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. 2012 Jul 15;11(14):2747-55.
doi: 10.4161/cc.21127. Epub 2012 Jul 15.

CDK4/6 Inhibition Antagonizes the Cytotoxic Response to Anthracycline Therapy

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Free PMC article

CDK4/6 Inhibition Antagonizes the Cytotoxic Response to Anthracycline Therapy

A Kathleen McClendon et al. Cell Cycle. .
Free PMC article

Abstract

Triple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.

Figures

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Figure 1. CDK4/6 inhibition yields a cooperative cytostatic effect with chemotherapy, but antagonizes cytotoxicity in RB-proficient TNBC cell lines. (A) Representative flow cytometry traces of cells treated with vehicle (Control), PD-0332991 (PD), and/or doxorubicin (DOX) for 24 h are shown, and average percent BrdU incorporation was quantified (***p < 0.0005). (B) Cells were treated for 24 h, and total protein lysates were immunoblotted as indicated. (C) Representative images of cells treated for 24 h and stained for p-γH2AX are shown, and average fold-increase in p-γH2AX compared with control was quantified (*p = 0.0074, **p < 0.005). (D) Cells were treated for 24 h, and total protein lysates were immunoblotted for the indicated pro-apoptotic factors.
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Figure 2. CDK4/6 inhibition does not modify the sensitivity of RB-deficient TNBC cell lines to cytotoxic chemotherapy. (A) Representative flow cytometry traces of cells treated with vehicle (Control), PD-0332991 (PD), and/or doxorubicin (DOX) for 24 h are shown, and average percent BrdU incorporation was quantified. (B) Cells were treated for 24 h, and total protein lysates were immunoblotted for the indicated proteins. (C) Representative images of cells treated for 24 h and stained for p-γH2AX are shown, and average fold-increase in p-γH2AX compared with control is shown (**p = 0.0033, ***p < 0.0005). (D) Cells were treated for 24 h, and total protein lysates were immunoblotted for the indicated pro-apoptotic factors.
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Figure 3. CDK4/6 inhibition antagonizes doxorubicin-mediated cytotoxicity in vivo in an RB-dependent manner. (A) Representative Ki67 staining in xenograft tumors treated with vehicle (Control), PD-0332991 (PD), and/or doxorubicin (DOX) is shown, and average percent Ki67-positive cells was quantified (**p = 0.0065; ***p < 0.0005). (B) Representative p-γH2AX staining in treated xenograft tumors is shown, and average percent p-γH2AX-positive cells was quantified (**p = 0.0032, ***p = 0.0008). (C) Representative phospho-histone H3 (pSer10) staining in treated xenograft tumors is shown, and average percent pSer10-positive cells was quantified (*p = 0.0055, **p = 0.005, ***p < 0.0005). (D) Representative cleaved caspase-3 (Casp3) staining in treated xenograft tumors is shown, and average percent Casp3-positive cells was quantified (*p = 0.023, **p < 0.003).
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Figure 4. CDK4/6 inhibition does not alter the cytotoxic response of RB-deficient TNBC tumors to doxorubicin. Representative images of Ki67, p-γH2AX, pSer10 and cleaved caspase-3 staining in RB-deficient MDA-MB-231 xenograft tumors treated with vehicle (Control), PD-0332991 (PD), and/or doxorubicin (DOX) are shown. Arrows highlight mitotic figures.
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Figure 5. CDK4/6 inhibition in combination with cytotoxic chemotherapy differentially impacts the long-term survival of RB-proficient and RB-deficient cells. RB-proficient (MDA-MB-231) and RB-deficient (MDA-MB-468) cells were treated with vehicle (Control), PD-0332991 (PD) and/or doxorubicin (DOX) for 24 h, allowed to recover in the absence of drug, and stained with crystal violet at the indicated time points post-treatment. Percentages of cell populations displaying clonal outgrowth post-treatment are displayed.

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