Critical roles for non-pRb targets of human papillomavirus type 16 E7 in cervical carcinogenesis

Abstract

High-risk human papillomaviruses (HPV) encode two oncogenes, E6 and E7, expressed in nearly all cervical cancers. In vivo, HPV-16 E7 has been shown to induce multiple phenotypes in the context of transgenic mice, including cervical cancer. E7 is a multifunctional protein known best for its ability to inactivate the tumor suppressor pRb. To determine the importance of pRb inactivation by E7 in cervical cancer, we pursued studies with genetically engineered mice. E7 expression in estrogen-treated murine cervix induced dysplasia and invasive cancers as reported previously, but targeted Rb inactivation in cervical epithelium was not sufficient to induce any cervical dysplasia or neoplasia. Furthermore, E7 induced cervical cancer formation even when the E7-pRb interaction was disrupted by the use of a knock-in mouse carrying an E7-resistant mutant Rb allele. pRb inactivation was necessary but not sufficient for E7 to overcome differentiation-induced or DNA damage-induced cell cycle arrest, and expression patterns of the E2F-responsive genes Mcm7 and cyclin E indicate that other E2F regulators besides pRb are important targets of E7. Together, these data indicate that non-pRb targets of E7 play critical roles in cervical carcinogenesis.

Figure 1

Early cervical phenotypes. Histologic cross sections of cervical epithelium from mice treated for 6 weeks with estrogen. Brown, positive staining; blue, hematoxylin counterstain. Dashed line, location of the basement membrane. A, pRb inactivation in murine cervix. pRb staining. B and C, BrdUrd incorporation in cervical epithelia. BrdUrd staining labels newly synthesized DNA. B, DNA synthesis is restricted to the lowest two cell layers in control epithelium, but E7 expression induces suprabasal DNA synthesis. Rb deletion in K14CreRb^flox/flox cervix fails to reproduce the effect of E7. C, E7 fails to induce suprabasal DNA synthesis in the presence of the Rb^ΔL allele. BrdUrd staining pattern in K14E7Rb^WT/WT cervix is indistinguishable from K14E7Rb^flox/flox cervix (data not shown).

Figure 2

Cervical carcinogenesis studies. Histologic cross sections of cervical epithelium from mice treated with estrogen for 6 months. A, E7 expression induces severe cervical phenotypes, including cancer. Control (Rb^flox/flox) cervix and pRb-deficient K14CreRb^flox/flox cervix show no dysplasia or cancers. B, Rb^WT/WT and Rb^ΔL/ΔL cervix do not develop dysplasia or invasive cancer, but both K14E7Rb^ΔL/ΔL and K14E7Rb^WT/WT cervixes develop invasive cancers. C, pRb immunohistochemical stain (brown) of invasive cancers from K14E7Rb^WT/WT and K14Rb^ΔL/ΔL mice. Arrows, nuclei of invasive cells. Blue, counterstained with hematoxylin.

Figure 3

Quantitative effects on DNA synthesis. Basal and suprabasal cells from BrdUrd-stained cervical epithelia were counted as BrdUrd positive or BrdUrd negative, and the frequency of BrdUrd-positive cells was graphed. A, BrdUrd incorporation in Rb^flox mice. *, P < 0.05, statistically significant change compared with Rb^flox/flox. B, BrdUrd incorporation in Rb^WT and Rb^ΔL mice. *, P < 0.05, statistically significant increase in suprabasal DNA synthesis in K14E7Rb^WT/WT mice compared with all other genotypes. C, E7 expression, but not Rb inactivation, prevents DNA damage–induced cell cycle arrest. Mice were exposed to 0 or 12 Gy ionizing radiation 24 hours before BrdUrd administration. BrdUrd staining frequency in basal cells is graphed. BrdUrd incorporation frequency is significantly reduced in irradiated Rb^flox/flox and K14CreRb^flox/flox mice versus unirradiated mice (P < 0.05). D, pRb inactivation is necessary for E7 to disrupt DNA damage–induced arrest. Mice were irradiated and analyzed as in (C). BrdUrd incorporation frequency is significantly reduced in irradiated Rb^WT/WT, Rb^ΔL/ΔL, and K14E7Rb^ΔL/ΔL mice versus unirradiated mice (P < 0.05).

Figure 4

Expression of E2F-responsive genes. A, cervical sections were stained for cyclin E. Brown, positive staining; blue, hematoxylin counterstain. Dashed line, location of the basement membrane. B, cervical cross sections of the indicated genotypes were stained for Mcm7. K14E7Rb^WT/WT cervix was identical to K14E7Rb^flox/flox, so is not shown.

Figure 5

Absence of CIN lesions in K14E7Rb^ΔL/ΔL cervix. Representative sections of K14E7Rb^WT/WT and K14E7Rb^ΔL/ΔL cervixes. Arrows, sites of cancer invasion into the dermis. In K14E7Rb^WT/WT cervix, cancer arises from epithelium containing many dysplastic changes (arrowheads), whereas in K14E7Rb^ΔL/ΔL cervix, cancer is observed without any dysplastic changes elsewhere in the cervical epithelium.