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. 2009;4(4):e5367.
doi: 10.1371/journal.pone.0005367. Epub 2009 Apr 28.

CTIP2 expression in human head and neck squamous cell carcinoma is linked to poorly differentiated tumor status

Gitali Ganguli-Indra  1 , Christine Wasylyk, Xiaobo Liang, Regine Millon, Mark Leid, Bohdan Wasylyk, Joseph Abecassis, Arup K Indra
Affiliations

CTIP2 expression in human head and neck squamous cell carcinoma is linked to poorly differentiated tumor status

Gitali Ganguli-Indra et al. PLoS One. 2009.

Erratum in

  • PLoS One. 2009;4(4). doi: 10.1371/annotation/b01c646f-14c7-4aac-8812-9ed4f847c857. Indra, Arup [corrected to Indra, Arup K]

Abstract

Background: We have demonstrated earlier that CTIP2 is highly expressed in mouse skin during embryogenesis and in adulthood. CTIP2 mutant mice die at birth with epidermal differentiation defects and a compromised epidermal permeability barrier suggesting its role in skin development and/or homeostasis. CTIP2 has also been suggested to function as tumor suppressor in cells, and several reports have described a link between chromosomal rearrangements of CTIP2 and human T cell acute lymphoblast leukemia (T-ALL). The aim of the present study was to look into the pattern of CTIP2 expression in Head and Neck Squamous Cell Carcinoma (HNSCC).

Methodology/principal findings: In the present study, we analyzed CTIP2 expression in human HNSCC cell lines by western blotting, in paraffin embedded archival specimens by immunohistochemistry (IHC), and in cDNA samples of human HNSCC by qRT-PCR. Elevated levels of CTIP2 protein was detected in several HNSCC cell lines. CTIP2 staining was mainly detected in the basal layer of the head and neck normal epithelium. CTIP2 expression was found to be significantly elevated in HNSCC (p<0.01), and increase in CTIP2 expression was associated with poorly differentiated tumor status. Nuclear co-localization of CTIP2 protein and cancer stem cell (CSC) marker BMI1 was observed in most, if not all of the cells expressing BMI1 in moderately and poorly differentiated tumors.

Conclusions/significance: We report for the first time expression of transcriptional regulator CTIP2 in normal human head and neck epithelia. A statistically significant increase in the expression of CTIP2 was detected in the poorly differentiated samples of the human head and neck tumors. Actual CTIP2, rather than the long form of CTIP2 (CTIP2(L)) was found to be more relevant to the differentiation state of the tumors. Results demonstrated existence of distinct subsets of cancer cells, which express CTIP2 and underscores the use of CTIP2 and BMI1 co-labeling to distinguish tumor initiating cells or cancer stem cells (CSCs) from surrounding cancer cells.

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Conflict of interest statement

Competing Interests: A provisional patent application has been filed related to CTIP2 expression pattern in human HNSCC.

Figures

Figure 1
Figure 1. Immunohistochemical analysis of CTIP2 in HNSCC.
(A) Expression in adjacent Normal epithelium is restricted to basal cell layers. (B) Expression in dysplasia is stronger than normal and extended to differentiated cell layers. (C–D) In well differentiated tumors, positive staining is restricted to the basal layers at the periphery of the keratinized horn pearl. (E–F) Homogeneous expression of CTIP2 was observed in tumor cells in moderately differentiated tumors. (G–H) Expression of stronger intensities (score 3) of CTIP2 was observed in poorly differentiated tumors. Arrows indicates the CTIP2 positive staining. Yellow dotted lines represents the margin of epidermis and dermis (A), and margin of undifferentiated tumor cell clusters (E) which is magnified in (F). The pictures in the insets (A, B, G and H) are magnified. (Original Magnification: 20×).
Figure 2
Figure 2. Staining intensities of CTIP2 by immuno-histochemistry in HNSCC.
Staining intensities were 0–3 as described in materials and method sections. (A) Staining intensities of CTIP2 in normal epithelium (NE), dysplasia and HNSCC. Plotted are mean measurements (±S.E.M). *p<0.05. (B) Staining intensities in different histological grades of HNSCC. Plotted are mean measurements (±S.E.M). *p<0.05. (C–D) Co-staining of CTIP2 and BMI1 protein. The insets are magnified. Yellow arrows show the CTIP2 and Bmi1 double positive cells. Yellow arrowhead indicates single positive cells. (E) CTIP2 expression in HNSCC cell lines by western blot analysis. CTIP2 antibody recognizes the two isoforms (Long and the actual CTIP2). Green box represents the normal cell lines.
Figure 3
Figure 3. Co-staining of CTIP2, cytokeratin 10 and Ki-67 using immunofluorescence.
(A) Co-staining of CTIP2 and differentiation marker cytokeratin 10 in dysplasia and in moderately differentiated tumor. CTIP2 in green, Cytokeratin 10 in red and DAPI in blue. Yellow dotted lines represents the margin of epidermis and dermis. (B) Co-staining of CTIP2 and a proliferation marker Ki-67 in poorly differentiated tumor. Insets are in yellow boxes and are magnified. Yellow arrows shows the CTIP2-Ki-67 double positive cells. (Original Magnification: 20×).
Figure 4
Figure 4. Scheme of CTIP2 transcripts and the expression pattern of CTIP2L and CTIP2 in HNSCC.
(A) Two transcripts of human CTIP2 are shown. CTIP2 with all the 4 exons, is the long form of CTIP2 (CTIP2 L), and, lacking exon 3 is the actual CTIP2. Primers for the long form were taken from Exon 2 for Forward and exon 3 for reverse, and for the shorter form, the forward from boundary of Exon 2 & 4 and the reverse from exon 4. Expression of the CTIP2 long (B) and CTIP2 (C), by qRT-PCR in HNSCC (n = 28) and 10 normal uvulas based on T size, Tumor staging, and differentiation status. Plotted are the Relative quantification levels normalized with RPLPO as S.E.M±. p values were not significant between the groups in (B) but a significant correlation of higher CTIP2 expression was observed with a poorer histological grade of the tumor, and a trend was noted in a relationship between expression and advanced T or clinical stage in (C) [P<0.05].
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