Constitutive CCND1/CDK2 activity substitutes for p53 loss, or MYC or oncogenic RAS expression in the transformation of human mammary epithelial cells

Abstract

Cancer develops following the accumulation of genetic and epigenetic alterations that inactivate tumor suppressor genes and activate proto-oncogenes. Dysregulated cyclin-dependent kinase (CDK) activity has oncogenic potential in breast cancer due to its ability to inactivate key tumor suppressor networks and drive aberrant proliferation. Accumulation or over-expression of cyclin D1 (CCND1) occurs in a majority of breast cancers and over-expression of CCND1 leads to accumulation of activated CCND1/CDK2 complexes in breast cancer cells. We describe here the role of constitutively active CCND1/CDK2 complexes in human mammary epithelial cell (HMEC) transformation. A genetically-defined, stepwise HMEC transformation model was generated by inhibiting p16 and p53 with shRNA, and expressing exogenous MYC and mutant RAS. By replacing components of this model, we demonstrate that constitutive CCND1/CDK2 activity effectively confers anchorage independent growth by inhibiting p53 or replacing MYC or oncogenic RAS expression. These findings are consistent with several clinical observations of luminal breast cancer sub-types that show elevated CCND1 typically occurs in specimens that retain wild-type p53, do not amplify MYC, and contain no RAS mutations. Taken together, these data suggest that targeted inhibition of constitutive CCND1/CDK2 activity may enhance the effectiveness of current treatments for luminal breast cancer.

Figure 1. Generation of a genetically-defined, stepwise model of HMEC transformation.

48R expressing shRNA targeting p16 (shp16), p53 (shp53), or both (shp16-shp53) were further infected with control virus (Vec), or viruses encoding RAS alone (RAS), MYC alone (MYC), or MYC and RAS together (M/R) and assessed at passage 19 for AIG. (A) The bar graph represents the average colony number per plate of quadruplicates. Error bars represent the standard deviation. (B) Pictorial representation of the HMEC transformation model and growth in soft agar. Normal cells (left) are transformed (right) by sequential inactivation of p16 and p53 using shRNA, and over-expression of MYC and oncogenic RAS.

Figure 2. Constitutive CCND1/CDK2 activity enhances the growth of 48R-shp16 cells despite elevated p53 and p21.

48R-shp16 cells were infected with viruses encoding CCND1/CDK2 (D1/K2), a shRNA targeting p53 (shp53), or control virus (Vec). (A) Western blot analysis comparing parental 48R passage 11 to derivative cells. (B) Population doublings of the parental 48R (diamonds), 48R-shp16 (squares), 48R-shp16-D1/K2 (crosses), 48R-shp16-shp53 (triangles), and 48R-shp16-shp53-D1/K2 (circles) cells. Cells were grown from passage 6 at the origin and infected with shp16 at passage 11 indicated by the arrow.

Figure 3. Constitutive CCND1/CDK2 activity replaces p53 inactivation in the transformation of HMEC.

48R-shp16 cells were infected with control virus (Vec), or viruses encoding CCND1/CDK2 (D1/K2), or shRNA targeting p53 (shp53), followed by virus encoding MYC and RAS (M/R). (A) Western blot analysis comparing parental 48R passage 11 to derivative cells. (B) Derivative cells from A were assessed for AIG. The bar graph represents the average colony number per plate of quadruplicates. Error bars represent the standard deviation.

Figure 4. Constitutive CCND1/CDK2 activity replaces either MYC or RAS in the transformation of HMEC.

48R-shp16-shp53 cells were infected with control virus (Vec), virus encoding RAS alone (RAS), MYC alone (MYC), or MYC and RAS together (M/R). Additionally, 48R-shp16-shp53 cells were infected with viruses encoding CCND1/CDK2 (D1/K2) followed by RAS, or viruses encoding MYC followed by CCND1/CDK2 (D1/K2). (A) Western blot analysis comparing parental 48R passage 11 to derivative cells. (B) Each derivative was plated in soft agar to assess AIG. The bar graph represents the average colony number per plate of quadruplicates. Error bars represent the standard deviation.

Figure 5. Constitutive CCND1/CDK2 activity does not replace multiple components of transformation.

48R-shp16-D1/K2 and 48R-shp16-shp53-D1/K2 cells were infected with control virus (Vec), virus encoding RAS alone (RAS), MYC alone (MYC), or MYC and RAS together (M/R). (A) Western blot analyses comparing parental 48R passage 11 to derivative cells. (B) Each derivative was plated in soft agar to assess AIG. The bar graph represents the average colony number per plate of quadruplicates. Error bars represent the standard deviation.

Figure 6. Cell derivatives capable of AIG demonstrate increased motility.

Representative pictures of wound healing scratch assay of each HMEC derivative generated to represent all combinations of CCND1/CDK2 (D1/K2) replacements in the transformation protocol. Solid black lines represent cell fronts. Numbers beneath pictures indicate percent closure of scratch at 24 hours. The ability of the cell derivatives to grow anchorage independently is represented above the pictures (+  =  growth, −  =  no growth, −/+  =  intermediate growth).

Figure 7. Constitutive CCND1/CDK2 activity replaces p53 inhibition, MYC expression, or oncogenic RAS expression in HMEC transformation.

Consistent HMEC transformation to AIG is achieved by stepwise expression of the four molecular perturbations surrounded by rectangles. We have shown that constitutive CCND1/CDK2 (D1/K2) activity can individually substitute for one of three of these events indicated by differing surrounding shapes. Constitutive CCND1/CDK2 activity did not replace any combinations of these events.