p14ARF Post-Transcriptional Regulation of Nuclear Cyclin D1 in MCF-7 Breast Cancer Cells: Discrimination between a Good and Bad Prognosis?

Abstract

As part of a cell’s inherent protection against carcinogenesis, p14ARF is upregulated in response to hyperproliferative signalling to induce cell cycle arrest. This property makes p14ARF a leading candidate for cancer therapy. This study explores the consequences of reactivating p14ARF in breast cancer and the potential of targeting p14ARF in breast cancer treatment. Our results show that activation of the p14ARF-p53-p21-Rb pathway in the estrogen sensitive MCF-7 breast cancer cells induces many hallmarks of senescence including a large flat cell morphology, multinucleation, senescence-associated-β-gal staining, and rapid G1 and G2/M phase cell cycle arrest. P14ARF also induces the expression of the proto-oncogene cyclin D1, which is most often associated with a transition from G1-S phase and is highly expressed in breast cancers with poor clinical prognosis. In this study, siRNA knockdown of cyclin D1, p21 and p53 show p21 plays a pivotal role in the maintenance of high cyclin D1 expression, cell cycle and growth arrest post-p14ARF induction. High p53 and p14ARF expression and low p21/cyclin D1 did not cause cell-cycle arrest. Knockdown of cyclin D1 stops proliferation but does not reverse senescence-associated cell growth. Furthermore, cyclin D1 accumulation in the nucleus post-p14ARF activation correlated with a rapid loss of nucleolar Ki-67 protein and inhibition of DNA synthesis. Latent effects of the p14ARF-induced cellular processes resulting from high nuclear cyclin D1 accumulation included a redistribution of Ki-67 into the nucleoli, aberrant nuclear growth (multinucleation), and cell proliferation. Lastly, downregulation of cyclin D1 through inhibition of ER abrogated latent recurrence. The mediation of these latent effects by continuous expression of p14ARF further suggests a novel mechanism whereby dysregulation of cyclin D1 could have a double-edged effect. Our results suggest that p14ARF induced-senescence is related to late-onset breast cancer in estrogen responsive breast cancers and/or the recurrence of more aggressive breast cancer post-therapy.

Sphingosine Kinase 1 Isoform-Specific Interactions in Breast Cancer

“…Downstream consequences of inhibiting DPP with Ala-BoroPro was evaluated by observing the effect on p21, a cell cycle regulator in MCF-7 cells (38), and the trypan blue exclusion cell proliferation assay (51). The Ala-BoroPro inhibitor had little or no effect on p21 protein expression in cell lines stably expressing the SK43 and SK51, whereas Ala-BoroPro increased SK51 expression under the same experimental conditions as shown by Western blot ( Figure 8A).…”

“…Cells were fixed and stained as described previously (38). FLAG antibody was used for SK1 detection, and Alexafluor 488 and 594 and Hoechst 33342 were used for visualization.…”

Annexin/S100A Protein Family Regulation through p14ARF-p53 Activation: A Role in Cell Survival and Predicting Treatment Outcomes in Breast Cancer

“…Our laboratory has demonstrated that activation of the p53-p21 pathway by p14ARF, in addition to rapid induction of cell cycle arrest, initiated a change in cellular metabolism consistent with a metabolically active senescence-like phenotype most likely to be important in cell survival and recurrence [16]. In our studies, the cancer cells maintained cell viability, and a sub-set of these cells retained the ability to proliferate [16, 17]. Dormant and senescent cells may be more resistant to cancer treatments so the more we understand about the behavior of these cells, the more likely we will be able to understand how cancers develop resistance to current treatments and, importantly, how they recur after treatment.…”

“…We have previously reported that activation of the p14ARF-p53 pathway in MCF-7 cells leads to cell cycle arrest, without inducing apoptosis, and, additionally, induces a metabolically active senescent-like phenotype [17]. To further explore the underlying mechanisms that lead to cell cycle arrest and metabolic/phenotypic changes we used SILAC LC-MS/MS methodology to determine the proteomic profile of MCF-7 cells post activation of p14ARF-p53 at 24 h and 72 h. Re-expression of p14ARF had no effect on the estrogen response in these cells (Fig 2 inset).…”

“…Protein isolation and western blot analysis were performed as described previously [17]. Primary antibodies used were p53 (DO-7, Dako, CA, USA), p21 (c-19, Santa Cruz), ANXA A1, ANXA A2 (Becton Dickinson) and β-actin (Abcam), followed by mouse secondary conjugated antibody (Abcam).…”