Concomitant expression of p16INK4a and p14ARF in primary breast cancer and analysis of inactivation mechanisms

Abstract

The INK4a/ARF locus encodes two tumour suppressor proteins, p16INK4a and p14ARF, which act in the two main cell-cycle control pathways, p16-Rb and p14-p53 respectively. The present study examined the mRNA expression of these genes by reverse transcription-polymerase chain reaction (RT-PCR), and the inactivation mechanisms that alter these levels, in 100 primary breast carcinomas. Furthermore, the interdependence of these mechanisms was examined, since it has been reported that p14ARF is altered in most tumours in concordance with p16INK4a. The results show that promoter hypermethylation, tested by methylation-specific PCR (MSP), was the major mechanism of inactivation of these genes and was present in 31 (31%) and 50 (50%) of the tumours that showed decreased p16INK4a and p14ARF expression, respectively. Hemizygous deletion was the second cause of down-regulation. Homozygous deletion was rare and mutation was absent. In most tumours overexpressing p16INK4a or p14ARF, no detectable inactivation mechanisms were observed. Finally, the results indicate that these proteins are often co-altered in primary breast tumours and that p16INK4a and p14ARF had non-independent behaviour, since they were silenced or overexpressed concomitantly with a significant correlation (p < 0.05).

p16INK4a promoter methylation and protein expression in breast fibroadenoma and carcinoma

“…31 In conclusion, the comparative analysis of p16 expression in breast carcinoma and fibroadenoma suggest a role for p16 in breast malignancy although promoter methylation, differently from others tumors, cannot be considered a marker of carcinogenesis. Because p16 point mutations were very rarely observed 6,8 and the protein is massively present in carcinoma epithelium, we propose that in breast cancer, similarly to non-small cell lung cancer, 32 p16 loss of function occurs by sequestration of the protein in the cytoplasm rather than by mutational or transcriptional inactivation.…”

“…Even though homozygous deletions of p16 are seen in 40 -60% of breast cancer cell lines, 6,7 neither homozygous deletions nor point mutations are observed frequently in primary breast carcinomas suggesting that these alterations might have been acquired in culture. 6,8 Similar to many other genes, p16 INK4a is commonly inactivated by hypermethylation of its CpG-rich promoter region. 9 -11 This important cancer-related modification has also been detected in breast carcinoma, although the reported prevalence of p16 promoter methylation in this tumor is discordant among different studies.…”

Tumor escape in a Wnt1-dependent mouse breast cancer model is enabled by p19Arf/p53 pathway lesions but not p16Ink4a loss

“…The INK4A/ARF locus critically regulates both the p53 and Rb tumor suppressor pathways and frequently is disrupted in human breast cancers (28)(29)(30). To investigate the impact of Ink4a/Arf deficiency on mammary tumor onset and subsequent tumor escape, a null Ink4a/Arf allele that disrupts both the p16 Ink4a and p19 Arf tumor suppressors (31) was introduced into the MTB/TWNT model via the germline to generate cohorts of Ink4a/Arf +/+ , Ink4a/Arf +/-, and Ink4a/Arf -/-female mice.…”

mRNA of placental origin is readily detectable in maternal plasma

“…Recently, a number of investigators have shown that in addition to DNA, RNA is also present in the plasma of human subjects, particularly those with cancer (15)(16)(17)(18). The inherent lability of RNA has made these observations rather surprising.…”