Loss of Keratin 17 induces tissue-specific cytokine polarization and cellular differentiation in HPV16-driven cervical tumorigenesis in vivo

Abstract

Despite preventive human papilloma virus (HPV) vaccination efforts, cervical cancer remains a leading cause of death in women worldwide. Development of therapeutic approaches for cervical cancer are hampered by a lack of mechanistic insight during tumorigenesis. The cytoskeletal protein Keratin 17 (KRT17;K17) is robustly expressed in a broad array of carcinomas, including in cervical tumors, where it has both diagnostic and prognostic value. In this study, we have established multiple functional roles for K17 in the promotion of cervical tumorigenesis in vivo using the established HPV16^tg mouse model for cervical squamous cell carcinoma. In HPV16^tg/+;Krt17^-/-relative to HPV16^tg/+ reference female mice, onset of cervical lesions is delayed and closely paralleled by marked reductions in hyperplasia, dysplasia and vascularization. In addition, loss of Krt17 is associated with a cytokine polarization and recruitment of effector immune cells to lesion-prone cervical epithelia. Further, we observed marked enhancement of terminal differentiation in HPV16^tg/+;Krt17^-/-cervical epithelium accompanied by a stimulation and expansion in the expression of p63, a known basal/reserve cell marker in this tissue. Altogether, the data suggest that the loss of Krt17 may foster an overall protective environment for lesion-prone cervical tissue. In addition to providing new insights into the immunomodulatory and cellular mechanisms of cervical tumorigenesis, these findings may help guide the development of future therapies including vaccines.

Figure 1

Increased K17 expression correlates with cervical lesion progression. (a) H&E staining of transformation zone epithelium for wild-type (WT), HPV16^tg/+ and HPV16^tg/+;Krt17^−/− mice at 1-, 3- and 6 months post implantation. Bar = 100 μm. (b) Immunoblots for Cdkn2a in cervical tissue of HPV16^tg/+ and HPV16^tg/+;Krt17^−/− mice at 1-, 3- and 6 months post implantation. Each lane represents a distinct biological replicate. Actin serves as loading control. Densitometric quantification shown below. (c) Immunostaining for Cdkn2a (green) in transformation zone epithelium of WT, HPV16^tg/+ and HPV16^tg/+;Krt17^−/− mice at 6 months post implantation. Nuclei are stained with DAPI (blue). Dotted line separates epithelium (above line) from underlying stroma (below line). Bar = 100 μm. (d, e) Immunoblots for K17 in cervical tissue of (d) WT and (e) HPV16^tg/+ mice at 1-, 3- and 6 months post implantation. Each lane represents a distinct biological replicate. Actin serves as loading control. E7 indicates transgene expression. Densitometric quantification of K17 expression normalized to WT shown at right. (f) Immunostaining for K17 (green) in transformation zone epithelium of HPV16^tg/+ mice at 1-, 3- and 6 months post implantation. Nuclei are stained with DAPI (blue). Dotted line separates epithelium (above line) from underlying stroma (below line). Bar = 100 μm. All error bars are s.e.m. *P<0.05. NS; no significance.

Figure 2

Markers of cervical lesion progression are attenuated with the loss of K17. (a) Immunostaining for phospho-histone H3 (PH3; green) in transformation zone epithelium of HPV16^tg/+ and HPV16^tg/+;Krt17^−/− mice at 3 months post implantation. Nuclei are stained with DAPI (blue). Dotted line separates epithelium (above line) from underlying stroma (below line). Bar = 100 μm. Quantification of PH3⁺ cells per field at right. (b) Immunostaining for PECAM-1 (green) in underlying stroma of transformation zone epithelium of HPV16^tg/+ and HPV16^tg/+;Krt17^−/− mice at 3 months post implantation. Dotted line separates epithelium (above line) from underlying stroma (below line). Bar = 100 μm. Quantification of PECAM-1 pixel intensity per stromal area at right. (c) Quantification of toluidine blue-positive cells per field (images not shown) of transformation zone epithelium of WT, HPV16^tg/+ and HPV16^tg/+;Krt17^−/− mice at 3 months post implantation. (d) Thickness (in μm) of transformation zone epithelium (left) and ectocervical epithelium (right) of WT, HPV16^tg/+ and HPV16^tg/+;Krt17^−/− mice at 3 months post implantation. All error bars are s.e.m. *P<0.05.

Figure 3

Inflammation is increased in cervical tissue in the absence of K17. (a, b) Normalized expression for gene transcripts in cervical tissue of HPV16^tg/+ and HPV16^tg/+;Krt17^−/− mice at (a) 1 month post implantation and at (b) 3 months post implantation. (c–e) Immunostaining for (c) Cd4, (d) F4/80 and (e) Cd11b in transformation zone epithelium of WT, HPV16^tg/+ and HPV16^tg/+;Krt17^−/− mice at 3 months post implantation. Nuclei are stained with DAPI (blue). Dotted line separates epithelium (above line) from underlying stroma (below line). Arrowheads denote intraepithelial immunostained cells. Bar = 100 μm. Graphs at right depict the number of intraepithelial immune cells per field across genotypes. All error bars are s.e.m. *P<0.05.

Figure 4

Differentiation of cervical epithelia is induced in the absence of K17. (a) Normalized expression for S100a8 and S100a9 gene transcripts in cervical (left) and ear (right) tissue of HPV16^tg/+ and HPV16^tg/+;Krt17^−/− mice at 3 months post implantation. (b) Immunoblots for S100a8 in cervical (top) and ear (bottom) tissues of WT, HPV16^tg/+ and HPV16^tg/+;Krt17^−/− mice at 3 months post implantation. Each lane represents a distinct biological replicate. Actin serves as loading control. Densitometric quantification below. (c) Normalized expression for differentiation markers in cervical tissue of HPV16^tg/+ and HPV16^tg/+;Krt17^−/− mice at 1 month (left) and 3 months (right) post implantation. (d) Immunoblots for K1 and filaggrin in cervical tissue of HPV16^tg/+ and HPV16^tg/+;Krt17^−/− mice at 1 month post implantation. Each lane represents a distinct biological replicate. Actin serves as loading control. Densitometric quantification below. (e, f) Immunostaining for K1 in (e) transformation zone epithelium and (f) ear skin epidermis of HPV16^tg/+ and HPV16^tg/+;Krt17^−/− mice at 1 month post implantation. Nuclei are stained with DAPI (blue). Dotted line separates epithelium (above line) from underlying stroma (below line). Bar = 100 μm. All error bars are s.e.m. *P<0.05. NS; no significance.

Figure 5

p63 expression is expanded throughout suprabasal cervical epithelium in the absence of K17. (a, b) Normalized expression for Sfn gene transcript in (a) cervical and (b) ear tissue of HPV16^tg/+ and HPV16^tg/+;Krt17^−/− mice at 1 month post implantation. (c) Immunostaining for p63α in transformation zone epithelium of WT, HPV16^tg/+ and HPV16^tg/+;Krt17^−/− mice at 1 month (left) and 3 months (right) post implantation. Nuclei are stained with DAPI (blue). Dotted line separates epithelium (above line) from underlying stroma (below line). Bar = 100 μm. Graphs below depict the number of suprabasal cells immunopositive for p63 per field across genotypes. All error bars are s.e.m. *P<0.05.