Clinico-Pathological Correlation of β-Catenin and Telomere Dysfunction in Head and Neck Squamous Cell Carcinoma Patients

Abstract

Background: Tumorigenesis is a complex process of accumulated alteration in function of multiple genes and pathways. Wnt signalling pathway is involved in various differentiation events during embryonic development and is conserved in various species.

Objective: A multicentre collaborative initiative is undertaken to study the occurrence, prognosis and molecular mechanism of HNSCC (Head and Neck Squamous Cell Carcinoma) which is highly prevalent in eastern parts of India. From a large cohort of HNSCC tissue repository, 67 cases were selected for multi-parametric investigation.

Results: 67 cases showed stable β-catenin expression. We have seen correlation, if any, of the transcription factor - β-catenin, telomere maintenance and shelterin complex proteins - TRF2, Rap1 and hTert with respect to tumor differentiation and telomere dysfunction. Immunohistochemistry of β-catenin protein showed stable and high expression in tumor when compared to stroma. MDSCC (Moderately Differentiated Squamous cell carcinoma) cases expressed nuclear expression of β-catenin in invasive fronts and showed increased genomic instability. Higher frequency of Anaphase bridges was observed ranging from <3% in normal cut margin to 13% in WDSCC (Well differentiated squamous cell carcinoma) and 18% in MDSCC (Moderately differentiated Squamous cell carcinoma). There was significant decrease in telomere length in MDSCC (<4) when compared to the normal cut margin samples (<7). Quantitative Real Time-PCR confirmed a significant correlationship between stable β-catenin expression and poor clinical and pathological outcome.

Conclusion: The Stabilisation and accumulation of β-catenin was significant and correlated well with de-differentiation process as well as prognosis and therapy outcome of the patients in the cohort. Expression status of molecular markers such as β-catenin, hTert, TRF2 and RAP1 correlate significantly with the process of tumorigenesis and prognosis and may play a role in therapeutic management of Head and neck patients.

Keywords: Dedifferentiation; Genomic instability; HNSCC; MDSCC; WDSCC.; Wnt/β-catenin.

Figure A

Flow chart showing the experimental methodology and study design.

Figure 1

The expression level of proteins β-catenin, hTert, Rap1 and TRF2 probed by respective antibodies in western blotting. (A) Western blot analysis shows expression level of the β-catenin, TRF2, Rap1 and hTert in human samples in compared with GAPDH as loading controls. (B) Graphical representation of increased protein levels of β-catenin, hTert, Rap1 and TRF2 in 33 patient sample cohort which was found to be stable in tumor tissue as well as their cut margin irrespective of their differentiation criteria of categorization analysed by VisionWorksLS^TM. Error bars represent SE.(C) Immunohistochemistry validation of β-catenin showed section specific marked stabilization of the signals. (I) Non-infiltrated regions showed no signal of tumorigenic β-catenin.(II) Regions showing infiltrated tumor cells having membranous β-catenin expression.(III) Regions tumor showing nuclear localization of the β-catenin as can be seen in the inset.

Figure 2

A comparative analysis of β-catenin, TRF2, Rap1 expression in WDSCC and MDSCC by Immunohistochemistry. (A) Image representing the Haematoxylin and Eosin (H& E) stain in well differentiated region. (B) Image representing the Haematoxylin and Eosin (H& E) stain in moderately differentiated regions. (C) Image representing stable membranous expression of β-catenin in WDSCC. (D) Image representing a nuclear translocation (inset) of β-catenin in MDSCC. (E) Image representing stable TRF2 expression in WDSCC. (F) Image representing a cytoplasmic translocation of TRF2 in MDSCC regions. (G) Image showing stable expression of Rap1 in the nucleus in WDSCC. (H) Image showing equally stable expression of Rap1 in the nucleus in MDSCC.

Figure 3

Incidence of anaphase bridges as an indicator of telomere dysfunction was found to be higher in higher grades of HNSCC. (A) Average frequency of anaphase bridge formation in all the grades of tumors. ***P<0.0001. (B) Anaphase bridges in Haematoxylin and eosin (H&E) stained tumor paraffin sections.

Figure 4

PNA-FISH revealed reduced telomere length in higher grades of tumor tissue sample. (A) Telomere length measured on paraffin tissue sections by PNA FISH. Normal sections showed 7.7 as an average fluorescent intensity, WDSCC sections showed 4.8 fluorescent intensity, and MDSCC sections showed 3.3 fluorescent intensity as telomere length on an average when compared with the paired adjacent tissues. Bars indicate SE. (B) Representative pictures of tissue FISH in the paraffin sections of Normal, WDSCC and MDSCC tumors showing telomere signals using the telomere specific Cy-3 labelled PNA probe. *P<0.05. The fluorescent intensity was calculated as arbitrary fluorescent intensity by Zeiss ISIS software.

Figure 5

qPCR analysis of the β-catenin gene expression as a factor of fold change in mRNA for cut margin (CM) and diseased tissues of 12 randomly chosen HNSCC cases are shown. P32 belonged to lower grade leukoplakia against which the expressions of the rest of the cases were compared. Upregulation of β-catenin in the cut margin region was observed in cases P31, P25, P29, P56, P62, P28 and P57 as compared to their respective counterparts which is suggestive of diseased cells infiltration expressing β-catenin into the cut margin area.

Figure 6

Schematic representation of the regulatory proteins involved in the process of tumorigenesis and linking three important events such as tumor development, dedifferentiation and genome instability in HNSCC.