Calmodulin-Like Protein 5 (CALML5) Expression in Squamous Cell Carcinoma of Esophagus and Oropharynx

Abstract

Squamous cell carcinoma (SCC), a common malignancy affecting the skin, vagina, uterine cervix, anus, larynx, and upper digestive tract, is characterized by significant disruption of cell-cell adhesion in stratified squamous epithelium during tumorigenesis, progression, and metastasis. CALML5, a stratified epithelial-specific protein linked to desmosomal junctions, plays a key role in cell adhesion and is notably downregulated in human papillomavirus (HPV)-associated cervical SCC. Esophageal and pharyngeal cancers, commonly with a squamous cell phenotype, have distinct etiologies: oropharyngeal carcinoma is strongly associated with HPV, whereas esophageal carcinoma is linked to environmental factors such as smoking, alcohol, and diet. To investigate the role of CALML5 in these cancers, we performed immunohistochemical analyses on clinical samples and explored its regulatory mechanisms using in vitro studies with human esophageal SCC cell lines. Our findings revealed that CALML5 expression is suppressed in early-stage esophageal SCC but reactivated at invasive sites in well to moderately differentiated SCC undergoing keratinization. In specialized SCC with sarcomatoid component, CALML5 reactivation occurred alongside aberrant KLF4 expression, highlighting its context-dependent role in tumor progression. Conversely, while HPV-unrelated oropharyngeal SCC exhibited patterns similar to esophageal SCC, HPV-related oropharyngeal SCC consistently showed suppressed CALML5 expression due to impaired KLF4 nuclear translocation. These results suggest that CALML5 functions as a tumor suppressor in HPV-associated cervical SCC but may be reactivated in non-HPV-associated invasive SCC, emphasizing its complex role in SCC pathogenesis and the need for careful interpretation of its expression in clinical contexts.

Keywords: Calmodulin-Like Protein 5; human papillomavirus; immunohistochemistry; squamous cell carcinoma; upper alimentary tract.

Fig. 1.

H&E stain (A and B), and immunohistochemical evaluation of CALML5 (C), p63 (D), ZNF750 (E), and KLF4 (F) in normal or nonneoplastic esophageal squamous epithelium. (A) Normal esophageal mucosal tissue and (B) magnified view of the basal portion (black box in A) (H&E stain). The following immunohistochemical images (C-D-E and F) correspond to magnified area (B). (C) CALML5 immunohistochemical staining. The magnified image within white box in the upper left corner shows CALML5-positive intercellular bridges (white arrow). (D) p63 immunohistochemical staining. (E) ZNF750 immunohistochemical staining. (F) KLF4 immunohistochemical staining. Bars = 150 μm.

Fig. 2.

H&E stain (A) and immunohistochemical evaluation of CALML5 (B), p63 (C), ZNF750 (D), KLF4 (E) in noninvasive esophageal carcinoma. In typical cases of noninvasive esophageal carcinoma, tumor cells proliferate in a downward-pressuring bulky fashion, while retaining the basement membrane (A, H&E stain). (B) CALML5 immunohistochemical staining. (C) p63 immunohistochemical staining. (D) ZNF750 immunohistochemical staining. (E) KLF4 immunohistochemical staining. Area within black box magnified in the upper right corner (B–E). Asterisks indicate the nontumorous areas. Bars = 150 μm.

Fig. 3.

H&E stain (A) and immunohistochemical evaluation of CALML5 (B), p63 (C), ZNF750 (D), KLF4 (E) in a case of invasive esophageal carcinoma (poorly differentiated or undifferentiated) showing sarcomatous feature. (A) H&E stain of a specimen from a case of invasive esophageal squamous cell carcinoma with poorly differentiated or undifferentiated morphology. (B) CALML5 immunohistochemical staining. (C) p63 immunohistochemical staining. (D) ZNF750 immunohistochemical staining. (E) KLF4 immunohistochemical staining. Bars = 100 μm.

Fig. 4.

Comparison of CALML5, ZNF750, and KLF4 expression in p16-negative (A–E) and p16-positive oropharyngeal carcinoma (F–J). (A, F) H&E staining. Immunostaining of the area in the black box (A) of the oropharyngeal carcinoma with negative p16 (B) is shown in C–E. (C) CALML5 immunohistochemical staining. (D) ZNF750 immunohistochemical staining. (E) KLF4 immunohistochemical staining. Immunostaining of the area in the black box (F) of the oropharyngeal carcinoma with positive p16 (G) is shown in H–J. (H) CALML5 immunohistochemical staining. (I) ZNF750 immunohistochemical staining. (J) KLF4 immunohistochemical staining. Magnified images are shown in the lower right corner (D, I, J). Bars = 100 μm.

Fig. 5.

Analysis of CALML5 (A), ZNF750 (B) and KLF4 (C) expression in nuclear and cytoplasmic fractions by Western blotting of various squamous cell carcinoma cell lines (A) Western blot analysis of CALML5. Immunoblot signals are detected as a 16 kDa band (indicated by yellow arrowheads). (B) Western blot analysis of ZNF750. Immunoblot signals are detected as a 77 kDa band. (C) Western blot analysis of KLF4. Immunoblot signals are detected as a 55 kDa band.

Fig. 6.

Effects of KLF4 nuclear transport (A) inhibition on CALML5 mRNA Expression (B). (A) KLF4 immunostaining is shown in the upper panels. DAPI staining is shown in the middle panels. Merged image of KLF4 and DAPI is shown in the bottom panels. (B) CHX and MG132, both inhibitors of nuclear translocation of KLF4, administered to TE-9 cells, and RNA collected after 24 hr. CALML5 expression was compared by Realtime PCR. Data are presented as the means ± standard deviation (SD), n = 4. *p < 0.05, one-way ANOVA.

Fig. 7.

Schema for regulation of CALML5 expression. (A) Squamous carcinoma showing a basal cell-like phenotype. In the noninvasive esophageal carcinoma, both ZNF750 and KLF4 are decreased, and their nuclear localization is nullified, as observed in the basal cell layer of normal esophageal squamous epithelium (shown as basaloid phenotype). (B) Invasive squamous carcinoma with sarcomatoid component. In poorly differentiated squamous cell carcinoma with sarcomatous feature, CALML5 expression, previously lost, is restored in a small number of cases. CALML5 expression restoration may be due to aberrantly expressed KLF4 in tumor cells sarcomatous feature. (C) Human papillomavirus (HPV)-associated squamous carcinoma. The inhibition of KLF4 nuclear translocation by HPV proteins prevents the activation of CALML5 expression.