[Show abstract][Hide abstract] ABSTRACT: BRCA1 promoter methylation has been detected in DNA from peripheral blood cells of both breast cancer patients and cancer-free females. However, the pathological significance of this epigenetic change in white blood cells (WBC) remains an open question. In this study, we hypothesized that if constitutional BRCA1 methylation reflects an elevated risk for developing breast cancer (BC), WBC that harbor methylated BRCA1 in both cancer-free females and BC patients should exhibit similar molecular changes.
BRCA1 promoter methylation was examined by methylation-specific PCR in WBC from 155 breast cancer patients and 143 cancer-free females. Furthermore, The Human Breast Cancer EpiTect Methyl II Signature PCR Array and The Human Breast Cancer RT2 ProfilerTM PCR Array were used to study the methylation status and the expression profile of several breast cancer-related genes, respectively. In addition, we used label-free MS-based technique to study protein expression in plasma.
We have shown that 14.2% of BC patients and 9.1% of cancer-free females (carriers) harbored methylated BRCA1 promoter in their WBC. Interestingly, 66.7% of patients harbored methylated BRCA1 promoter in both WBC and tumors. Importantly, we have shown the presence of epigenetic changes in 9 other BC-related genes in WBC of both patients and carriers. Additionally, BRCA1 and 15 other important cancer -related genes were found to be differentially expressed in WBC from patients and carriers as compared to controls. Furthermore, we have shown that the carriers exhibited a unique plasma protein pattern different from those of BC patients and controls, with 10 proteins similarly differentially expressed in patients and carriers as compared to controls.
The present results suggest the presence of a strong link between aberrant methylation of the BRCA1 promoter in WBC and breast cancer -related molecular changes, which indicate the potential predisposition of the carriers for developing breast cancer. This informs the potential use of the aberrant methylation of BRCA1 promoter in WBC as a powerful non-invasive molecular marker for detecting predisposed individuals at a very early age.
BMC Cancer 11/2014; 14(1):830. · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Breast cancer (BC) is the most common malignancy and the leading cause of cancer-related death amongst women worldwide. The risk factors of this disease are numerous, and their prevalence varies between racial and ethnic groups as well as geographical regions. Therefore, we sought to delineate the association of socio-demographic, reproductive and life-style related risk factors with breast cancer in the Arab population.
Unmatched case-control study was conducted in the kingdom of Saudi Arabia using 534 cases of histologically confirmed breast cancer and 638 controls. Controls were randomly selected from primary health care visits and were free of breast cancer. Unconditional logistic regression analysis was performed to estimate odds ratios (ORs) and to examine the predictive effect of each factor on risk for BC. All study participants were interviewed by trained interviewers at hospital (cases) or at primary health care centers (controls).
A total of 1172 women were eligible for this study, of which 281 (24.0%) were aged ≤35 years, 22.9% illiterate, 43.6% employed, 89.5% married, and 38.1% were obese. Grade III tumors constituted 38.4% of cases. Tumor stage I was 7.5%; II, 50.7%; II, 30.9%; IV, 11.1%. We have shown strong association between breast cancer among Arab females and obesity (OR =2.29, 95% CI 1.68-3.13), positive family history of breast cancer (OR =2.31, 95% CI 1.60 – 3.32), the use of hormonal replacement therapy (OR =2.25, 95% CI 1.65 – 3.08), post-menopause (OR =1.72, 95% CI 1.25 – 2.38), lack of education (OR =9.09, 95% CI 5.88 – 14.29), and never breastfeed (OR =1.89, 95% CI 1.19 – 2.94).
These results indicate the presence of classical risk factors established in the western countries, and also some specific ones, which may result from genetic and/or environmental factors. Thereby, these findings will be of great value to establish adequate evidence-based awareness and preventative measures in the Arab world.
[Show abstract][Hide abstract] ABSTRACT: MMiR-141 and miR-146b-5p are two important tumor suppressor microRNAs, which control several cancer-related genes and processes. In the present report, we have shown that these microRNAs bind specific sites at the 3[prime]-untranslated region (UTR) of the mRNA-binding protein AUF1, leading to its down-regulation. This inverse correlation between the levels of these microRNAs and AUF1 has been identified in various osteosarcoma cell lines. Additionally, we present clear evidence that AUF1 promotes mesenchymal features in osteosarcoma cells, and that miR-141 and miR-146b-5p suppress this pro-metastatic process through AUF1 repression. Indeed, both microRNAs suppressed the invasion/migration and proliferation abilities of osteosarcoma cells through inhibiting the AKT protein kinase in an AUF1-dependent manner. We have also shown that AUF1 binds to and stabilizes the mRNA of the AKT activator phosphoinositide-dependent kinase-1 (PDK1). Furthermore, miR-141 and miR-146b-5p positively regulate the epithelial markers (E-cadherin and Epcam) and represses the mesenchymal markers (N-cadherin, Vimentin, Twist2 and ZEB1). These effects were mediated via the repression of the epithelial-to-mesenchymal (EMT)-inducer ZEB1 through targeting AUF1, which binds the 3[prime]UTR of the ZEB1 mRNA and reduces its turnover. These results indicate that at least some tumor suppressor functions of miR-141 and miR-146b-5p are mediated through the repression of the oncogenic potentials of AUF1. Thereby, these 3[prime]-UTR-directed post-transcriptional gene expression regulators constitute promising new targets for diagnostic and/or therapeutic interventions.
Journal of Biological Chemistry 09/2014; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The development and spread of mammary carcinomas require synergetic interplay between tumor cells and their microenvironment through paracrine secretions, which are still not well defined. We have shown here that the interleukin-6 (IL-6), either recombinant or secreted from highly invasive breast cancer cells, down-regulates the tumor suppressor proteins p16INK4A, p21WAF1 and p53, and activates breast stromal fibroblasts in a paracrine manner. The formation of myofibroblasts requires p16INK4A down-regulation and the activation of the JAK2/STAT3 pathway. Indeed, the transcription factor STAT3 positively controls the expression of the three major myofibroblast markers SDF-1, [alfa]-SMA and TGF-[beta1], and mediates IL-6-related down-regulation of p16INK4A, p21WAF1 and p53, as well as the activation of stromal fibroblasts. Importantly, these effects were mediated through STAT3-dependent up-regulation of the mRNA binding protein AUF1, whose promoter contains 3 canonical STAT3 binding sites. AUF1 binds the SDF-1, [alfa]-SMA, TGF-[beta1] and IL-6 mRNAs and reduces their turnover. Consequently, specific AUF1 down-regulation inhibits IL-6-dependent activation of breast stromal fibroblasts, whilst AUF1 ectopic expression of p37AUF1 activated these cells and enhanced their paracrine induction of epithelial-to-mesenchymal transition in breast cancer cells, which shows a non-cell-autonomous oncogenic function of AUF1. Together, these results demonstrate a major role of IL-6 in activating breast stromal fibroblasts through STAT3-dependent AUF1 induction.
Journal of Biological Chemistry 09/2014; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Active cancer-associated fibroblasts (CAFs) or myofibroblasts play important roles not only in the development and progression of breast carcinomas, but also in their prognosis and treatment. Therefore, targeting these cells through suppressing their supportive procarcinogenic paracrine effects is mandatory for improving the current therapies that are mainly targeting tumor cells. To this end, we investigated the effect of the natural and pharmacologically safe molecule, caffeine, on CAF cells and their various procarcinogenic effects.
We have shown here that caffeine up-regulates the tumor suppressor proteins p16, p21, p53 and Cav-1, and reduces the expression/secretion of various cytokines (IL-6, TGF-β, SDF-1 and MMP-2), and down-regulates α-SMA. Furthermore, caffeine suppressed the migratory/invasiveness abilities of CAF cells through PTEN-dependent Akt/Erk1/2 inactivation. Moreover, caffeine reduced the paracrine pro-invasion/-migration effects of CAF cells on breast cancer cells. These results indicate that caffeine can inactivate breast stromal myofibroblasts. This has been confirmed by showing that caffeine also suppresses the paracrine pro-angiogenic effect of CAF cells through down-regulating HIF-1αand its downstream effector VEGF-A. Interestingly, these effects were sustained in absence of caffeine.
The present findings provide a proof of principle that breast cancer myofibroblasts can be inactivated, and thereby caffeine may provide a safe and effective prevention against breast tumor growth/recurrence through inhibition of the procarcinogenic effects of active stromal fibroblasts.
PLoS ONE 03/2014; 9(3):e90907. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Breast cancer is a major health problem that threatens the lives of millions of women worldwide each year. Most of the chemotherapeutic agents that are currently used to treat this complex disease are highly toxic with long-term side effects. Therefore, novel generation of anti-cancer drugs with higher efficiency and specificity are urgently needed.
Breast cancer cell lines were treated with eugenol and cytotoxicity was measured using the WST-1 reagent, while propidium iodide/annexinV associated with flow cytometry was utilized in order to determine the induced cell death pathway. The effect of eugenol on apoptotic and pro-carcinogenic proteins, both in vitro and in tumor xenografts was assessed by immunoblotting. While RT-PCR was used to determine eugenol effect on the E2F1 and survivin mRNA levels. In addition, we tested the effect of eugenol on cell proliferation using the real-time cell electronic sensing system.
Eugenol at low dose (2 muM) has specific toxicity against different breast cancer cells. This killing effect was mediated mainly through inducing the internal apoptotic pathway and strong down-regulation of E2F1 and its downstream antiapoptosis target survivin, independently of the status of p53 and ERalpha. Eugenol inhibited also several other breast cancer related oncogenes, such as NF-kappaB and cyclin D1. Moreover, eugenol up-regulated the versatile cyclin-dependent kinase inhibitor p21WAF1 protein, and inhibited the proliferation of breast cancer cells in a p53-independent manner. Importantly, these anti-proliferative and pro-apoptotic effects were also observed in vivo in xenografted human breast tumors.
Eugenol exhibits anti-breast cancer properties both in vitro and in vivo, indicating that it could be used to consolidate the adjuvant treatment of breast cancer through targeting the E2F1/survivin pathway, especially for the less responsive triple-negative subtype of the disease.
BMC Cancer 12/2013; 13(1):600. · 3.32 Impact Factor
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[Show abstract][Hide abstract] ABSTRACT: p16INK4a is a tumor suppressor protein involved in several stress-related cellular responses, including apoptosis. Recent lines of evidence indicate that p16INK4a is also a modulator of gene expression. However, the molecular mechanisms underlying this novel function are still obscure. Here, we present clear evidence that p16INK4a modulates the levels of various microRNAs, with marked positive effect on miR-141 and miR-146b-5p. This effect is mediated through the formation of p16/CDK4/Sp1 heterocomplex, which binds to Sp1 consensus-binding motifs present in the promoters of miR-141 and miR-146b-5p, and enables their transcription. In addition, we have shown that p16INK4a interacts with Sp1 through the fourth ankyrin repeat, which is crucial for Sp1 binding to the miR-141 and miR-146b-5p promoters and their transcriptional activation. The physiologic importance of this association was revealed by the inability of cancer-related p16INK4a mutants to interact with Sp1. Moreover, we have shown p16/CDK4/Sp1-dependent up-regulation of miR-141 and miR-146b-5p following UV-induced DNA damage, and the role of these 2 microRNAs in mediating p16-related induction of apoptosis in response to this genotoxic stress. Together, these results indicate that p16INK4a associates with CDK4 not only to inhibit the cell cycle but also to enable the transcription of 2 important onco-microRNAs, which act as downstream effectors.
Journal of Biological Chemistry 10/2013; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is evidence that normal breast stromal fibroblasts (NBFs) suppress tumour growth while cancer-associated fibroblasts (CAFs) promote tumourigenesis through functional interactions with tumour cells. Little is known about the biology and the carcinogenic potential of stromal fibroblasts present in histologically normal surgical margins (TCFs). Therefore, we first undertook gene expression analysis on five CAF/TCF pairs from breast cancer patients and three NBFs (derived from mammoplasties). This comparative analysis revealed variation in gene expression between these three categories of cells, with a TCF-specific gene expression profile. This variability was higher in TCFs than in their paired CAFs and also NBFs. Cytokine arrays show that TCFs have a specific secretory cytokine profile. In addition, stromal fibroblasts from surgical margins expressed high levels of α-SMA and SDF-1and exhibited higher migratory/invasiveness abilities. Indirect co-culturing showed that TCF cells enhance the proliferation of noncancerous mammary epithelial cells, and the epithelial to mesenchymal transition of breast cancer cells. Moreover, TCF and CAF cells increased the level of PCNA, MMP-2 and the phosphorylated/activated form of Akt in normal breast luminal fibroblasts in a paracrine manner. Furthermore, TCFs were able to promote the formation and growth of humanized orthotopic breast tumours in nude mice. Interestingly, these TCF phenotypes and the extent of their effects were intermediate between NBFs and CAFs. Together, these results indicate that stromal fibroblasts located in noncancerous tissues exhibit a tumour-promoting phenotype, indicating that their presence post-surgery may play important roles in cancer recurrence.
The Journal of Pathology 09/2013; · 7.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Carcinomas initiate and progress due to genetic and epigenetic alterations in epithelial cells. However, recently, these alterations have also been reported in stromal fibroblasts. The gain-of-function mutations in the PI3K p110 catalytic subunit (PIK3CA) have been identified in many cancers with a current global incidence of 26% (18-40%) in breast carcinomas. We analyzed the mutational frequency of PIK3CA of three hotspots (exons 1, 9, and 20) in 81 primary invasive breast cancers (BC) and 25 cultured breast cancer-associated fibroblast (CAF) samples by Sanger sequencing in Arab breast cancer patients. Associations between the incidence of any PIK3CA mutation and several clinicopathologic characteristics were assessed using chi-square tests for categorical or t-test for continuous variables. Furthermore, survival curves were estimated using the Kaplan-Meier method with the log rank test to evaluate the significance of their differences. We identified a total of 21 PIK3CA missense mutations with a frequency of 25.9%. The majority of the mutations, 17 out of 21 (81%), were in exon 20 (p.His1047Arg, p.His1047Lys, p.Thr1025Ala, p.Gly1049Arg, p.Asp1056Asn) while the remainder, 4 out of 21 (19%) were in exon 9 (p.Glu545Lys). PIK3CA mutations were significantly associated with lower grade and hormone receptor positivity. Although there was a favorable trend in overall survival for patients whose tumor harbored PIK3CA mutations, the difference was not statistically significant (P = 0.10). However, we did not detect any somatic mutations in CAFs. Furthermore, we have shown a high prevalence (8.2-fold) of a silent variant (SNP, rs17849079) in the Arab breast cancer population compared with disease-free individuals.
Cancer biology & therapy 08/2013; 14(10). · 3.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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).
PLoS ONE 07/2013; 8(7):e70133. · 3.53 Impact Factor
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[Show abstract][Hide abstract] ABSTRACT: 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 p16(INK4A) 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 p16(INK4A)-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.
Neoplasia (New York, N.Y.) 06/2013; 15(6):631-40. · 5.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Breast cancer constitutes a major health problem for women worldwide. However, its incidence varies between populations and geographical locations. These variations could be diet-related, since there are several carcinogenic compounds in the modern diet, while natural products contain various anti-cancer elements. Several lines of evidence indicate that, in addition to their clear preventive effect, these compounds could also be used as therapeutic agents. In the present report we have shown that oleuropein, a pharmacologically safe natural product of olive leaf, has potent anti-breast cancer properties. Indeed, oleuropein exhibits specific cytotoxicity against breast cancer cells, with higher effect on the basal-like MDA-MB-231 cells than on the luminal MCF-7 cells. This effect is mediated through the induction of apoptosis via the mitochondrial pathway. Moreover, oleuropein inhibits cell proliferation by delaying the cell cycle at S phase and up-regulated the cyclin-dependent inhibitor p21. Furthermore, oleuropein inhibited the anti-apoptosis and pro-proliferation protein NF-κB and its main oncogenic target cyclin D1. This inhibition could explain the great effect of oleuropein on cell proliferation and cell death of breast cancer cells. Therefore, oleuropein warrants further investigations to prove its utility in preventing/treating breast cancer, especially the less-responsive basal-like type.
Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 12/2012; 53. · 2.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stromal fibroblasts, the most abundant and probably the most active cellular component of breast cancer-associated stroma, become active and promote angiogenesis through paracrine effects. However, it still unclear how these processes are regulated. Here, we have shown that down-regulation of the tumor suppressor p16(INK4A) protein enhances the migration/invasion abilities of breast stromal fibroblasts, which form dendritic network of extensions into matrigel. Furthermore, we present clear evidence that p16(INK4A) represses the expression/secretion of the proangiogenesis protein vascular endothelial growth factor A (VEGF-A). Consequently, p16(INK4A)-deficient breast stromal fibroblasts and mouse embryonic fibroblasts enhanced endothelial cell differentiation into capillary-like structures in a paracrine manner. This effect was suppressed by adding bevacizumab, a specific VEGF-A inhibitor. Additionally, p16(INK4A)-defective mouse embryonic fibroblasts enhanced angiogenesis in breast cancer xenografts in mice. Furthermore, we have shown that p16(INK4A) suppresses the Akt/mammalian target of rapamycin (mTOR) signaling pathway and its downstream effector hypoxia-inducible factor 1-alpha (HIF-1α), which transactivates VEGF-A. Consequently, Akt inactivation suppressed both the p16(INK4A)-dependent autocrine effect on fibroblast migration/invasion and the paracrine effect on angiogenesis, showing the important role of this protein kinase in mediating the various effects related to p16(INK4A) deficiency. These results indicate that p16(INK4A) is an efficient inhibitor of the migration/invasion abilities of breast stromal fibroblasts and also their paracrine proangiogenic effects, through inhibition of Akt. Therefore, pharmacologic restoration of p16(INK4A) level in stromal fibroblasts may be exploited as therapeutic strategy to help eradicate tumor cells and/or prevent their recurrence, through suppressing cell non-autonomous procarcinogenic mediators.
Neoplasia (New York, N.Y.) 12/2012; 14(12):1269-77. · 5.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: While camel urine (CU) is widely used in the Arabian Peninsula to treat various diseases, including cancer, its exact mechanism of action is still not defined. The objective of the present study is to investigate whether camel urine has anti-cancer effect on human cells in vitro.
The annexinV/PI assay was used to assess apoptosis, and immunoblotting analysis determined the effect of CU on different apoptotic and oncogenic proteins. Furthermore, flow cytometry and Elispot were utilized to investigate cytotoxicity and the effect on the cell cycle as well as the production of cytokines, respectively.
Camel urine showed cytotoxicity against various, but not all, human cancer cell lines, with only marginal effect on non-tumorigenic epithelial and normal fibroblast cells epithelial and fibroblast cells. Interestingly, 216mg/ml of lyophilized CU inhibited cell proliferation and triggered more than 80% of apoptosis in different cancer cells, including breast carcinomas and medulloblastomas. Apoptosis was induced in these cells through the intrinsic pathway via Bcl-2 decrease. Furthermore, CU down-regulated the cancer-promoting proteins survivin, β-catenin and cyclin D1 and increased the level of the cyclin-dependent kinase inhibitor p21. In addition, we have shown that CU has no cytotoxic effect against peripheral blood mononuclear cells and has strong immuno-inducer activity through inducing IFN-γ and inhibiting the Th2 cytokines IL-4, IL-6 and IL-10.
CU has specific and efficient anti-cancer and potent immune-modulator properties in vitro.
Journal of ethnopharmacology 08/2012; 143(3):819-25. · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cancer-associated fibroblasts (CAFs), the most abundant and probably the most active cellular component of breast cancer-associated stroma, promote carcinogenesis through paracrine effects; however, the molecular basis remains elusive. We have shown here that p16(INK4A) expression is reduced in 83% CAFs as compared with their normal adjacent counterparts cancer-free tissues isolated from the same patients. This decrease is mainly due to AUF1-dependent higher turnover of the CDKN2A mRNA in CAFs. Importantly, p16(INK4A) downregulation using specific siRNA activated breast fibroblasts and increased the expression/secretion levels of stromal cell-derived factor 1 (SDF-1) and matrix metalloproteinase (MMP)-2. Consequently, media conditioned with these cells stimulated the proliferation of epithelial cells. Furthermore, the migration/invasion of breast cancer cells was also enhanced in an SDF-1-dependent manner. This effect was mediated through inducing an epithelial-mesenchymal transition state. By contrast, increase in p16(INK4A) level through ectopic expression or AUF1 downregulation, reduced the secreted levels of SDF-1 and MMP-2 and suppressed the pro-carcinogenic effects of CAFs. In addition, p16(INK4A)-defective fibroblasts accelerated breast tumor xenograft formation and growth rate in mice. Importantly, tumors formed in the presence of p16(INK4A)-defective fibroblasts exhibited higher levels of active Akt, Cox-2, MMP-2 and MMP-9, showing their greater aggressiveness as compared with xenografts formed in the presence of p16(INK4A)-proficient fibroblasts. These results provide the first indication that p16(INK4A) downregulation in breast stromal fibroblasts is an important step toward their activation.Oncogene advance online publication, 2 July 2012; doi:10.1038/onc.2012.270.
[Show abstract][Hide abstract] ABSTRACT: Breast cancer has a major impact on the health of women worldwide. In the Kingdom of Saudi Arabia (KSA), breast cancer incidence is on the increase and is characterized by early onset and aggressiveness. Owing to the importance of the TP53 gene in breast carcinogenesis, we analyzed the possible link between TP53 single nucleotide polymorphisms (SNPs) and the risk of breast cancer in Saudi women by direct sequencing of the TP53 gene exon 4 from 100 breast cancer tissues. The proportion of the polymorphic forms of SNP72 in the Saudi breast cancer patients were: Arg/Arg (RR), 39%; Pro/Pro (PP), 36%; and Arg/Pro (RP), 25%. The frequencies of these forms in disease-free Saudi women were 7.59, 22.22 and 60.19%, respectively. This indicates that the RR form of the codon 72 polymorphism is a potential risk factor, whereas the RP form is a protection factor against breast cancer among Saudi women (p=0.0001). Moreover, the results have shown that the p53 R72P SNP is significantly associated with the early onset of breast cancer in the Saudi population (p=0.0138). However, the codon 47 polymorphism appears to have no role in this disease among Saudi women. These results indicate that the TP53 gene could play a major role in breast carcinogenesis and the early onset of the disease among Saudi women.
[Show abstract][Hide abstract] ABSTRACT: Survivin, an important anti-apoptotic protein, is highly expressed in most cancers, which generally arise in cells of older individuals. We have shown here accumulation of survivin and phospho-survivin in aged normal human skin fibroblasts and mice organs. This age-related accumulation of survivin was due to protein stabilization through association with the molecular chaperone Hsp90 protein, which was also up-regulated during aging. Interestingly, Hsp90 binds preferentially to phospho-survivin, which explains its higher stability. In addition, we provide clear evidence that aged cells exhibit apoptosis resistance when challenged with UV light, cisplatin, γ-rays or H(2)O(2) as compared to their younger counterparts. In response to γ-rays and H(2)O(2), the levels of Bcl-2 and both forms of survivin were up-regulated in old cells, but not in their corresponding young ones. This repression of survivin and phospho-survivin in young cells is p53 dependent. Importantly, survivin inhibition/down-regulation with flavopiridol or specific shRNAs increased the apoptotic response of old fibroblasts to various genotoxic agents, and restored the pro-apoptotic Bax/Bcl2 ratio and the increase in the levels of cleaved caspase-3 and PARP in old cells. These results show the role of survivin in the age-dependent resistance of human fibroblasts, and provide new insights into the molecular mechanisms that underlie the complex relationship between aging, apoptosis, and cancer.
[Show abstract][Hide abstract] ABSTRACT: The cyclin-D/CDK4,6/p16(INK4a)/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.
We have shown here that the tumor suppressor p16(INK4a) protein positively controls the expression of cyclin D1 and E2F1 in both human and mouse cells. p16(INK4a) stabilizes the mRNAs of the corresponding genes through negative regulation of the mRNA decay-promoting AUF1 protein. Immunoprecipitation of AUF1-associated RNAs followed by RT-PCR indicated that endogenous AUF1 binds to the cyclin D1 and E2F1 mRNAs. Furthermore, AUF1 down-regulation increased the expression levels of these genes, while concurrent silencing of AUF1 and p16(INK4a), using specific siRNAs, restored normal expression of both cyclinD1 and E2F1. Besides, we have shown the presence of functional AU-rich elements in the E2F1 3'UTR, which contributed to p16/AUF1-mediated regulation of E2F1 post-transcriptional events in vivo. Importantly, genome-wide gene expression microarray analysis revealed the presence of a large number of genes differentially expressed in a p16(INK4a) -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.
These findings show that the cyclin-dependent kinase inhibitor p16( INK4a) is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1.
PLoS ONE 07/2011; 6(7):e21111. · 3.53 Impact Factor