Pancreatic cancers accumulate multiple genetic alterations, including frequent mutations in the KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), TP53 (encoding the p53 protein), CDKN2A (also known as p16 or p16INK4a), and SMAD4 (SMAD family member 4, also known as DPC4; deleted in pancreatic carcinoma locus 4) genes 92–94
. A summary of these four mutations in PA cell lines is presented in . Information on the genotype of these cell lines provide a background for understanding how alterations in these pathways contribute to the growth characteristics, tumorigenicity, and chemosensitivity. The relationship of genotype to phenotype is still unclear, as there are few studies available that directly assess the effect of these mutations on cell behavior. There is some evidence that the mutational status of KRAS, TP53, CDKN2A/p16, and SMAD4/DPC4 do not correlate with either the grade of differentiation 95
or the biological behavior 96
of pancreatic cancer cell lines. However, one group found that in vivo
metastatic behavior was associated with p53 status, suggesting that genotype and phenotype may be related 88
Genotype. The four most common mutations in pancreas cancer.
KRAS mutations are very common in pancreatic cancer, occurring in almost all primary tumors, and are present early in the progression of the disease 97–99
. The RAS family members (H-, K- and N-RAS) are guanine nucleotide binding proteins that transmit signals from cell surface receptors by cycling from an inactive GDP-bound state to an active GTP-bound state. Mutations in codons 12, 13, or 61 inhibit the GTPase activity of RAS, leading to oncogenic RAS protein that is constitutively activated in its GTP-bound state, inducing multiple signaling pathways 100
. Of clinical significance are the findings that activating mutations of KRAS activate the Raf/mitogen-activated protein kinase pathway and the Akt/protein kinase B pathway, resulting in the up-regulation of COX-2 transcription and stabilization of its mRNA, respectively63
. Loukopoulos et al.
directly measured the four most common mutations using sequence analysis in ten pancreatic cancer cell lines 88
. Mutations were found in the second base of codon 12 of KRAS in all cell lines but two (Hs 766T, BxPC-3) 88
. In a similar study by Berrozpe and co-workers, KRAS codon 12 mutations were found in 14 of 17 pancreatic cancer cell lines analyzed while Hs 766T, BxPC-3, and SW979 were determined to be wild-type 101
. In these and other studies which looked exclusively at exon 1 of KRAS, no mutations were found for Hs 766T. In a subsequent assay assessing the activation state of RAS by measuring the percentage of RAS bound to GTP, Hs 766T was found to contain a high level of activated RAS, similar to cell lines containing a codon 12 mutation. Sequencing of KRAS exon 2 revealed an activating mutation in codon 61 of Hs 766T 102
. There is a consensus that BxPC-3 cells contain wild-type RAS and are not RAS-activated. Consequently, although BxPC-3 is one of the most commonly used PA cell lines, it is probably not representative of the majority of pancreatic cancers.
Inactivation of the CDKN2A/p16 tumor suppressor gene is thought to be an early event in the progression of pancreas cancers, since CDKN2A/p16 inactivation can be found in up to 40% of precursor PanIN (Pancreatic Intraepithelial Neoplasia) lesions 103, 104
. The p16 pathway is disrupted, either by mutation, homozygous deletion, or promoter methylation, in up to 98% of all pancreatic carcinomas 105
. In a recent examination of 25 primary ductal adenocarcinomas, p16 was inactivated or mutated in 80% of tumors 106
. The most common cause of p16 inactivation was aberrant promoter methylation, seen in 52% of cases. Sequence mutations (16%) and homozygous deletions (12%) were also found. Correspondingly, p16 is also inactivated in many pancreas cancer cell lines. Using PCR and direct sequencing of exons 1 and 2, Loukopoulos found alterations of CDKN2A/p16 in 7 of 10 cell lines 88
. Capan-1 and PANC-1 contained homozygous deletions, while HPAF-II had an in-frame deletion and HPAC had a mutation in exon 2. In each case, sequence analysis detected only the mutated allele, indicating a loss of the normal allele which is important for loss of tumor suppressor function. CFPAC-1 contained wild-type sequence but did not express protein, as shown by western blotting. This is in agreement with previous reports that the CDKN2A/p16 promoter is methylated in CFPAC-1 cells 107
. Similarly, BxPC-3 showed a wild-type sequence but undetectable protein product. This may be explained by the presence of a homozygous deletion in exons 2–3 108–110
. AsPC-1, Capan-2 and Hs 766T were reported to be wild-type for the CDKN2A/p16 sequence. However, AsPC-1 has also been shown in other reports to have either a homozygous deletion of CDKN2A/p16 exons 2–3110
and/or a frameshift mutation 107–109
. Capan-2 does express p16 protein, however this protein was shown by other groups to be inactivated by an insertion in codons 11 and 12 108, 109
. There is also disagreement on the status of Hs 766T, which was shown to be wild-type for CDKN2A/p16 but has also been found to contain possible mutations 108, 109
. Considering these discrepancies, it is possible that all of these cell lines are lacking functional CDKN2A/p16. Additionally, taking into account epigenetic changes such as methylation, CDKN2A/p16 deficiency may be the most common occurrence in the development of pancreas cancer.
Mutations in the TP53 tumor suppressor gene are common in many types of human tumors, including more than 50% of pancreatic adenocarcinomas, where they occur late in the tumorigenesis process 94
. Berrozpe et al. 101
reported TP53 mutations in 26% of primary pancreas cancers and metastases. Interestingly, mutations were much more common in cell lines, with 15 of 17 pancreatic cancer cell lines showing mutations. Moore found TP53 mutations in 95% of the cell lines tested 107
. In the Loukopoulos study, TP53 mutations were missense in eight of ten cell lines, with Capan-2 and HPAC being wild-type 88
. As seen with CDKN2A/p16, only the mutant p53 allele was detected, indicating loss of the wild-type allele. Capan-2 has been reported by several groups to be wild-type for TP53, but it should be noted that Berrozpe found a 200-bp deletion 101
. In support of Capan-2 possessing wild-type TP53 is a study showing that radiation was able to induce elevated TP53 and p21WAF1/CIP1 protein expression in Capan-2 cells, suggesting the presence of a functional TP53 response. This response to radiation was not seen in PANC-1 or MIA PaCa-2 cells, which contain TP53 mutations 111
. There is also some discrepancy on the TP53 status of Hs 766T, with some groups finding mutations while others report wild-type sequence. In one case, the presence of a mutation was detected between codons 225–282, but the actual mutation was not sequenced 101
. Overall, mutations in TP53 were very common in PA cell lines. TP53 and CDKN2A/p16 both play significant roles in G1/S cell cycle checkpoint control and maintenance of genome integrity after DNA damage. The high frequency of loss of CDKN2A/p16 and TP53 underscores the importance of abrogation of the G1/S cell cycle checkpoint in the progression of pancreatic cancer.
SMAD4/DPC4, a member of the transforming growth factor β family and also a tumor suppressor, is inactivated in approximately 48 – 55% of invasive pancreatic adenocarcinomas 112, 113
. Accordingly, SMAD4/DPC4 inactivation has been found at a similar rate in PA cell lines. BxPC-3, CFPAC-1, and Hs 766T have all been shown to lack SMAD4/DPC4 protein due to homozygous deletions 88, 110, 112, 114, 115
. Capan-1 cells possess a point mutation in SMAD4/DPC4 that result in loss of expression 88, 113
. However, no SMAD4/DPC4 alterations have been found in Capan-2, MIA PaCa-2, PANC-1, or SU.86.86 88, 107, 110
. Divergent results have been seen for AsPC-1, with some groups finding wild-type sequence 88, 107, 114
while others have reported a non-conservative point mutation 113
or homozygous deletion 110
. In a comprehensive analysis by Moore, using direct sequencing as well as methylation-specific PCR to test for the four mutations in 22 pancreatic cancer cell lines, inactivation of SMAD4/DPC4 was always found along with alterations in the three other genes 107
. This supports data from a study on the molecular pathogenesis of pancreas adenocarcinoma which shows that loss of SMAD4/DPC4 occurs late in the progression towards invasive cancers 97
To summarize, there is a consensus that KRAS is activated in 10 of 11 cell lines, with BxPC-3 being the wild-type exception. SMAD4/DPC4 is clearly inactivated in 4 of the 11 cell lines. AsPC-1 was the only cell line with divergent results for SMAD4/DPC4. The status of the tumor suppressor genes TP53 and CDKN2A/p16 are more inconsistent. The three cell lines AsPC-1, Capan-2, and Hs 766T showed variable alterations in these genes. It is possible that these cells have acquired additional alterations during routine culturing. It is also been suggested that heterogeneous populations in the original tumor could provide a source of different genetic variants 110
. With this in mind, researchers should be aware of the potential for discrepancies in the mutational status of cell lines currently being used in each individual laboratory versus that reported in the literature.