Wakkas Fadhil

Embryonic NANOG Activity Defines Colorectal Cancer Stem Cells and Modulates through AP1‐ and TCF‐dependent Mechanisms

Embryonic NANOG (NANOG1) is considered as an important regulator of pluripotency while NANOGP8 (NANOG‐pseudogene) plays a role in tumorigenesis. Herein, we show NANOG is expressed from both NANOG1 and NANOGP8 in human colorectal cancers (CRC). Enforced NANOG1‐expression increases clonogenic potential and tumor formation in xenograft models, although it is expressed only in a small subpopulation of tumor cells and is colocalized with endogenous nuclear β‐cateninHigh. Moreover, single NANOG1‐CRCs form spherical aggregates, similar to the embryoid body of embryonic stem cells (ESCs), and express higher levels of stem‐like Wnt‐associated target genes. Furthermore, we show that NANOG1‐expression is positively regulated by c‐JUN and β‐catenin/TCF4. Ectopic expression of c‐Jun in murine ApcMin/+‐ESCs results in the development of larger xenograft tumors with higher cell density compared to controls. Chromatin immunoprecipitation assays demonstrate that c‐JUN binds to the NANOG1‐promoter via the octamer M1 DNA element. Collectively, our data suggest that β‐Catenin/TCF4 and c‐JUN together drive a subpopulation of CRC tumor cells that adopt a stem‐like phenotype via the NANOG1‐promoter. STEM Cells2012;30:2076–2087

Concomitant mutations and splice variants in KRAS and BRAF demonstrate complex perturbation of the Ras/Raf signalling pathway in advanced colorectal cancer

Background and aims: KRAS and BRAF mutations occur in colorectal cancers (CRCs) and are considered mutually exclusive methods of activating the RAS/RAF/MEK/ERK pathway. This pathway is a therapeutic target and KRAS mutation may predict tumour responsiveness. The purpose of this study was to investigate the relationship between KRAS and BRAF mutations in 24 CRC cell lines and 29 advanced CRCs. Methods: KRAS and BRAF mutations were detected using high resolution melting and sequencing. Expression of mutations was confirmed by reverse transcription- PCR (RT-PCR) and sequencing. CpG island methylator phenotype (CIMP) was tested by methylation-specific PCR. Results: KRAS or BRAF mutation occurred in 79% of cell lines and 59% of CRCs. In the cell lines, KRAS mutations occurred in 54% of cases (with 62% in codons 12/13 and 38% in other codons). Four cell lines had a homozygous mutation. Only heterozygous BRAF mutations were detected in 29% cell lines. The V600E mutation occurred most commonly and was associated with CIMP+ status (p = 0.005). Mutations at codons 529 and 581 were also found and, in one case, BRAF and KRAS mutation co-occurred. Unexpectedly, BRAF splice variants (with a predicted kinase-dead protein) were found in 5/24 (21%) cell lines. In advanced CRCs, KRAS mutations occurred in 48% of cases (64% codons 12/13, 36% other codons) and BRAF mutations in 10% (66% V600E, 33% exon 11). A compound KRAS/BRAF mutation was not seen. Conclusions: Disrupted Ras/Raf signalling is common in CRC. Homozygous KRAS mutations and concomitant KRAS/BRAF mutations may be indicative of a gene dosage effect. The significance of BRAF splice variants is uncertain but may represent another layer of complexity. Finally, if KRAS mutation is to be used for predictive testing, then the whole gene may need to be screened as mutations occur outside codons 12/13.

Comparative analysis of pyrosequencing and QMC-PCR in conjunction with high resolution melting for KRAS/BRAF mutation detection

Mutation detection is important in cancer management. Several methods are available of which high resolution melting (HRM) analysis and pyrosequencing are the most versatile. We undertook a comparative analysis of these techniques. The methods are:

Quick-multiplex-consensus (QMC)-PCR followed by high-resolution melting: a simple and robust method for mutation detection in formalin-fixed paraffin-embedded tissue

Background Mutation detection in tumours will become increasingly important in pathological diagnosis as ‘predictive’ mutations are identified. A cheap and reliable test that works on formalin-fixed paraffin-embedded (FFPE) tissue is required. Methods The quick-multiplex-consensus (QMC)-PCR protocol was developed to be used with high-resolution melting (HRM) analysis. The assay was compared with Sanger sequencing. Robustness of the assay was tested in DNA from FFPE tissue. Results QMC-PCR with HRM could detect a minimum of 2.5% of mutant alleles (compared with 20% detectable for Sanger sequencing). Ten mutation hotspots in KRAS, BRAF, PIK3CA and CDC4 were screened in 29 cell lines with 100% sensitivity and specificity. Forty-three FFPE colorectal tumours were sequenced for hotspots in KRAS and PIK3CA and then screened by QMC-PCR. There was 100% sensitivity, although, of 21 mutations detected by QMC-PCR, 16 were confirmed by sequencing (71% specificity, positive predictive value 76%). All 43 samples were then screened for mutations in all 10 hotspots. Of 430 tests, 43 (10%) showed aberrant melting and 36 were confirmed mutant (positive predictive value 84%). As our technique is more sensitive than direct sequencing, the remaining seven tests are probably sequencing false-negatives. Precision tests showed that there was little intra-assay and interassay variation. Conclusions QMC-PCR with HRM is a simple, robust and inexpensive technique which had greater sensitivity than Sanger sequencing. It allows multiple mutation hotspots to be rapidly screened and is thus highly suited to mutation detection in DNA derived from FFPE tissues.

Cten signals through integrin-linked kinase (ILK) and may promote metastasis in colorectal cancer

CTEN/TNS4 is an oncogene in colorectal cancer (CRC), which can induce cell motility although its mechanistic basis of activity and the clinical implications of Cten expression are unknown. As Cten is in complex with integrins at focal adhesions, we hypothesised that it may interact with integrin-linked kinase (ILK). Through forced expression and knockdown of Cten in HCT116 and SW620 (respectively, showing low and high Cten expression), we showed that Cten could regulate ILK. However, inhibition of ILK after forced expression of Cten abrogated the motility-inducing effects of Cten, thereby demonstrating that the Cten–ILK interaction was functionally relevant. Combined knockdown of Cten and ILK had no additive effects on cell motility compared with knockdown of each individually. In order to investigate the clinical implications of Cten expression, a series of 462 CRCs were evaluated by immunohistochemistry. High expression of Cten was associated with advanced Dukes’ stage (P<0.001), poor prognosis (P<0.001) and distant metastasis (P=0.008). The role of Cten in metastasis was tested by (a) intrasplenic injection of CRC cells stably transfected with a Cten expression vector into nude mice and (b) testing a series of primary human CRCs and their metastases by immunohistochemistry. Compared with controls, mice injected with cells expressing Cten developed larger tumours in the spleen (P<0.05) and liver (P<0.05). In the human cases, compared with primary tumours, the metastatic deposits had a significantly higher frequency of nuclear localisation of Cten (P=0.002). We conclude that Cten expression is of prognostic significance in CRC, and we delineate a Cten–ILK pathway controlling cell motility and possibly promoting metastasis.

The utility of diagnostic biopsy specimens for predictive molecular testing in colorectal cancer

Fadhil W, Ibrahem S, Seth R, AbuAli G, Ragunath K, Kaye P & Ilyas M 
(2012) Histopathology
The utility of diagnostic biopsy specimens for predictive molecular testing in colorectal cancer

Immunostaining for mismatch repair (MMR) protein expression in colorectal cancer is better and easier to interpret when performed on diagnostic biopsies.

Sir: Colorectal cancer (CRC) is a genetically heterogeneous disease and tumours can be classified into those showing chromosomal instability (CIN) and those showing microsatellite instability (MSI). The distinction is important because, as well as genetic implications and prognostic differences, evidence suggests that MSI tumours are relatively resistant to 5-fluorouracilbased therapies (which underpin chemotherapy in CRC) but, conversely, they may be more sensitive to Topoisomerase II inhibitors (such as irinotecan). MSI in tumours can be detected by polymerase chain reaction (PCR) on DNA extracted from tumour samples or it can be inferred from immunohistochemistry (IHC). The latter is based on the proven rationale that MSI reflects defective mismatch repair (MMR) function which can be caused by loss of expression of any one of the four main MMR proteins (MLH1, PMS2, MSH2 and MSH6). IHC for the MMR proteins is usually undertaken on a single representative tumour block from the surgical resection specimen, and diagnosis is

STAT3 paradoxically stimulates β-catenin expression but inhibits β-catenin function

Wnt signalling and the signal transducer and activator of transcription 3 (STAT3) are oncogenic signalling pathways which are deregulated in colorectal cancer (CRC). Here we investigated the interaction of these two pathways. Firstly, we investigated biochemical interaction by inhibiting STAT3 and β‐catenin (through gene knock‐down and dominant‐negative TCF4 expression) in nine CRC cell lines. β‐catenin inhibition did not affect STAT3 levels, whereas STAT3 knock‐down resulted in reduced β‐catenin mRNA and protein levels. The reduction in β‐catenin protein was not prevented by proteasome inhibition, and IL6‐induced STAT3 activation resulted in increased β‐catenin mRNA. This suggests that STAT3 positively regulates β‐catenin (at a transcriptional level) and evaluation of 44 CRCs by immunostaining supported this by showing an association between nuclear STAT3 expression and nuclear β‐catenin (P = 0.022). We tested the functional interaction between STAT3 and Wnt signalling by knocking down STAT3 and β‐catenin individually and in combination. Knock‐down of β‐catenin and STAT3 individually inhibited cell proliferation (P < 0. 001 for each) through G1 arrest. However, simultaneous knock‐down of STAT3 and β‐catenin had a significantly weaker effect than knock‐down of β‐catenin alone (P < 0.01). Knock‐down of STAT3 and β‐catenin, individually and together, inhibited cell motility (P < 0.001) without evidence of interaction. We conclude that STAT3 regulates β‐catenin but β‐catenin does not regulate STAT3. The STAT3/β‐catenin interaction is complex but may reduce the proliferative activity of β‐catenin possibly by taking β‐catenin protein beyond the optimal level. This may indicate biological differences in tumours where both STAT3 and β‐catenin are activated compared to those where only one is activated.

N_LyST: a simple and rapid screening test for Lynch syndrome

Aims We sought to use PCR followed by high-resolution melting analysis to develop a single closed-tube screening panel to screen for Lynch syndrome. This comprises tests for microsatellite instability (MSI), MLH1 methylation promoter and BRAF mutation. Methods For MSI testing, five mononucleotide markers (BAT25, BAT26, BCAT25, MYB, EWSR1) were developed. In addition, primers were designed to interrogate Region C of the MLH1 promoter for methylation (using bisulphite-modified DNA) and to test for mutations in codon 600 of BRAF. Two separate cohorts from Nottingham (n=99, 46 with MSI, 53 being microsatellite stable (MSS)) and Edinburgh (n=88, 45 MSI, 43 MSS) were tested. Results All the cases (n=187) were blind tested for MSI and all were correctly characterised by our panel. The MLH1 promoter and BRAF were tested only in the Nottingham cohort. Successful blinded analysis was performed on the MLH1 promoter in 97 cases. All MSS cases showed a pattern of non-methylation while 41/44 cases with MSI showed full methylation. The three cases with MSI and a non-methylated pattern had aberrations in MSH2 and MSH6 expression. BRAF mutation was detected in 61% of MSI cases and 11% of MSS cases. Finally, 12 cases were blind screened by using the whole panel as a single test. Of these, five were identified as MSS, four as MSI/non-LS and three as MSI/possible LS. These results were concordant with the previous data. Conclusion We describe the Nottingham Lynch Syndrome Test (N_LyST). This is a quick, simple and cheap method for screening for Lynch syndrome.

DNA content analysis of colorectal cancer defines a distinct ‘microsatellite and chromosome stable’ group but does not predict response to radiotherapy

Colorectal cancers (CRC) are thought to have genetic instability in the form of either microsatellite instability (MSI) or chromosomal instability (CIN). Recently, tumours have been described without either MSI or CIN, that is, microsatellite and chromosome stable (MACS) CRCs. We investigated the (i) frequency of the MACS‐CRCs and (ii) whether this genotype predicted responsiveness to neoadjuvant chemoradiotherapy. To examine the frequency of MACS‐CRCs, DNA content (ploidy) was examined in 89 sporadic microsatellite‐stable CRCs using flow cytometry. The tumours were also screened for mutations in KRAS/BRAF/TP53/PIK3CA by QMC‐PCR. To examine the value of tumour ploidy in predicting response to chemoradiotherapy, DNA content was tested in a separate group of 62 rectal cancers treated with neoadjuvant chemoradiotherapy. Fifty‐one of 89 CRCs (57%) were aneuploid and 38 (43%) were diploid. There was no significant association between mutations in TP53/KRAS/BRAF/PIK3CA and ploidy. Testing of association between mutations revealed only mutual exclusivity of KRAS/BRAF mutation (P < 0.001). Of the 62 rectal cancers treated with neoadjuvant chemoradiotherapy, 22 had responded (Mandard tumour regression grade 1/2) and 40 failed to respond (Grade 3–5). Twenty‐five of 62 (40%) tumours were diploid, but there was no association between ploidy and response to therapy. We conclude that MACS‐CRCs form a significant proportion of microsatellite‐stable CRCs with a mutation profile overlapping that of CRCs with CIN. A diploid genotype does not, however, predict the responsiveness to radiotherapy.