Parkin enhances the expression of cyclin-dependent kinase 6 and negatively regulates the proliferation of breast cancer cells

Abstract

Although mutations in the parkin gene are frequently associated with familial Parkinsonism, emerging evidence suggests that parkin also plays a role in cancers as a putative tumor suppressor. Supporting this, we show here that parkin expression is dramatically reduced in several breast cancer-derived cell lines as well as in primary breast cancer tissues. Importantly, we found that ectopic parkin expression in parkin-deficient breast cancer cells mitigates their proliferation rate both in vitro and in vivo, as well as reduces the capacity of these cells to migrate. Cell cycle analysis revealed the arrestment of a significant percentage of parkin-expressing breast cancer cells at the G1-phase. However, we did not observe significant changes in the levels of the G1-associated cyclin D1 and E. On the other hand, the level of cyclin-dependent kinase 6 (CDK6) is dramatically and selectively elevated in parkin-expressing breast cancer cells, the extent of which correlates well with the expression of parkin. Interestingly, a recent study demonstrated that CDK6 restrains the proliferation of breast cancer cells. Taken together, our results support a negative role for parkin in tumorigenesis and provide a potential mechanism by which parkin exerts its suppressing effects on breast cancer cell proliferation.

FIGURE 1.

Parkin expression is down-regulated in breast cancer cells. A, bar graph depicting the real-time quantification of parkin mRNA levels in various breast cancer cell lines and a normal breast cell line Hs578BST (BST). The non tumor-derived HEK 293 cells were used as an additional control (*, p < 0.05, **, p < 0.001, Student's t test). B, representative anti-parkin immunoblot showing the relative expression of endogenous parkin protein in various lines. The blot was stripped and reprobed with anti-actin to reflect loading variations. C, graphs showing the Ct value of parkin expression in normal and cancer tissues (left panel), and parkin immunoreactivity scores derived from 6 pairs of adjacent benign and malignant breast cancer tissues. Statistical analysis was carried up using GraphPad Prism, Version 4.0 software (SanDiego, CA). Wilcoxon matched pair test was used to compare the difference between the adjacent benign and malignant breast cancer tissues (*, p < 0.05).

FIGURE 2.

Overexpression of parkin in MCF7 cells mitigates their proliferation rate. A, anti-FLAG and anti-parkin immunoblots of total lysates prepared from MCF7 cells (−), vector control (V), and the parkin-expressing stable clones (5, 7, & 11). Equal loading of the different lysates was verified by anti-actin immunoblotting. B, graphical depiction of the percentage of cells undergoing cell proliferation, as measured by cell population number, in MCF7 vector control and parkin-expressing stable cell lines (left), or parkin-expressing cells in the presence of control or parkin siRNA (right). Inset, immunoblots showing the expression of parkin in cells transduced with control (lane 1) or parkin siRNA (lane 2) (top panel), and the respective actin levels (bottom panel). C, BrdU assay of MCF7 vector control and parkin-expressing stable cell lines. D, representative images showing apparent decreased ability of parkin-expressing MCF7 cells in forming colonies in soft agar compared with MCF7-vector control. E, graph showing the volume (left and middle panels) and mass (right panel) of tumor generated by different cell types in NOD-SCID mice (n = 9) at day 14 and day 28. The experiment was duplicated with essentially the same result (*, p < 0.05; **, p < 0.001, Student's t test).

FIGURE 3.

Parkin-expressing MCF7 cells exhibits slower migration. A, phase-contrast images showing the re-colonization of cells into the wound area at 60 h. B, graphical depiction of the distance moved by the various cell types (as indicated) into the wound area. (*, p < 0.05, Student's t test). C, bar graph showing the ratio between parkin-expressing and control cells that migrated to the underside of the matrigel-coated transwell after 24 h of incubation (*, p < 0.05, Student's t test).

FIGURE 4.

Accumulation of parkin-expressing MCF7 cells at G1 phase. A, graphical depiction of flow cytometry-based cell cycle analysis of control and parkin-expressing MCF7 cells. B, bar graphs showing the percentage of control (vector) or parkin-expressing MCF cells (parkin) in various phases of the cell cycle.

FIGURE 5.

Levels of cyclins and mitogenic components in control and parkin-expressing MCF7 cells. A, anti-cyclin E; B, anti-cyclin D1; C, anti-phosphorylated Erk 1/2 and anti-Erk1/2; D, anti-phosphorylated Akt and anti-Akt immunoblots in vector control (V3) and parkin-expressing (PK5, -7 & -11) MCF7 cells. Equal loading of the different lysates was verified by anti-actin immunoblotting.

FIGURE 6.

CDK6 expression is enhanced in parkin-expressing MCF 7 cells. A, left, immunoblots showing the levels of CDK6 and parkin in control (vector) or parkin-expressing MCF7 cells, as indicated. Right, bar graph showing the relative difference in CDK6 expression among the MCF7 clones examined as determined by densitometric quantification. B, as in A except that parkin is substituted with a catalytically-impaired T415N mutant. Two clonal populations of MCF7 cells stably expressing this mutant are indicated as TN2 and TN3. C, representative confocal images showing CDK6 staining in MCF7 cells transiently transfected with parkin or CHIP, as indicated.