The CDK4/6 inhibitor PD0332991 reverses epithelial dysplasia associated with abnormal activation of the cyclin-CDK-Rb pathway

Abstract

Loss of normal growth control is a hallmark of cancer progression. Therefore, understanding the early mechanisms of normal growth regulation and the changes that occur during preneoplasia may provide insights of both diagnostic and therapeutic importance. Models of dysplasia that help elucidate the mechanisms responsible for disease progression are useful in highlighting potential targets for prevention. An important strategy in cancer prevention treatment programs is to reduce hyperplasia and dysplasia. This study identified abnormal upregulation of cell cycle-related proteins cyclin D1, cyclin-dependent kinase (CDK)4, CDK6, and phosphorylated retinoblastoma protein (pRb) as mechanisms responsible for maintenance of hyperplasia and dysplasia following downregulation of the initiating viral oncoprotein Simian virus 40 (SV40) T antigen. Significantly, p53 was not required for successful reversal of hyperplasia and dysplasia. Ligand-induced activation of retinoid X receptor and PPARγ agonists attenuated cyclin D1 and CDK6 but not CDK4 or phosphorylated pRb upregulation with limited reversal of hyperplasia and dysplasia. PD0332991, an orally available CDK4/6 inhibitor, was able to prevent upregulation of cyclin D1 and CDK6 as well as CDK4 and phosphorylated pRb and this correlated with a more profound reversal of hyperplasia and dysplasia. In summary, the study distinguished CDK4 and phosphorylated pRb as targets for chemoprevention regimens targeting reversal of hyperplasia and dysplasia.

FIGURE 1. Dysplastic and hyperplastic histology was progressively reversed over 14 days when TAg was downregulated at 4- but not 7-months of age

Representative H&E sections illustrate phenotypic reversal of ductal cells in the submandibular salivary gland accompanying TAg downregulation in 4- (A) but not 7- (B) month-old Tet-op-TAg^MMTV-tTA (tTa/TAg) mice over a 14-day timecourse. Arrows point to dysplastic ductal epithelial cells with nuclear pleomorphism and hyperchromasia and arrowheads point to striated ductal epithelial cells with normal nuclear appearance. The histological phenotype at both ages consists of varying percentages of hyperplastic and dysplastic (C), in transition (D), and normal-like (E) structures resembling those of wild-type mice (F). Stacked bar graphs shows distribution of hyperplastic and dysplastic, in transition and normal-like structures before and 14 days after TAg downregulation in 4- (G) and 7-month-old (H) mice. Timepoint (days): 0; prior to TAg downregulation, 2, 6, 10 and 14 indicate the number of days of doxycycline administration to downregulate TAg. m= age in months. Magnification H&E images = 60X. Size bar = 10 µm. *, **, ***:p<0.05, Student’s t test (two-tailed). Cohort sizes at different timepoints: 4m: 0(n = 4), 2(n = 4, 6(n = 4), 10(n = 4), and 14(n=5); 7m: 0(n = 3), 2(n = 3), 6(n = 3), 10(n=3), 14(n = 5).

FIGURE 2. Comparison of protein expression patterns when TAg was downregulated in 4- and 7-month-old mice

Representative western blots illustrate the different expression patterns of cell cycle regulatory proteins accompanying TAg downregulation in submandibular salivary tissue in 4- (A) and 7- (B) month-old Tet-op-TAg^MMTV-tTA mice over a 14-day timecourse. (C–G) Line graphs illustrate mean relative steady-state protein levels in 4- (dotted lines) and 7- (solid lines) month-old mice before and 2, 4, 6, 10 and 14 days after initiating doxycycline to downregulate TAg. Relative protein expression levels at different timepoints were compared by Student t test (two-tailed). (C) Cyclin D1, CDK6 and CDK4 expression levels were significantly higher in 7- as compared to 4-month-old mice at the 10 (Cyclin D1: ^4-fold (p<0.02); CDK6: ^2-fold (p<0.01); CDK4: ^3-fold (p<0.01) higher) and 14 (Cyclin D1: ^8-fold (p<0.01); CDK6: ^4-fold (p<0.01); CDK4: ^22-fold (p< 0.05) higher) day timepoints. (D) Phosphorylated pRb expression levels were 18-fold lower at the 0 timepoint (*p<0.05) and significantly higher at the 10 (^24-fold (p<0.01) and 14 (^68-fold (p<0.018) day timepoints in 7- as compared to 4-month-old mice. Total pRb levels were 5-fold lower in 7- as compared to 4-month-old mice at the 6-day timepoint (*p<0.05). (E) Expression levels of DP-1 were 2-fold lower at the 0 timepoint (*p<0.005) and significantly higher at the 2 (^3-fold (p<0.04), 6 (^2-fold (p<0.01), 10 (^3-fold (p<0.05) and 14 (^6-fold (p<0.01) day timepoints in 7- as compared to 4-month-old mice. E2F-1 expression was 29-fold higher in the 7- as compared to 4-month-old mice at the 14-day timepoint (^p<0.05). (F) Expression levels of p21 and p27 were significantly lower in 7- as compared to 4-month-old mice at the 2 (p21: *2-fold (p<0.05), p27: *7-fold (p<0.03), 6 (p21: *3-fold (p<0.01), p27: *6-fold (*p<0.05), 10 (p21: *10-fold (p<0.04), p27: 5-fold (p<0.01), and 14 (p21: 13-fold (p<0.01), p27: 6-fold (p<0.01) day timepoints. (G) Expression levels of Cyclin E were ^2-fold higher at 10 days (p<0.05) and CDK2 levels were ^2- fold higher at 14 days (p<0.05) in 7- as compared to 4-month-old mice. Relative protein expression levels determined by quantification of western blots using densitometry. Mean and SEM indicated for each timepoint. m= age in months. Asterisks (*) indicate statistically significantly lower and carets (^) statistically significantly higher relative expression levels in 7- as compared to 4-month-old mice at the same timepoint (both p<0.05, Student’s t test (two-tailed)., Cohort sizes and timepoints as described (Fig 1).

FIGURE 3. Comparison of protein expression patterns in salivary ductal cells when TAg was downregulated in 4- and 7-month-old mice

Representative histological sections of submandibular salivary gland from 4- and 7-month-old Tet-op-TAg^MMTV-tTA mice. Immunohistochemical detection of Cyclin D1 (A), CDK6 (B), CDK4 (C), CDK2 (D), pRb (G), DP-1 (H), and E2F-1 (I) was higher and detection of p21 (E) and p27 (F) lower in 7- as compared to 4-month-old mice 14 days after administration of doxycycline to downregulate TAg (panels outlined in black). No differences in expression of total pRb (J) or Cyclin E (K) were found. Digital photographs taken at ×40 and cropped to pre-set dimensions. Size bar = 20 µm. m= age in months. Cohort sizes and timepoints as described (Fig 1).

FIGURE 4. p53 was not required for dysplasia reversal in 4-month-old mice

(A) Stacked bar graphs showing distribution of hyperplastic and dysplastic, in transition and normal-like structures before and 14 days after TAg downregulation in submandibular salivary glands of 4-month-old Tet-op-TAg^{MMTV-tTA/p53−/−} (tTa/TAg/p53+/−) mice. (B) Bar graphs comparing relative protein expression levels in submandibular salivary tissue 14 days after TAg downregulation in 4-month-old Tet-op-TAg^{MMTV-tTA/p53−/−} and Tet-op-TAg^MMTV-tTA (tTA/TAg) and 7-month-old Tet-op-TAg^MMTV-tTA mice. Absence of p53 was associated with significant reductions in relative expression levels of p21 (p<0.01), p27 (p<0.01) and total pRb (p<0.05). No significant differences in expression levels of Cyclin D1, CDK6, CDK4, Cyclin E, CDK2, phosphorylated pRb, DP-1 or E2F-1 were found between 4-month-old Tet-op-TAg^{MMTV-tTA/p53−/−} and Tet-op-TAg^MMTV-tTA mice. There were significant differences in expression of Cyclin D1, CDK6, CDK4, CDK2, pRb, phosphorylated pRb, DP-1 and E2F-1 between 7-month-old Tet-op-TAg^MMTV-tTA and 4-month-old Tet-op-TAg^{MMTV-tTA/p53−/−} and Tet-op-TAg^MMTV-tTA mice. Relative protein expression levels determined by quantification of western blots using densitometry. Mean and SEM indicated. Timepoints=0 (A) and 14 (A,B) days after administration of doxycycline to downregulate TAg. m= age in months. *:p<0.05, Student’s t test (two-tailed).Cohort sizes by genotype at different timepoints: (A) 4m tTA/TAg/p53−/−: 0(n=5), 14(n=4), (B) 4m tTA/TAg/p53−/− : 14(n=3), 4m tTA/TAg: 14(n=5), 7mtTA/TAg: 14(n=5).

FIGURE 5. Treatment with RXRα and PPARγ ligands UAB30 and rosiglitazone did not change percentage of normal-like structures but did alter expression levels of Cyclin D1, CDK6, phosphorylated Rb and E2F1 in 7-month-old mice

Stacked bar graphs comparing distribution of hyperplastic and dysplastic, in transition and normal-like structures in submandibular salivary glands from untreated control and 14 day-UAB30-treated (A) and in untreated control and 28 day-UAB30+ROSI-treated (B) 7-month-old Tet-op-TAg^MMTV-tTA- (tTA/TAg) mice in which TAg was coincidentally downregulated by doxycycline. (C) Representative western blots illustrate expression levels of cell cycle regulatory proteins in submandibular salivary tissue of untreated control and 28-day-UAB30+ROSI-treated 7-month-old Tet-op-TAg^MMTV-tTA mice that coincidentally received doxycycline to downregulate TAg. (D) Bar graphs comparing relative protein expression levels in submandibular salivary tissue 28 days after TAg downregulation in control untreated and UAB30+ROSI-treated 7-month-old TAg^MMTV-tTA mice. Expression levels of Cyclin D1 were reduced 2-fold (p<0.01), CDK6 were reduced 3-fold (p<0.02), phosphorylated pRb were reduced 3-fold (p<0.05) and E2F-1 were reduced 4-fold (p<0.01) in the UAB30+ROSI-treated mice. Relative protein expression levels determined by quantification of western blots using densitometry. Mean and SEM indicated. (E) Boxplots comparing relative RNA expression levels in submandibular salivary tissue from 7-month-old TAg^MMTV-tTA control untreated mice versus 14-day-UAB30-treated, 28-day-UAB-treated and 28-day-UAB30+ROSI-treated mice with coincident TAg downregulation. Significant increases in expression of Fabp4, Adrp, Pdk4 were found in all three treatment groups as compared to no treatment controls (p<0.05). Timepoints: 14 or 28 days after administration of doxycycline to downregulate TAg. m= age in months.. *:p<0.05, Student’s t test (two-tailed) (D) or Mann Whitney U test (E). Cohort sizes by treatment group and timepoint: (A) 7m control 14(n=5), UAB30 14(n=10), (B) 7m control 28(n=5), UAB30+ROSI 28(n=9), UAB30(n=5, data not shown) (C,D) 7m control 28(n=3), UAB30+ROSI 28(n=3), (E) 7m control (n=3), UAB30 14(n=3), UAB30 28(n=3), UAB30+ROSI 28(n=3).

FIGURE 6. CDK4/6 inhibitor, PD0332991, successfully reversed dysplasia in 7-month-old mice

(A) Stacked bar graphs showing distribution of hyperplastic and dysplastic, in transition and normal-like structures in submandibular salivary glands from untreated control and 10-day-PD0332991-treated 7-month-old Tet-op-TAg^MMTV-tTA mice in which TAg was coincidentally downregulated by doxycycline. (B) Representative western blots illustrate expression levels of cell cycle regulatory proteins in submandibular salivary tissue in untreated control and 10-day-PD0332991-treated 7-month-old Tet-op-TAg^MMTV-tTA mice coincidentally receiving doxycycline to downregulate TAg. Control and PD0332991-treated samples were run on the same blot and cropped to show representative lanes (C) Bar graphs comparing relative expression levels of proteins 10 days after TAg downregulation in control untreated and PD0332991-treated 7-month-old mice. Expression levels of Cyclin D1 were reduced 3-fold (p<0.04), CDK6 were reduced 5-fold (p<0.003), CDK4 were reduced 11-fold (p<0.002), phosphorylated pRb were reduced 17-fold (p=0.05), DP-1 were reduced 6-fold (p<0.02), E2F-1 were reduced 2-fold (p<0.04), p21 were increased 4-fold (p<0.05) and p27 increased 6-fold (p=0.04), all significantly changes, in the PD0332991-treated mice as compared to controls. Relative protein expression levels determined by quantification of western blots using densitometry. Mean and SEM indicated. (D) Representative histological sections of submandibular salivary gland from untreated control and PD0332991-treated 7-month-old Tet-op-TAg^MMTV-tTA mice illustrated. Immunohistochemical detection of Cyclin D1, CDK6, CDK4, phosphorylated pRb, DP-1, and E2F-1 was lower and detection of p21 (E) and p27 (F) higher in PD0332991-treated as compared to untreated control mice (panels outlined in black). Digital photographs taken at ×40 and all cropped to pre-set dimensions. Size bar = 20 µm. Rx=PD0332991-treated. Timepoint=10 days after downregulation of TAg; m=months. *,**:p<0.05, Student’s t test (two-tailed). Cohort sizes by treatment group and timepoint: 7m control 10(n=3), PD0332991 10(n=4).