Loss of p 15 ( INK 4 b ) Expression in colorectal cancer is Linked to Ethnic Origin

Colorectal cancers remain to be a common cause of cancer-related death. Early-onset cases as well as those of various ethnic origins have aggressive clinical features, the basis of which requires further exploration. The aim of this work was to examine the expression patterns of p15INK4b and SMAD4 in colorectal carcinoma of different ethnic origins. Fifty-five sporadic colorectal carcinoma of Egyptian origin, 25 of which were early onset, and 54 cancers of Finnish origin were immunohistochemically stained with antibodies against p15INK4b and SMAD4 proteins. Data were compared to the methylation status of the p15INK4b gene promotor. p15INK4b was totally lost or deficient (lost in ≥ 50% of tumor cell) in 47/55 (85%) tumors of Egyptian origin as compared to 6/50 (12%) tumors of Finnish origin (p=7e-15). In the Egyptian cases with p15INK4b loss and available p15INK4b promotor methylation status, 89% of cases which lost p15INK4b expression were associated with p15INK4b gene promotor hypermethylation. SMAD4 was lost or deficient in 25/54 (46%) tumors of Egyptian origin and 28/48 (58%) tumors of Finnish origin. 22/54 (41%) Egyptian tumors showed combined loss/deficiency of both p15INK4b and SMAD4, while p15INK4b was selectively lost/deficient with positive SMAD4 expression in 24/54 (44%) tumors. Loss of p15INK4b was associated with older age at presentation (>50 years) in the Egyptian tumors (p=0.04). These data show for the first time that p15INK4b loss of expression marks a subset of colorectal cancers and ethnic origin may play a role in this selection. In a substantial number of cases, the loss was independent of SMAD4 but rather associated with p15INK4b gene promotor hypermethylation and old age which could be related to different environmental exposures.


Introduction
p15 INK4b (CDKN2B) is a tumor suppressor gene located, together with two other related genes ARF and p16 INK4a (CDKN2A) within a 35 kb stretch on chromosome 9p21.The INK4a/ARF/INK4b locus is deleted in a variety of tumors including melanoma, pancreatic adenocarcinoma, glioblastoma, certain leukemias, non-small cell lung cancer, and bladder carcinoma (Kim Sharpless, 2006;Nakamura et al., 2011).The binding of the INK4 proteins to the cyclin dependent kinases CDK4 and CDK6 abrogates the binding of these kinases to D-type cyclins, thus inhibiting CDK4/6-mediated phosphorylation of retinoblastoma (pRb) protein and its family members.Hypophosphorylated Rb-family proteins potently bind E2F transcription factors to exert a G1 cell-cycle arrest (Kim and Sharpless, 2006).Deregulation of pRb pathway is common in human cancers, but direct alterations of the pRb protein or its closely associated molecules are rarely observed in colorectal cancer apart from the infrequent

Ethnic Origin
Wael Mohamed Abdel-Rahman 1 *, Taina Tuulikki Nieminen 2 , Soheir Shoman 3 , Saad Eissa 3 , Paivi Peltomaki 2 loss of p16 INK4a expression associated with promoter methylation (Cheng et al., 2006;Joensuu et al., 2008).Colorectal cancers, however, undertake a more drastic upstream manoeuvre to deregulate the pRb-mediated cell cycle control through eliminating the growth inhibitory SMAD proteins, such as SMAD3 and SMAD2 which form a heterodimeric complex with SMAD4.The SMAD3/SMAD4 (or SMAD2/SMAD4) dimer then migrates to the nucleus, where it teams up with MIZ-l to induce expression of the p15 INK4b also relieves p15 INK4b from the MYC-induced repression by down regulating the MYC gene expression (Warner et al., 1999;Seoane et al., 2001).More recently, SMAD/ STAT3 signaling pathway was shown to play a role in epithelial-to-mesenchymal transition during colorectal carcinogenesis (Zhu et al., 2013) and single nucleotide polymorphism (SNIP) variations within one of the SMADs (SMAD7 colorectal cancers (Nassiri et al., 2013).
We r e c e n t l y f o u n d r e m a r k a b l e f r e q u e n t hypermethylation of the p15 INK4b gene promoter in colorectal carcinoma of Egyptian origin (Nieminen et al., 2012).Conversely, hypermethylation of p15 INK4b was reported mainly in glial tumors, leukemias, myelodysplasia (Esteller et al., 2001), hepatocellular carcinoma (Zekri Ael et al., 2013) and, more recently, in peripheral blood of leukemia patients (Bodoor et al., 2014), but was not a (Cheng et al., 2006;Nieminen et al., 2012).Interestingly, p15 INK4b methylation was detected in 68% of colorectal cancer specimens of Chinese origin (Xu et al., 2004) and in 26% colorectal cancers from Japan (Ishiguro et al., 2006).Egyptian colorectal carcinoma is surprisingly young age disease with high proportion of rectal and advanced stage cancers.The p15 INK4b methylation data could explain these clinical differences and link them to exposure to environmental toxins, since gene methylation may be related to different environmental exposures.
Here, we characterized sporadic colorectal cancers of Egyptian and Finnish origins for expression of p15 INK4b and its closely related upstream protein SMAD4 by immunohistochemistry staining and correlated the results with the clinico-pathological and gene methylation data available on this series.

Patients and samples
This study was performed on a consecutive series of 55 Egyptian carcinoma and 54 cancers of Finnish origin (Table 1).These cases were selected from a bigger series (Nieminen et al., 2012) according to the availability of immunohistochemistry tissue specimens as explained previously (Nieminen et al., 2012).DNA was extracted from paraffin-embedded specimens by standard techniques.Mutation screening, microsatellite instability (MSI), methylation analyses and p53 immunohistochemistry were performed in previous studies (Joensuu et al., 2008;Nieminen et al., 2012).The work was conducted at Helsinki under the approval of the institutional review boards of the Helsinki University Central Hospital.

Immunohistochemistry
Four-micrometer sections from formalin-fixed to distilled water then sections were subject to heat-induced target retrieval in 1 mM ethylenediaminetetraacetic acid (EDTA) buffer pH 8.0 for 5 minutes at 750 W followed by 5 minutes at 450 W in a microwave oven.After cooling, the slides were washed in Tris-buffered saline/Tween 20 ph 7.2 and subsequent staining steps were performed manually with the Dako EnVision+ System, Peroxidase (DAB), according to manufacturer's instructions (Dako, Glostrup, Denmark).Additionally, after blocking endogenous peroxidase activity, and prior to incubation with the primary antibody, the sections were incubated with 10% normal (non-immune) goat serum (Dako, Glostrup, Denmark) for 30 minutes.The primary antibodies were: anti p15 INK4b mouse monoclonal antibody clone15P06 used at dilution 1:25 and anti SMAD4 rabbit monoclonal antibody clone EP618Y at dilution 1:200.Both antibodies were purchased from Abcam (Cambridge, UK).Primary antibody incubation was for 2 hours at room temprature.Paired tumor and normal mucosa were in the same section and the normal tissues were used as internal reference for evaluation of staining results.

Interpretation of staining results
Interpretation of staining results was performed by experienced histopathologist (W M A-R) SMAD4 staining was cytoplasmic in normal mucosa and neoplastic cells.Tumors showing positive staining in more than 50% of neoplastic cells were considered positive, tumors showing staining in less than 50% of neoplastic cells were less than 2% of neoplastic were considered negative.The cut-off level of 50% was according to Sakellariou et al (Sakellariou et al., 2008).p15 INK4b expression was nuclear and a scoring scale similar to the one described above with a 50% cut-off level was employed according to the published literature (Oda et al., 2005;Endo et al., 2011).

Statistical analysis
Fisher's exact probability test was used to evaluate differences between groups.Analyses were performed using MS Excel and/or VassarStats Web-based statistical program http://faculty.vassar.edu/lowry/VassarStats.html.All reported p values were two-tailed and p values < 0.05

Relationship between p15 INK4b expression and pathological and molecular features
Table 2 shows p15 INK4b expression in relation to the clinico-pathological and molecular features of the Egyptian tumors.Significant correlation was found between older age at presentation (>50 years) and the loss of p15 INK4b between p15 INK4b expression and microsatellite instability

Discussion
Prompted by our finding of remarkable p15 INK4b promoter methylation in colorectal cancers of Egyptian origin (Nieminen et al., 2012), we have analysed the immunohistochemical expression of p15 INK4b and SMAD4 in colorectal cancers of Egyptian and Western origins with the purpose to exploit these markers in diagnosis and personalized medicine.The results of the present study lead us to speculate that the loss of p15 INK4b protein expression marks the development of subsets of colorectal cancers of Eastern origins.This is supported by the methylation data on colorectal cancers of Chinese (Xu et al., 2004) and Japanese origin (Ishiguro et al., 2006).the tumors examined of both Egyptian and Finnish origin consistent with the published literature (Royce et al., 2010;Ahn et al., 2011).SMAD4 is a potential upstream inducer of p15 INK4b (see introduction).Hence, some cases of p15 INK4b   to explain the remarkable loss of p15 INK4b in the Egyptian tumors since it was not associated with similar p15 INK4b loss in the Finn cancers.
The loss of p15 INK4b expression was reported in nonepithelial malignancies including leukemias, malignant peripheral nerve sheeth tumors, meningioma, and Simon et al., 2001;Endo et al., 2011).Furthermore, in expression studies of p15 INK4b in epithelial cancers showed and co-workers reported p15 INK4b loss in more than 70% of advanced gastric cancers, especially the intestinal subtype (Sakellariou et al., 2008).Consistent with our data, no correlation was observed between p15 INK4b and pathological or survival data apart from tendency to affect male gender and distal location within the stomach (Sakellariou et al., 2008).In cutaneous squamous cell carcinoma, p15 INK4b protein expression was absent in relationship between clinicopathologic variables of the patients (age, sex and tumor grade) and p15 INK4b protein expression (Moad et al., 2009).More recently, Holm et al reported loss of p15 INK4b in 82% of vulvar squamous cell   invasiveness.However, they could not establish p15 INK4b as independent prognostic markers (Holm et al., 2013).Interestingly, the loss p15 INK4b in the Egyptian series was associated with its gene promoter methylation and with old age at onset suggesting a potential causal relationship.While tumor suppressor promoter methylation is known to increase with age (Fraga et al., 2007), aging alone seems less frequent methylation in the Finnish series (Figure 1).Paun et al 2010 demonstrated that environmental toxins such as smoking were associated with gene methylation in the normal rectal mucosa and with the presence of colorectal adenomas.These methylated genes were potentially involved in early stages of adenoma formation and the authors speculated that the observed epigenetic alterations in these markers may be caused in part by the effects of smoking and/or age (Paun et al., 2010).Exposures to environmental toxicants and toxins might cause epigenetic changes (O'Hagan, 2013;Coppede et al., 2014;Senut et al., 2014) and it is clear that many different adverse environmental factors are likely to exist in the East compared to the West as discussed previously (Nieminen et al., 2012).Our data, together with the available literature (Belinsky et al., 2004;Marsit et al., 2006) suggest a link between environmental exposures, epigenetic changes and experimental models and large series of clinical samples.

Figure 1 .
Figure 1.Diagrammatic Comparison between the SMAD4 and p15 INK4b Results from the Egyptian Tumors (top) and Finnish Tumors (bottom).Black boxes expression, while the black boxes under methylation indicate methylated p15 INK4b promotor.The hatched/grey boxes indicate that data were not available for these cases.The number of cases in each category is indicated on the top

Figure 2 .
Figure 2. p15 INK4b and SMAD4 Immunohistochemistry. A, positive nuclear staining of p15 INK4b in carcinoma; B, loss of p15 INK4b staining in carcinoma; C, positive cytoplasmic staining of SMAD4 in carcinoma; D, loss of SMAD4 expression in carcinoma compared to normal mucosa (upper right corner).