Tumor-suppressor genes (TSG) are often deleted or transcriptionally suppressed in cancer. PGR codes for progesterone receptor (PR), a transcription factor whose function depends on its ligand. Although PR expression is often undetectable in cervical cancer, its relevance to the endocrine-related etiology of this prevalent gynecological disease remains unclear. In this study, we show that the deletion of one Pgr allele in cervical epithelium promoted spontaneous cervical cancer in human papilloma viral oncogene-expressing transgenic mice as efficiently as the ablation of both Pgr alleles. We also show that tumors arising in the transgenic mice with one or both Pgr alleles did not express PR or expressed at the reduced levels compared with the normal epithelium. PR status correlated with estrogen receptor α (ERα) status in the mouse model and the Cancer Genome Atlas (TCGA) dataset. TCGA data analyses revealed that PGR expression significantly decreased in cervical cancer and that the biallelic deletion of PGR was rare. Furthermore, low PGR expression was associated with poor prognosis in young patients with cervical cancer. These discoveries point to PGR as a haploinsufficient TSG in the uterine cervix. They also raise the possibility that the restoration of PGR expression may improve the survival rate.
The decreased expression of PR may increase the risk of cervical cancer in human papillomavirus–infected women.
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Genetically engineered mouse models have been a powerful tool to discover novel tumor-suppressor genes (TSG) and define their molecular mechanisms. Functions of TSGs are frequently impaired in cancers by mutations or transcriptional repression. The removal of one allele of many haploinsufficient TSGs, such as Dicer1, Fbw7, and Pten, is sufficient to promote cancers in mice, and they have similar mono-allelic mutations in human cancers (1). Unlike TSGs with biallelic deletions, re-expression or reactivation of haploinsufficient TSGs may be exploited as therapeutic targets.
Cervical cancer remains the third most frequent cancer and the third leading cause of cancer-related deaths among women worldwide (2). Although human papillomavirus (HPV) vaccines and the Pap test are effective in preventing cervical cancer, they are not readily available to women in low-income countries and those of low socioeconomic status in developed countries. Multiple full-term pregnancies and the use of oral contraceptives for longer than 5 years increases the risk of cervical cancer in HPV-infected women (3, 4). Although these results implicate female sex hormones in cervical cancer, retrospective studies attempting to determine individual roles of estrogen (E2) and progesterone (P4) have been inconclusive (5). E2 and P4 activate estrogen receptor α (ERα) and progesterone receptor (PR), respectively. Both receptors are ligand-dependent transcription factors that belong to the nuclear receptor superfamily.
HPV16 E6 and E7 oncoproteins are responsible for cervical cancer. HPV transgenic mice expressing these oncoproteins develop cervical cancer at high penetrance when treated with E2 for 6 months (6). We have previously shown that the cancer-promoting action of E2 depends on stromal ERα rather than epithelial ERα (7, 8). In contrast, we discovered that P4 inhibits proliferation and induces apoptosis in the cervical epithelium in an epithelial PR-dependent manner (9). Activation of PR by synthetic progesterone medroxyprogesterone acetate (MPA) was effective in treating and preventing cervical cancer in the HPV transgenic mouse model (10). In the present study, we show that the deletion of one PR-coding Pgr allele sensitizes HPV transgenic mice to cervical cancer without E2 treatment. Our analyses of cervical cancer tissues from the mouse model and the Cancer Genome Atlas (TCGA) database reveal that PR is expressed at the lower level in cancer than normal epithelium. Furthermore, PGR heterozygosity is common in human cervical cancer, and low PGR expression is associated with poor overall survival in young patients with cervical cancer. Our results support that PGR is a haploinsufficient TSG in the uterine cervix and a promising cervical cancer therapeutic target.
Materials and Methods
Animals and reagents
Experimental mice were generated by mating K14E7/Pgrf/f males with K14E6/Wnt7aCre/Pgrf/+ females. All mouse strains were previously described and summarized in Supplementary Table S1. Cervical tissues were harvested at 8 to 9 months of age without any treatment. The University of Houston Institutional Animal Care and Use Committee approved all procedures performed on mice. Antibodies and chemicals are described in Supplementary Table S2.
Tissue processing, histological staining, and analyses
Mouse cervical tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and serially sectioned throughout the cervix at a 5-μm thickness. Every tenth slide was stained with hematoxylin and eosin (H&E). Blinded histopathological analyses with the help of a pathologist and cancer measurement were carried out as described in Supplementary Materials and Methods.
IHC and apoptosis assay
Antigens were retrieved by incubating slides in pepsin solution for 5 minutes for p16Ink4a or microwaving sections for 20 minutes in 10 mmol/L sodium citrate buffer (pH 6.0) for the others. Slides were incubated with primary antibodies diluted in the blocking buffer, as described in Supplementary Table S2. After extensive washes in PBS, slides were subsequently incubated with a secondary antibody. Nuclei were stained with Hoechst 33258 solution (10 μg/mL) for 30 seconds. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was carried out with ApopTag Fluorescein in situ apoptosis detection kit according to the manufacturer's instruction. Slides were mounted with a gelvatol mounting medium.
Clinical data analysis
TCGA-Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (CESC) data were used for survival analysis and the expression of PGR and ESR1 mRNA as described in Supplementary Materials and Methods. The Genotype-Tissue Expression (GTEx) Cervix cohort was also used for the latter.
The deletion of one or both Pgr alleles sensitizes HPV transgenic mice to spontaneous cervical carcinogenesis
Although PR activation by MPA suppresses cervical cancer, E2 treatment results in similar cancer incidences in K14E7/Pgr+/+ and K14E7/Pgr−/− (10). We reasoned that it was due to the low level of P4 (i.e., minimal PR activation) because the treatment with exogenous E2 keeps mice at a continuous estrus-like stage (i.e., no P4 surges; ref. 11). We sought to determine whether cervical cancer would arise at a high frequency without E2 treatment if the PR signaling pathways were inactive. We used the Wnt7aCre transgene (referred to as Cre hereafter), which deletes Pgr specifically in the cervical epithelium (9). We generated K14E6/K14E7 (E6/E7) double transgenic or K14E7 (E7) single transgenic mice on the Pgrf/+, Cre/Pgrf/+, and Cre/Pgrf/f background and aged them without any treatments. As expected, cervical cancer did not occur in non-transgenic (NTG)/Pgrf/+ and NTG/Cre/Pgrf/f control mice that did not express any HPV oncogenes (Table 1). Cervical cancer incidence was significantly higher in E7/Pgrf/+ (33.3%) and E6/E7/Pgrf/+ (28.6%) than NTG/Pgrf/+ control (P < 0.002). Notably, the incidence of cervical cancer significantly increased in E7/Cre/Pgrf/f (61.7%) and E6/E7/Cre/Pgrf/f (78.6%) compared with E7/Pgrf/+ and E6/E7/Pgrf/+, respectively (P < 0.009; Table 1). Unexpectedly, the incidence of cervical cancer significantly increased in E7/Cre/Pgrf/+ (62.0%) and E6/E7/Cre/Pgrf/+ (64.3%) compared with E7/Pgrf/+ and E6/E7/Pgrf/+, respectively (P < 0.05; Table 1). Also, cancer incidences in E7/Cre/Pgrf/+ and E6/E7/Cre/Pgrf/+ were not significantly different from those of E7/Cre/Pgrf/f and E6/E7/Cre/Pgrf/f (Table 1). These results indicated that the deletion of only one Pgr allele was sufficient to inhibit the tumor-suppressive activity of PR in the cervix.
|Genotypes .||HPV oncogenes .||Epithelial Pgr status .||Group size, n .||No Disease .||CIN .||Cancer .||Cancer incidence (%) .|
|E6/E7/Cre/Pgrf/f||E6 and E7||null||14||0||3||11||78.6a|
|Genotypes .||HPV oncogenes .||Epithelial Pgr status .||Group size, n .||No Disease .||CIN .||Cancer .||Cancer incidence (%) .|
|E6/E7/Cre/Pgrf/f||E6 and E7||null||14||0||3||11||78.6a|
Note: NTG/Pgrf/+ and NTG/Pgrf/f were pooled and shown as NTG/Pgrf/+. E7/Cre/Pgrf/− and E7/Cre/Pgrf/f showed the similar disease burden. They were pooled and shown as E7/Cre/Pgrf/f. E7/Pgrf/− and E7/Cre/Pgrf/+ showed the similar disease burden. They were pooled and shown as E7/Cre/Pgrf/+.
Abbreviations: Het, heterozygote; wt, wild-type.
P < 0.05 compared with cancer incidence in E6/E7/Pgrf/+ or E7/Pgrf/+.
bP < 0.002 compared with cancer incidence in NTG/Pgrf/+.
PR is not expressed in the majority of cervical cancers arising in Pgr-sufficient mice
Next, we asked whether the level of PR expression in cancers correlated with the Pgr genotype. We analyzed all cancers for PR expression by IHC. As expected, PR was not expressed in all cancers in E7/Cre/Pgrf/f and E6/E7/Cre/Pgrf/f mice (Fig. 1A). To our surprise, PR was undetectable in the majority of cancers arising in E7/Pgrf/+ (58.8%, n = 17), E7/Cre/Pgrf/+ (56.9%, n = 51), E6/E7/Pgrf/+ (61.5%, n = 13), and E6/E7/Cre/Pgrf/+ (83.3%, n = 18; Fig. 1A; see PR− cancer). The percentage of PR-negative cancer was highest in E6/E7/Cre/Pgrf/+ mice, but the difference did not reach statistical significance. The remaining cancers expressed PR (Fig. 1A; see PR+ cancer). Cancer-associated stroma was positive for PR in all mice. Like cancers arising in E2-treated mice (6), all cervical cancers were microscopic and well-differentiated regardless of genotypes and PR status (Supplementary Fig. S1A). Dysplastic epithelia adjacent to PR-negative cancers did not express PR in both E7/Pgrf/+ and E7/Cre/Pgrf/+ (Fig. 1B). PR expression was similar in PR-positive cancer and nearby dysplastic epithelium, but it was reduced compared with the normal epithelium distant from cancer (Fig. 1B). H&E staining of corresponding epithelia is shown in Supplementary Fig. S1B. These results supported that PR expression was lost or downregulated in a precancer stage. ERα is necessary for the expression PR in the cervix (9). ERα expression was undetectable in 29 of 33 (87.9%) PR-negative cancers (Fig. 1C). The remaining PR-negative cancers and all PR-positive cancers (n = 20) expressed ERα (Fig. 1C). There was a strong positive correlation between the PR and ERα status (rϕ = 0.86, P = 5.25 × 10−11). These results suggested that ERα-dependent and -independent mechanisms were involved in PR downregulation.
PR-negative cancers are larger than PR-positive cancers
The expression of p16Ink4a and Mcm7, biomarkers for HPV+ cervical cancer, was increased in both PR− and PR+ cervical cancer in E7 and E6/E7 mice compared with a cancer in NTG/Cre/Pgrf/+ (Supplementary Fig. S2A and S2B). To assess an effect of PR on cancer growth, we divided cancers in each genotype according to the PR status. In both E7/Pgrf/+ and E7/Cre/Pgrf/+, the largest and total cancer areas were significantly larger for PR− than PR+ cervical cancer (Fig. 2A). The size of PR− cancer in these mice was not different from that in E7/Cre/Pgrf/f, indicating that the PR status rather than the Pgr genotype was predictive of cancer size. We observed a similar trend in E6/E7/Pgrf/+ and E6/E7/Cre/Pgrf/+ mice, but the difference did not reach statistical significance (Supplementary Fig. S3A). When pooled from all genotypes, however, PR− cancers had significantly greater individual and total cancer areas than PR+ cancers (Supplementary Fig. S3B). Larger cancers could be due to either earlier development (i.e., growth for a longer period) and/or faster growth. We could not evaluate the former possibility because diseases were diagnosed at end points. To test the latter, we determined proliferation and apoptosis rate in PR− and PR+ cancer. To control for hormonal conditions, we compared a PR− carcinoma with a PR+ cancer in the same mouse. A fraction of Ki67+ cells was always higher in PR− than PR+ cancer (Fig. 2B). In all five paired comparisons, the rate of TUNEL+ cells was lower in PR− than PR+ cervical cancer (Fig. 2C). These findings suggested that, albeit the decreased expression in PR+ cancers and failure to inhibit cancer development, PR was still active in suppressing tumor growth.
The level of PGR expression decreases in cervical cancer
We sought to determine whether our findings in the mouse model were relevant to patients. Using publicly available GTEx and TCGA datasets, we first analyzed PGR expression in cancerous and normal cervical tissues. The mean log-normalized transcripts per million (TPM) value of PGR indicated an approximately 55-fold reduction in the cancer tissue compared with the normal tissue (Fig. 3A). A similar analysis showed an 11-fold decrease in ESR1 mRNA levels in cervical cancer compared with the normal cervix (Supplementary Fig. S4A). Decreased expression of ESR1 and PGR in cervical cancer has been observed in an independent cohort (12). There was a modest, significant positive correlation between transcript levels of ESR1 and PGR in the TCGA dataset (Fig. 3B). To determine whether PGR copy number correlated with the expression level, we analyzed GISTIC-thresholded copy-number variation (CNV) data from TCGA. Notably, PGR expression levels were not significantly different among all CNV groups (Fig. 3C). Although homozygous deletion was infrequent (5 of 292 cancers), 47.3% and 37.0% had no copy-number change and heterozygous PGR deletion, respectively, and 41 cancers (14.1%) had an increased copy number (Supplementary Fig. S4B).
Low PGR expression is associated with poor prognosis in cervical cancer
Menopause is diagnosed after women have gone one year without a menstrual period. The average age of menopause is 51 in the United States. Because progesterone levels are low without a menstrual cycle, we divided TCGA patients into two age groups, 31 to 50 years of age (young) and 50+ years of age (old) for survival analyses. In the young cohort, patients with high PGR expression had a better 18-month overall survival than those with low PGR expression (hazard ratio = 0.203; Fig. 3D). On the contrary, the survival benefit of high PGR status was absent in the old patient group (hazard ratio = 0.639). These results suggest that a progesterone surge during the menstrual cycle protects patients with cervical cancer with high PGR expression.
We showed that the deletion of one Pgr allele promoted cervical cancer (Table 1) and that more than one third of TCGA patient samples had heterozygous deletion of PGR (Fig. 3C). Regardless of the PR-coding gene copy number, levels of PR decreased in cervical cancer compared with the normal epithelium in both mouse models and clinical samples (Fig. 1A and B; Fig. 3C). These results strongly support that PGR is a dose-dependent, haploinsufficient TSG in cervical cancer. PR similarly suppresses endometrial cancer (13). It would be interesting to see whether PGR is also haploinsufficient in this malignancy.
Dose-dependent TSGs fail to suppress tumorigenesis when expression drops below a threshold level, and nullizygotes for most of them are more susceptible to cancer than heterozygotes (1). However, Pgr null and heterozygotes had similar cancer incidence (Table 1). In this regard, Pgr was similar to a handful of haploinsufficient TSGs. Dmp1+/− and Dmp1−/− mice display similarly accelerated Kras-driven lung tumorigenesis (14). The deletion of either one or both Trp53bp1 alleles augments tumorigenesis to a similar degree in a glioma mouse model (15). In E7/Cre/Pgrf/+ (i.e., epithelial Pgr heterozygote) mice, PR expression was lower in cancer than the normal epithelium (Fig. 1A and B). We postulate that a threshold level for Pgr is just below 50% of the normal level.
The expression of PR is undetectable in 60% to 80% of cervical cancer (10, 16, 17). In our spontaneous cervical cancer models, 57%–83% of cancers arising in Pgr-sufficient mice did not express PR (Fig. 1A). TCGA data analyses showed that PGR expression decreased in cervical cancer (Fig. 3A). Multiple mechanisms could be responsible for the reduced expression of PR. The promoter of PGR is hypermethylated in cervical cancer tissues compared with normal tissues (18). PGR is a direct transcriptional target of ERα (19), and the expression of ESR1 and PGR are correlated (Fig. 1C and Fig. 3B). Overexpression of ERα restores the expression of PGR mRNA in HeLa cervical cancer cells (20). These observations support that transcriptional repression contributes, at least in part, to the reduced PGR expression in cervical cancer. Although we cannot rule out post-transcriptional and post-translational mechanisms, we do not favor the mechanism of loss of heterozygosity because percentages of PR-negative cancers were similar between Pgrf/+ and Cre/Pgrf/+ mice (Fig. 1A) and because one or more PGR copies were retained in 98.4% of human cancers (Fig. 3C). High stromal PR expression is associated with better survival of patients with cervical cancer (21), suggesting that stromal PR also has the anti-cervical cancer activity and that PR signaling in cervical cancer is more complex than currently appreciated.
In summary, our results demonstrate that PGR is downregulated in cervical cancer and suggest that reactivation of PGR expression may improve the survival rate of patients with cervical cancer. Our spontaneous cervical cancer model demonstrated the development of PR-negative cervical cancer for the first time. Further studies are warranted to better understand the mechanism of PGR downregulation during cervical carcinogenesis in vivo, and our mouse model provides a valuable tool for such studies.
No disclosures were reported.
Y. Park: Conceptualization, data curation, investigation, visualization, methodology, writing-original draft, writing-review and editing. S. Baik: Investigation, visualization, methodology. C. Ho: Data curation, investigation, visualization, methodology, writing-original draft, writing-review and editing. C.-Y. Lin: Data curation, investigation, methodology, writing-review and editing. S.-H. Chung: Conceptualization, data curation, supervision, funding acquisition, investigation, visualization, methodology, writing-review and editing.
We thank Dr. Roger E. Price for consultation on histopathology. We also thank Drs. Richard Behringer and John Lydon for providing us with Wnt7aCre and Pgrf/f mice, respectively. This work was supported in part by National Institutes of Health grant R01 CA188646, Cancer Prevention and Research Institute of Texas grant RP180275, and University of Houston Large Core Equipment grants (to S.-H. Chung).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.