedback manner to sustain poorly-differentiated tumors. Taken together, our study provides a mechanistic understanding for how Ihh signaling promotes HCC tumorigenesis specifically in obese mice. We propose that therapeutic targeting of the Hh pathway offers benefit for patients with dietary / NAFLD-driven steatotic HCC.

Aberrant Notch signalling has been linked to many cancers including choroid plexus (CP) tumours, a group of rare and predominantly paediatric brain neoplasms. We developed animal models of CP tumours, by inducing sustained expression of Notch1, that recapitulate properties of human CP tumours with aberrant NOTCH signalling. Whole-transcriptome and functional analyses showed that tumour cell proliferation is associated with Sonic Hedgehog (Shh) in the tumour microenvironment. Unlike CP epithelial cells, which have multiple primary cilia, tumour cells possess a solitary primary cilium as a result of Notch-mediated suppression of multiciliate differentiation. A Shh-driven signalling cascade in the primary cilium occurs in tumour cells but not in epithelial cells. Lineage studies show that CP tumours arise from monociliated progenitors in the roof plate characterized by elevated Notch signalling. Abnormal SHH signalling and distinct ciliogenesis are detected in human CP tumours, suggesting the SHH pathway and cilia differentiation as potential therapeutic avenues.

Long non-coding RNAs (lncRNAs) may contribute to carcinogenesis and tumor progression by regulating transcription and gene expression. The role of lncRNAs in the regulation of thyroid cancer progression is being extensively examined. Here, we analyzed three lncRNAs that were overexpressed in papillary thyroid carcinomas, long intergenic non-protein coding RNA, regulator of reprogramming (Linc-ROR, ROR) PVT1 oncogene (PVT1), and HOX transcript antisense intergenic RNA (HOTAIR) to determine their roles in thyroid tumor development and progression. ROR expression has not been previously examined in thyroid carcinomas. Tissue microarrays (TMAs) of formalin-fixed paraffin-embedded tissue sections from 129 thyroid cases of benign and malignant tissues were analyzed by in situ hybridization (ISH), automated image analysis, and real-time PCR. All three lncRNAs were most highly expressed in the nuclei of PTCs. SiRNA experiments with a PTC cell line, TPC1, showed inhibition of proliferation with siRNAs for all three lncRNAs while invasion was inhibited with siRNAs for ROR and HOTAIR. SiRNA experiments with ROR also led to increased expression of miR-145, supporting the role of ROR as an endogenous miR-145 sponge. After treatment with TGF-β, there was increased expression of ROR, PVT1, and HOTAIR in the PTC1 cell line compared to control groups, indicating an induction of their expression during epithelial to mesenchymal transition (EMT). These results indicate that ROR, PVT1, and HOTAIR have important regulatory roles during the development of PTCs.

Although long non-coding RNAs (lncRNAs) predominately reside in the nucleus and exert their functions in many biological processes, their potential involvement in cytoplasmic signal transduction remains unexplored. Here, we identify a cytoplasmic lncRNA, LINK-A (long intergenic non-coding RNA for kinase activation), which mediates HB-EGF-triggered, EGFR:GPNMB heterodimer-dependent HIF1α phosphorylation at Tyr 565 and Ser 797 by BRK and LRRK2, respectively. These events cause HIF1α stabilization, HIF1α-p300 interaction, and activation of HIF1α transcriptional programs under normoxic conditions. Mechanistically, LINK-A facilitates the recruitment of BRK to the EGFR:GPNMB complex and BRK kinase activation. The BRK-dependent HIF1α Tyr 565 phosphorylation interferes with Pro 564 hydroxylation, leading to normoxic HIF1α stabilization. Both LINK-A expression and LINK-A-dependent signalling pathway activation correlate with triple-negative breast cancer (TNBC), promoting breast cancer glycolysis reprogramming and tumorigenesis. Our findings illustrate the magnitude and diversity of cytoplasmic lncRNAs in signal transduction and highlight the important roles of lncRNAs in cancer.