Sex-dependent development of Kras-induced anal squamous cell carcinoma in mice

Abstract

Anal squamous cell carcinoma (SCC) will be diagnosed in an estimated 9,080 adults in the United States this year, and rates have been rising over the last several decades. Most people that develop anal SCC have associated human papillomavirus (HPV) infection (~85–95%), with approximately 5–15% of anal SCC cases occurring in HPV-negative patients from unknown etiology. This study identified and characterized the Kras-driven, female sex hormone-dependent development of anal squamous cell carcinoma (SCC) in the LSL-KrasG12D; Pdx1-Cre (KC) mouse model that is not dependent on papillomavirus infection. One hundred percent of female KC mice develop anal SCC, while no male KC mice develop tumors. Both male and female KC anal tissue express Pdx1 and Cre-recombinase mRNA, and the activated mutant KrasG12D gene. Although the driver gene mutation KrasG12D is present in anus of both sexes, only female KC mice develop Kras-mutant induced anal SCC. To understand the sex-dependent differences, KC male mice were castrated and KC female mice were ovariectomized. Castrated KC males displayed an unchanged phenotype with no anal tumor formation. In contrast, ovariectomized KC females demonstrated a marked reduction in anal SCC development, with only 15% developing anal SCC. Finally, exogenous administration of estrogen rescued the tumor development in ovariectomized KC female mice and induced tumor development in castrated KC males. These results confirm that the anal SCC is estrogen mediated. The delineation of the role of female sex hormones in mediating mutant Kras to drive anal SCC pathogenesis highlights a subtype of anal SCC that is independent of papillomavirus infection. These findings may have clinical applicability for the papillomavirus-negative subset of anal SCC patients that typically respond poorly to standard of care chemoradiation.

Pdx1 expression in hematopoietic cells activates Kras-mutation to drive leukemia in KC (Pdx1-Cre ; LSL-KrasG12D/+) mice

“…However, additional studies are required to specifically address this current limitation. Positive control (+) consisted of splenocytes from Vav-Cre ; LSL-Kras G12D/+ mice (as previously described) 3 and negative control (-) was tail from C57BL/6J mice. These are shown along with a ladder demonstrating the 250 and 500 bp markers.…”

“…Activated Kras genotyping was performed as previously published. 1,3 All mice were housed under identical conditions and are congenic on a C57BL/6J background (backcrossing > 15 generations). Both male and female mice of all strains were used.…”

“…To determine the penetration of Kras G12D mutation in thymuses of WT, KC and bone marrow chimeric mice, we followed the genotyping method for Kras G12D mutation previously described. 3 Genomic DNA was isolated from formalin fixed paraffin embedded thymus slides using ReliaPrep FFPE gDNA MiniPrep System (Promega, Madison, WI). The DNA was then amplified using polymerase chain reaction (PCR) with the following probes: 5’-GGGTAGGTGTTGGGATAGCTG-3’ (OL8403) and 5’-CCGAATTCAGTGACTACAGATGTACAGAG-3’ (OL8404) with conditions previously published.…”

“…PCR of genomic DNA from thymus of KC mice without T-ALL (Thymus) reveals wild type Kras (WT Kras). In KC mice with T-ALL, the active form of Kras-mutant allele (L-Kras G12D/+ ) is present in the thymic tumors (tumor), indicating Cre-mediated excision of Lox-Stop sequence.Positive control (+) consisted of splenocytes from Vav-Cre ; LSL-Kras G12D/+ mice (as previously described)3 and negative control (-) was tail from C57BL/6J mice. These are shown along with a ladder demonstrating the 250 and 500 bp markers.…”

“…DNA isolated from thymus of CD45.1 recipients of C57BL6/J bone marrow (1, 2, 3), and KC bone marrow(4,5,6) and demonstrated wild-type Kras and activated Kras-mutation, respectively (E). Positive control (PC) consisted of splenocytes from Vav-Cre ; LSL-Kras G12D/+ mice (as previously described)3 and negative control (NC) was tail from C57BL/6J mice.…”