p16INK4A Positively Regulates Cyclin D1 and E2F1 through Negative Control of AUF1

/pRB/E2F pathway, a key regulator of the critical G1 to S phase transition of the cell cycle, is universally disrupted in human cancer. However, the precise function of the different members of this pathway and their functional interplay are still not well defined. -dependent manner, and several of these genes are also members of the AUF1 and E2F1 regulons. We also present evidence that E2F1 mediates p16-dependent regulation of several pro- and anti-apoptotic proteins, and the consequent induction of spontaneous as well as doxorubicin-induced apoptosis. is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1

p16(INK4A) positively regulates p21(WAF1) expression by suppressing AUF1-dependent mRNA decay.

BACKGROUND: p16(INK4a) and p21(WAF1) are two independent cyclin-dependent kinase inhibitors encoded by the CDKN2A and CDKN1A genes, respectively. p16(INK4a) and p21(WAF1) are similarly involved in various anti-cancer processes, including the regulation of the critical G1 to S phase transition of the cell cycle, senescence and apoptosis. Therefore, we sought to elucidate the molecular mechanisms underlying the link between these two important tumor suppressor proteins. METHODOLOGY/PRINCIPAL FINDINGS: We have shown here that the p16(INK4a) protein positively controls the expression of p21(WAF1) in both human and mouse cells. p16(INK4a) stabilizes the CDKN1A mRNA through negative regulation of the mRNA decay-promoting AUF1 protein. Immunoprecipitation of AUF1-associated RNAs followed by quantitative RT-PCR indicated that endogenous AUF1 binds to the CDKN1A mRNA in a p16(INK4A)-dependent manner. Furthermore, while AUF1 down-regulation increased the expression level of the CDKN1A mRNA, the concurrent knockdown of AUF1 and CDKN2A, using specific silencing RNAs, restored the normal expression of the gene. Moreover, we used EGFP reporter fused to the CDKN2A AU-rich element (ARE) to demonstrate that p16(INK4A) regulation of the CDKN1A mRNA is AUF1- and ARE-dependent. Furthermore, ectopic expression of p16(INK4A) in p16(INK4A)-deficient breast epithelial MCF-10A cells significantly increased the level of p21(WAF1), with no effect on cell proliferation. In addition, we have shown direct correlation between p16(INK4a) and p21(WAF1) levels in various cancer cell lines. CONCLUSION/SIGNIFICANCE: These findings show that p16(INK4a) stabilizes the CDKN1A mRNA in an AUF1-dependent manner, and further confirm the presence of a direct link between the 2 important cancer-related pathways, pRB/p16(INK4A) and p14(ARF)/p53/p21(WAF1)

p16 controls the transcription of the <i>CDKN1A</i> mRNA through AUF1.

<p>(A) Cell lysates were prepared from <i>CDKN2A</i>-shRNA expressing cells and their control counterparts and AUF1 mouse monoclonal antibody and mouse IgG (used as control) were utilized for immunoprecipitation. Co-precipitated RNA was purified and used for quantitative RT-PCR reactions. (B) Total RNA was purified from p16 proficient (HFSN1 and EH1) and p16-deficient (U2OS and HFSN1 expressing <i>CDKN2A</i>-shRNA) cells, expressing either AUF1-siRNA or control-siRNA. Transcripts for the indicated genes were amplified by quantitative RT-PCR. (C) U2OS cells were transfected with EGFP reporter containing either the wild-type (WT) or the mutated (Mut) <i>CDKN1A</i> ARE, while U2OS containing AUF1-siRNA as well as EH1 cells were transfected with the <i>CDKN1A</i>-WT-ARE construct. GFP fluorescence was measured 24 hrs post-transfection. Error bars represent means ± S.D. *: <i>p</i>-value < 0.05.</p

p16 positively controls p21 expression in epithelial cells.

<p>MCF-10A cells were transfected with a plasmid bearing the <i>CDKN2A</i>-ORF or a control plasmid. (A) 2x10⁴ cells were plated in E-16 plate for 48 hrs and cell proliferation was monitored using the RTCA-DP system. Cell index (CI) represents cell impedance measurement, which represents quantitative information about cell number. (B) p16 and Ki-67 immunostaining. Upper panel: phase contrast microscopy, lower panel: Labeling Index for Ki-67 and p16 staining was determined for at least 500 cells per data point and expressed as mean ± S.D of triplicate determinations. (C) Whole cell lysates were prepared from the indicated cells and were used for immunoblotting utilizing antibodies against the indicated proteins. (D) Total RNA was purified and used for qRT-PCR amplification using specific primers for the indicated genes.</p

Curcumin Triggers p16-Dependent Senescence in Active Breast Cancer-Associated Fibroblasts and Suppresses Their Paracrine Procarcinogenic Effects

Activated cancer-associated fibroblasts (CAFs) or myofibroblasts not only facilitate tumor growth and spread but also affect tumor response to therapeutic agents. Therefore, it became clear that efficient therapeutic regimens should also take into account the presence of these supportive cells and inhibit their paracrine effects. To this end, we tested the effect of low concentrations of curcumin, a pharmacologically safe natural product, on patient-derived primary breast CAF cells. We have shown that curcumin treatment upregulates p16INK4A and other tumor suppressor proteins while inactivates the JAK2/STAT3 pathway. This reduced the level of alpha-smooth muscle actin (α-SMA) and the migration/invasion abilities of these cells. Furthermore, curcumin suppressed the expression/secretion of stromal cell-derived factor-1 (SDF-1), interleukin-6 (IL-6), matrix metalloproteinase-2 (MMP-2), MMP-9, and transforming growth factor-β, which impeded their paracrine procarcinogenic potential. Intriguingly, these effects were sustained even after curcumin withdrawal and cell splitting. Therefore, using different markers of senescence [senescence-associated β-galactosidase (SA-β-gal) activity, Ki-67 and Lamin B1 levels, and bromodeoxyuridine incorporation], we have shown that curcumin markedly suppresses Lamin B1 and triggers DNA damage-independent senescence in proliferating but not quiescent breast stromal fibroblasts. Importantly, this curcumin-related senescence was p16INK4A-dependent and occurred with no associated inflammatory secretory phenotype. These results indicate the possible inactivation of cancer-associated myofibroblasts and present the first indication that curcumin can trigger DNA damage-independent and safe senescence in stromal fibroblasts

p16 modulates p21 protein level in human and mouse cells.

<p>Whole cell extracts were prepared from different human and mouse cell lines and were used for immunoblotting analysis using the indicated antibodies (A, B, C and E). The histograms show the expression levels of the indicated proteins. Error bars indicate standard deviations of 3 different experiments. *: <i>p</i>-value < 0.05. (D) Cell cycle analysis of the indicated cells using flow cytometry.</p

p21 and p16 protein levels are correlative in cancer cells.

<p>Whole cell extracts were prepared from the indicated cells and used to assess the levels of p16, AUF1 and p21 by immunoblotting.</p

p16 modulates the <i>CDKN1A</i> mRNA level in human and mouse cells.

<p>(A) Total RNA was extracted from both p16-deficient and p16-proficient cells and used for qRT-PCR utilizing specific primers, and β-actin was used as internal control. Error bars indicate standard deviations of 3 different experiments. *: <i>p</i>-value < 0.05. (B) Cells were treated with actinomycin D and re-incubated for different periods of time as indicated. Total RNA was extracted and the <i>CDKN1A</i> mRNA level of was assessed by qRT-PCR using specific primers and normalized against β -actin. The graph shows the proportion of the <i>CDKN1A</i> mRNA remaining post-treatment, and the dotted lines indicate the <i>CDKN1A</i> mRNA half-life. Error bars indicate standard errors of 3 different experiments.</p

Ionizing radiation normalizes the features of active breast cancer stromal fibroblasts and suppresses their paracrine pro-carcinogenic effects

Background: Radiotherapy is an important therapeutic strategy for breast cancer patients through reducing the chances of recurrence and metastasis, which are fueled by cancer-associated fibroblasts (CAFs). Thereby, we addressed here the effect of various doses of X-rays on breast CAFs and their adjacent counterparts. Methods: We have used WST1 and annexin V-associated with flow cytometry to test the cytotoxic effects of X-rays. Immunoblotting and ELISA was used to assess the expression/secretion of various proteins. Immunohistochemistry was utilized to determine the level of β-galactosidase and Ki-67. Sphere formation assay was used to test the ability of breast cancer cells to form tumorspheres. Orthotopic tumor xenografts were also used to evaluate the effect of X-ray-treated breast stromal fibroblasts on breast cancer tumor growth in vivo. Results: Breast stromal fibroblasts showed high resistance to X-rays. While the low dose (5 Gy) inhibited cell proliferation and the active features of CAFs, the higher doses (16 and 50 Gy) promoted senescence. However, this was not accompanied by the senescence-associated secretory phenotype (SASP), but rather a reduction in the synthesis/secretion of various cancer-associated cytokines. Additionally, X-rays suppressed the features of active breast stromal fibroblasts, and their paracrine pro-carcinogenic effects. The ablative dose (16 Gy) inhibited the capacity of active stromal fibroblasts to promote the pro-metastatic processes epithelial-to-mesenchymal transition, the formation of cancer stem cells, as well as the growth of humanized orthotopic breast tumor xenografts. Conclusion: Together, these findings indicate that X-rays can normalize the features of active breast stromal fibroblasts through promoting senescence without SASP