Pathobiological role of cleft palate transmembrane protein 1 family proteins in oral squamous cell carcinoma

  • Keisuke Inoue
  • Kiichi Hatano
  • Yuki Hanamatsu
  • Chiemi Saigo
  • Yusuke Kito
  • Katsuaki Bunai
  • Toshiyuki Shibata
  • Tamotsu TakeuchiEmail author
Original Article – Cancer Research



Cleft palate transmembrane protein 1 (Clptm1) and its paralog protein, Cisplatin resistance-related protein 9 (CRR9) constitute a highly conserved protein family, from Caenorhabditis elegans to Homo sapiens. In the present study, we examined the clinicopathological and biological significance of Clptm1 and CRR9 expression in oral squamous cell carcinoma (OSCC).


Ninety-eight OSCC tissue specimens were immunohistochemically stained with specific antibodies to Clptm1 and CRR9. The immunoreactivity of Clptm1 and CRR9 was then correlated with clinicopathological factors, including the prognosis of patients. siRNA-mediated gene silencing of CRR9 followed by cell proliferation, Matrigel invasion, anoikis assay, and gelatin zymography were performed using cultured OSCC cells. Subsequently, immunohistochemical examination including double staining was performed to determine the correlation between CRR9 and Bcl-xL expression in OSCC cells.


Non-tumorous oral squamous cells exhibited vague, weak, or little cytoplasmic staining with anti-Clptm1 and CRR9 antibodies. By contrast, robust Clptm1 and CRR9 immunoreactivity was found at the cancer invasion front in 55 and 54 of the 98 OSCC tissue specimens, respectively. Notably, CRR9 immunoreactivity was associated with more than 5 mm of depth of invasion, poor prognosis of the patients, and smoking habits (P < 0.05). siRNA-mediated gene silencing of CRR9 did not alter the cell proliferation but decreased Matrigel invasion and impaired anoikis resistance in cultured Ca9-22 and SAS cells. CRR9 and anti-apoptotic Bcl-xL expression levels were correlated in pT1 OSCC tissue specimens.


Clptm1 and CRR9 were overexpressed in many OSCC tissues. In particular, CRR9 expression may promote tumor development and have a significant poor prognostic value in OSCC, possibly through conferring invasion ability and resistance to apoptotic stimuli possibly related to Bcl-xL expression. CRR9 could be a novel molecular target for patients with OSCC.


Oral squamous cell carcinoma Clptm1 CRR9 Outcome Anoikis 



This study was supported by grants from the Ministry of Education of Japan (Grant nos. KAKEN 15K08361, 15K19051, and 17K15642).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The present study was conducted in accordance with the ethical standards of the Helsinki Declaration in 1975, after approval of the Institutional Review Board of the Gifu University Graduate School of Medicine (specific approval number: 28–524).

Informed consent

The need for written informed consent was waived by the Institutional Review Board of the Gifu University Graduate School of Medicine. Instead, the Institutional Review Board requested us to inform the patients that they could refuse the use of their tissue specimens for this present study, if they did not want to participate.

Supplementary material

432_2019_2843_MOESM1_ESM.tif (518 kb)
Supplementary Fig. 1: Two independent anoikis assays followed by Annexin V/PI flow cytometry also demonstrated that down-regulation of CRR9 increased anchor-dependent apoptosis in Ca9-22 and SAS OSCC cells. Data using s37500 siRNA are shown. (TIF 517 KB)
432_2019_2843_MOESM2_ESM.tif (77 kb)
Supplementary Fig. 2: Two independent anoikis assays followed by Annexin V/PI flow cytometry also demonstrated that down-regulation of CRR9 increased anchor-dependent apoptosis in Ca9-22 and SAS OSCC cells. Data using s37500 siRNA are shown. (TIF 76 KB)
432_2019_2843_MOESM3_ESM.docx (19 kb)
Supplementary Table 1: Percentage identity of deduced amino acid sequence of human Clptm1, human CRR9, C. elegans CELE_C36B7.6, and CELE_T13H5.8 determined by the SIM program, with comparison matrix: BLOSUM30, Number of alignments computed: 30, Gap open penalty: 25, and Gap extension penalty: 0. Clptm1 has a close homology to CELE_C36B7.6, while CRR9 has homology to CELE_T13H5.8. (DOCX 18 KB)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Oral and Maxillofacial SurgeryGifu University Graduate School of MedicineYanagidoJapan
  2. 2.Department of Pathology and Translational ResearchGifu University Graduate School of MedicineYanagidoJapan

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