• HIF1α;
  • CUL2;
  • hypoxia;
  • mutation;
  • cancer;
  • microsatellite instability


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Park SW, Chung NG, Hur SY, Kim HS, Yoo NJ, Lee SH. Mutational analysis of hypoxia-related genes HIF1α and CUL2 in common human cancers. APMIS 2009; 117: 880–5.

Hypoxia is a general feature of solid cancer tissues. Hypoxia upregulates hypoxia-inducible factor 1α (HIF1α) that transactivates downstream genes and contributes to cancer pathogenesis. HIF1α is upregulated not only by hypoxia but also by genetic alterations in HIF1α-related genes, including VHL. Cullin 2 (CUL2) interacts with the trimeric VHL-elongin B-elongin C complex and plays an essential role in the ubiquitinated degradation of HIF1α. The aim of this study was to explore whether HIF1α and CUL2 genes are somatically mutated, and contribute to HIF1α activation in common human cancers. For this, we have analyzed the coding region of oxygen-dependent degradation domain of HIF1α in 47 colon, 47 gastric, 47 breast, 47 lung, and 47 hepatocellular carcinomas, and 47 acute leukemias by a single-strand conformation polymorphism assay. In addition, we analyzed mononucleotide repeat sequences (A8) in CUL2 in 55 colorectal and 45 gastric carcinomas with microsatellite instability (MSI). We found one HIF1α mutation (p.Ala593Pro) in the hepatocellular carcinomas (1/47; 2.1%), but none in other cancers. We found two CUL2 frameshift mutations in colon cancers (p.Asn292MetfsX20), which were exclusively detected in high MSI cancers (4.9%; 2/41). Our data indicate that somatic mutation of HIF1α is rare in common cancers, and somatic mutation of CUL2 occurs in a fraction of colorectal cancers (colorectal cancers with high MSI). The data suggest that neither HIF1α nor CUL2 mutation may play a central role in HIF1α activation in gastric, colorectal, breast, lung and hepatocellular carcinomas, and acute leukemias.

Mammalian cells in the body frequently meet hypoxic conditions, in which oxygen concentration does not meet the demand of cells (1, 2). As an adaptive response to hypoxia, mammalian cells are able to produce proteins involved in diverse processes such as angiogenesis, survival, and metabolism (1, 2). Hypoxia-inducible factor 1 (HIF1) is a master transcriptional factor complex that activates transcription of genes which function to enhance oxygen availability and to allow metabolic adaptation to hypoxia (3). HIF1 consists of an oxygen-sensitive α subunit (HIF1α) and a constitutively expressed β subunit (HIF1β). In normoxia, HIF1α is hydroxylated at two proline residues (Pro-402 and Pro-564) by the oxygen-dependent HIF prolyl hydroxylases (PHD) and the hydroxylated HIF1α is degraded by von Hippel Lindau (VHL) E3 ubiquitin ligase (4–6). In hypoxia, HIF1α is not hydroxylated, is accumulated in cells, and activates transcription of many genes involved in cell survival, angiogenesis, proliferation and metabolism, to answer to hypoxic conditions (1, 2). Cullin 2 (CUL2) interacts with the trimeric VHL-elongin B-elongin C complex and plays an essential role in the degradation of HIF1α by ubiquitination (4, 7).

Uncontrolled proliferation of cancer cells often results in hypoxia in cancer cell masses, and indeed up to 50–60% of solid cancers contain hypoxic tissue areas (2). As a result, HIF1α is over-expressed in a majority of human cancers (8–12). However, HIF1α is also overexpressed and activated in cancers under normal oxygen conditions by genetic alterations in genes involved in the oxygen-sensing pathway. The VHL gene is mutated and functionally inactivated in patients with the VHL syndrome and in some sporadic tumors, including hemangioblastoma and renal cell carcinoma (13–15). HIF1α somatic mutation p.Pro582Ser was identified in prostate cancers, and the mutation stabilized HIF1α (16). Of note, the same sequence variation was also identified in renal and head/neck cancers as a polymorphism (17, 18). Also, inherited genetic defects in succinate dehydrogenase and fumarate hydratase that are linked to HIF1α metabolism activate HIF1α (19, 20). These reports suggest a possibility that genetic alterations in genes encoding proteins in HIF1α-related signaling might be responsible for HIF1α activation in cancers.

By analyzing a public database, we found an A8 nucleotide repeat in exon 9 of CUL2. Many tumor suppressor genes harbor frameshift mutations at the nucleotide repeats in coding sequences in cancers with microsatellite instability (MSI) (21). The MSI is characterized by length alterations in repeated DNA sequences, and fractions of colorectal cancer (CRC) and gastric cancer (GC) are categorized as MSI-positive cancers (21).

To date, the data on HIF1α gene mutation except the p.Pro582Ser are lacking in human cancers. There is no report on CUL2 mutation in the cancers with MSI, either. In this study, we analyzed somatic HIF1α mutation in five types of common solid cancers and acute leukemias. In addition, we included mutation analysis of the A8 repeat in CUL2 gene in GC and CRC with MSI, because frameshift mutations in nucleotide repeats frequently occur in the cancers with MSI. We analyzed HIF1α and CUL2 genes together, because it appeared that alterations in both genes might deregulate HIF1α metabolism.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Tissue samples and microdissection

For the mutation analysis of HIF1α, methacarn-fixed tissues of 47 CRC, 47 GC, 47 breast cancers, 47 lung cancers, 47 hepatocellular carcinomas (HCC), and fresh bone marrow aspirates of 47 acute adulthood leukemias were randomly selected for the study. The CRC originated from the cecum (n = 1), ascending colon (n = 9), transverse colon (n = 2), descending colon (n = 2), sigmoid colon (n = 13), and the rectum (n = 20). The GC consisted of 22 diffuse-type, 18 intestinal-type, and seven mixed-type gastric adenocarcinomas by Lauren’s classification, and four early and 43 advanced gastric carcinomas according to the depth of invasion. The lung carcinomas consisted of 25 adenocarcinomas and 22 squamous cell carcinomas. The breast carcinomas consisted of seven ductal carcinomas in situ and 40 invasive ductal carcinomas. The HCC consisted of five grade I, 22 grade II, and 20 grade III cancers by Edmondson’s classification. The leukemias consisted of 37 acute myelogenous leukemias and 10 acute lymphoblastic leukemias.

For the mutation analysis of CUL2, methacarn-fixed tissues of 45 GC and 55 CRC with MSI were used for this study. The cancers consisted of 32 GC with high MSI (MSI-H), 13 GC with low MSI (MSI-L), 41 CRC with MSI-H, and 14 CRC with MSI-L according to the NCI criteria (21). The GC with MSI-H consisted of 15 diffuse-type, 13 intestinal-type, and four mixed-type carcinomas by Lauren’s classification. The GC with MSI-H consisted of three early (EGC) and 29 advanced GC (AGC). The TNM stages of the GC with MSI-H were 16 stage I, 10 stage II, five stage III, and one stage IV. The CRC with MSI-H originated from the cecum (n = 6), ascending colon (n = 23), transverse colon (n = 9), and the sigmoid colon (n = 3). The TNM stages of the CRC with MSI-H were six stage I, 14 stage II, 18 stage III, and three stage IV.

Malignant cells and normal cells of the cancer tissues were selectively procured from hematoxylin and eosin-stained slides using a 30G1/2 hypodermic needle affixed to a micromanipulator by the microdissection, as described previously (22, 23). DNA extraction was performed by a modified single-step DNA extraction method by proteinase K treatment. All of the patients with the cancers were Koreans. Approval was obtained from the Catholic University of Korea College of Medicine’s institutional review board for this study.

Single strand conformation polymorphism (SSCP) analysis

Degradation of HIF1α is controlled by an oxygen-dependent degradation (ODD) domain within HIF1α (amino acids 401–603) (5, 6). We designed six primer pairs that encompass DNA sequences encoding the ODD of HIF1α (Table 1). There is an A8 repeat in exon 9 of CUL2. We used a primer pair that could amplify the repeat (Table 1). Genomic DNA each from tumor cells and corresponding normal cells was amplified with the primer pairs. For the HIF1α, 235 tumors and their 235 paired normal tissues were analyzed, and for CUL2, 100 tumors and their 100 paired normal tissues were analyzed. Each PCR reaction was performed under standard conditions in 8 μl reaction mixture. Radioisotope ([32P]dCTP) was incorporated into PCR products for detection by SSCP autoradiogram. After SSCP, mobility shifts on the SSCP were determined by visual inspection. Other procedures of PCR and SSCP analysis were performed as described previously (22, 23). Direct DNA sequencing reactions were performed in the cases with the mobility shifts on the SSCP (tumor vs normal DNA: DNA from the normal cells of the patients with the mobility shifts). Sequencing of the PCR products was carried out using a capillary automatic sequencer (ABI Prism Genetic Analyzer; Applied Biosystems, Foster City, CA, USA). To confirm the mutation data of HIF1α and CUL2, we repeated the PCR-SSCP twice.

Table 1.   Primer sequences of HIF1A and CUL2 genes
GeneSequencesSize of PCR product (bp)


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

HIF1α mutation

Genomic DNAs isolated from the CRC, GC, breast cancers, lung cancers, HCC, and acute adulthood leukemias were analyzed for the detection of somatic mutations in the four exons (exon 9–12) and their splice sites of HIF1α gene by the PCR-SSCP assay. This area encompassed the coding sequences for the ODD domain of HIF1α (amino acids 401–603). On SSCP, all of the PCR products were clearly seen. The SSCP from the cancer tissues showed aberrantly migrating bands in one HCC compared to the wild-type bands from its normal tissue. There was no aberrantly migrating band of HIF1α in the breast cancers or lung cancers or CRC or GC or acute leukemias. Direct DNA sequencing analysis of the HCC with the aberrant bands in the SSCP led to identification of a HIF1α mutation (1/47; 2.1%) (Table 2 and Fig. 1A). The HIF1α mutation was a missense mutation in exon 12 (c.1777G>C), which would result in substitution of Ala by Pro at amino acid residue 593 (p.Ala593Pro) (Fig. 1A).

Table 2.   Summary of HIF1α and CUL2 mutations
Case no.Type of cancerSex/ageViral infection (HCC) or MSI status (CRC)Edmondson’s grade (HCC) or TNM (CRC) Nucleotide change (predicted amino acid change)
  1. HCC, hepatocellular carcinoma; MSI, microsatellite instability; CRC, colorectal cancer.

HCC 44HCCM/56Hepatitis BIIHIF1α c.1777G>C (p.Ala593Pro)
Scol 9CRC (ascending)M/55MSI-HIICUL2 c.875delA (p.Asn292MetfsX20)
Scol 18CRC (ascending)M/59MSI-HIIICUL2 c.875delA (p.Asn292MetfsX20)

Figure 1.  Representative single strand conformation polymorphism (SSCP) and DNA sequencing of HIF1 α and CUL2 genes in cancers. SSCP (upper) and DNA sequencing analysis (lower) of HIF1 α (A) and CUL2 (B) genes from tumors (lane T) and normal tissues (lane N). (A) SSCP of the PCR product of HIF1 α from a tumor (T) shows aberrant bands (arrows in lane T) compared with SSCP from normal tissues (N). Direct DNA sequencing analysis shows nucleotide substitution (G to C) in tumor tissue compared with normal tissues. (B) SSCP of the PCR product of CUL2 from a tumor (T) shows aberrant bands (arrows in lane T) compared with SSCP from normal tissues (N). Direct DNA sequencing analysis shows a heterozygous deletion of a nucleotide in tumor tissue DNA compared with normal tissues.

Download figure to PowerPoint

Normal DNA of the HCC patient showed no evidence of mutations by the SSCP (Fig. 1A), indicating the mutations detected had arisen somatically. The SSCP pattern of the HCC (Fig. 1A) showed weak bands of the wild-type allele and strong aberrant bands of the mutant allele. Also, the direct DNA sequencing revealed a stronger mutation sequence peak than the wild-type sequence peak (Fig. 1A). The HCC with the mutation was an Edmondson’s grade II cancer with hepatitis B virus infection. There was no significant difference of histologic or clinicopathlogic data of the tumor between the HCC with and without HIF1α mutation (Fisher’s exact test, p > 0.05).

CUL2 mutation

Genomic DNAs isolated from normal and tumor tissues of the 55 CRC and 45 GC through microdissection were analyzed for the detection of mutation in the CUL2 gene (exon 9) by the PCR-SSCP assay. Overall, the PCR-SSCP analysis identified aberrant bands in 2 of the 100 cancers analyzed (Table 2). None of the corresponding normal samples of these two cancers showed evidence of mutations by SSCP, indicating the aberrant bands had risen somatically (Fig. 1B). Direct DNA sequencing analysis showed that the two cancers with the aberrant bands had the same CUL2 mutation in exon 9 (Fig. 1B). The mutation was a frameshift mutation (c.875delA), which would result in premature stops of the amino acid synthesis (p.Asn292MetfsX20). Both of the mutations were detected in the CRC with MSI-H (4.9%; 2/41) (Table 2), but not in those with the GC with MSI-H or GC with MSI-L or CRC with MSI-L. We carefully inspected the clinicopathologic data of the patient (demographic data, grade, stage, and metastasis), but there was no significant association of the CUL2 mutations with them.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The aim of this study was to determine whether somatic mutations of HIF1α and CUL2 are present in common cancers. Earlier studies disclosed that genetic defects of VHL, succinate dehydrogenase, and fumarate hydratase activate HIF1α and promote tumor formation (13–15, 19, 20). Similarly, we explored a possibility that HIF1α and CUL2 genes are inactivated in common human cancers by somatic mutations. In the current study, we detected only one HIF1α mutation in HCC, but not in the other cancers. With regard to CUL2, we detected two frameshift mutations in CRC with MSI-H (4.9%; 2/41). These data indicate that somatic mutations of HIF1α encoding the ODD domain and the mononucleotide repeat in CUL2 are rare in HCC, GC, CRC, breast cancer, lung cancer, and acute leukemia. Also, the data suggest that activation of HIF1α by somatic mutation of HIF1α or CUL2 is not common in these cancers.

In Caenorhabditis elegans, a loss of cul1, a homolog of cul2, causes hyperplasia in certain cell lineages (24). Nearly 70% of cancer-disposing mutations in VHL abrogate elongin binding, suggesting that binding to elongin-CUL2 complexes contributes to the ability of VHL to suppress tumor growth in vivo (4). Human papillomavirus E7 oncoprotein interacts with CUL2, which contributes to degradation of the retinoblastoma tumor suppressor (25). Together, these observations suggest that CUL2 may be a candidate tumor suppressor gene. CUL2 consists of 745 amino acids, and the frameshift mutation (p.Asn292MetfsX20) would remove most of the amino acids (about 75% of the amino acids; 554 amino acids in the C-terminus). Although CUL2 mutation in the mononucleotide repeat was not common, the frameshift mutation of CUL2 may inactivate the tumor suppressor function of CUL2 in the affected cells.

As shown in Fig. 1A, the wild-type signals were much weaker than the mutant signals in both SSCP and direct DNA sequencing of the HCC with the HIF1α mutation. Even with the use of microdissection, about 5% of the procured tumor cells were admixed with normal cells in this HCC (data not shown). Given the invariable contamination of normal cells in primary tumors, the HIF1α mutation appears to be homozygous or hemizygous. However, the functional significance of the HIF1α mutation (p.Ala593Pro) remains unknown. Oxygen-dependent hydroxylation of HIF1α occurs at both Pro-402 and Pro-564 in the ODD. These proline residues are conserved within the Leu-X-Leu-Ala-Pro motif (4–6). In addition, Leu-574 is known as a molecular determinant of Pro-564 hydroxylation (5). The p.Ala593Pro mutation is apart from these amino acids or motifs, and it appears less likely that the mutation directly inhibits prolyl hydroxylations.

Hypoxia is a fundamental physiologic stimulus for all organisms, and HIF family members are crucial mediators of the oxygen-signaling pathway. In addition to HIF1α, other HIFs, including HIF2α, HIF3α, ARNT, and ARNT-2, mediate transcription responses to oxygen deprivation (1, 2). Also, in addition to CUL2, many other molecules, including VHL, elongins B and C, and RBX1, are needed to constitute a functional E3 ubiquitin protein ligase complex (1, 2). It is evident that HIF is activated in many human cancers (8–12). Besides HIF1α and CUL2, whether other genes encoding the mediators of the oxygen-signaling pathway are genetically altered should be further studied. Also, it is possible that HIF activation is caused by alterations in protein expression of the components in the oxygen-signaling pathway. Therefore, it is imperative to analyze the expression status of these proteins in cancer tissues together with the genetic alterations


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This study was supported by a grant from the National Cancer Control R&D Program, Korea (0820080).


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  • 1
    Semenza GL. HIF-1, O(2), and the 3 PHDs: how animal cells signal hypoxia to the nucleus. Cell 2001;107:13.
  • 2
    Rankin EB, Giaccia AJ. The role of hypoxia-inducible factors in tumorigenesis. Cell Death Differ 2008;15:67885.
  • 3
    Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A 1995;92:55104.
  • 4
    Pause A, Lee S, Worrell RA, Chen DY, Burgess WH, Linehan WM, et al. The von Hippel-Lindau tumor-suppressor gene product forms a stable complex with human CUL-2, a member of the Cdc53 family of proteins. Proc Natl Acad Sci USA 1997;94:215661.
  • 5
    Kageyama Y, Koshiji M, To KK, Tian YM, Ratcliffe PJ, Huang LE. Leu-574 of human HIF-1alpha is a molecular determinant of prolyl hydroxylation. FASEB J 2004;18:102830.
  • 6
    Huang LE, Gu J, Schau M, Bunn HF. Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc Natl Acad Sci USA 1998;95:798792.
  • 7
    Lonergan KM, Iliopoulos O, Ohh M, Kamura T, Conaway RC, Conaway JW, et al. Regulation of hypoxia-inducible mRNAs by the von Hippel-Lindau tumor suppressor protein requires binding to complexes containing elongins B/C and Cul2. Mol Cell Biol 1998;18:73241.
  • 8
    Talks KL, Turley H, Gatter KC, Maxwell PH, Pugh CW, Ratcliffe PJ, et al. The expression and distribution of the hypoxia-inducible factors HIF-1alpha and HIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages. Am J Pathol 2000;157:41121.
  • 9
    Schindl M, Schoppmann SF, Samonigg H, Hausmaninger H, Kwasny W, Gnant M, et al. Overexpression of hypoxia-inducible factor 1alpha is associated with an unfavorable prognosis in lymph node-positive breast cancer. Clin Cancer Res 2002;8:18317.
  • 10
    Birner P, Schindl M, Obermair A, Plank C, Breitenecker G, Oberhuber G. Overexpression of hypoxia-inducible factor 1alpha is a marker for an unfavorable prognosis in early-stage invasive cervical cancer. Cancer Res 2000;60:46936.
  • 11
    Osada R, Horiuchi A, Kikuchi N, Yoshida J, Hayashi A, Ota M, et al. Expression of hypoxia-inducible factor 1alpha, hypoxia-inducible factor 2alpha, and von Hippel-Lindau protein in epithelial ovarian neoplasms and allelic loss of von Hippel-Lindau gene: nuclear expression of hypoxia-inducible factor 1alpha is an independent prognostic factor in ovarian carcinoma. Hum Pathol 2007;38:131020.
  • 12
    Shibaji T, Nagao M, Ikeda N, Kanehiro H, Hisanaga M, Ko S, et al. Prognostic significance of HIF-1 alpha overexpression in human pancreatic cancer. Anticancer Res 2003;23:47217.
  • 13
    Iliopoulos O, Levy AP, Jiang C, Kaelin WG Jr, Goldberg MA. Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein. Proc Natl Acad Sci U S A 1996;93:105959.
  • 14
    Gnarra JR, Glenn GM, Latif F, Anglard P, Lerman MI, Zbar B, et al. Molecular genetic studies of sporadic and familial renal cell carcinoma. Urol Clin North Am 1993;20:20716.
  • 15
    Sprenger SH, Gijtenbeek JM, Wesseling P, Sciot R, van Calenbergh F, Lammens M, et al. Characteristic chromosomal aberrations in sporadic cerebellar hemangioblastomas revealed by comparative genomic hybridization. J Neurooncol 2001;52:2417.
  • 16
    Fu XS, Choi E, Bubley GJ, Balk SP. Identification of hypoxia-inducible factor-1alpha (HIF-1alpha) polymorphism as a mutation in prostate cancer that prevents normoxia-induced degradation. Prostate 2005;63:21521.
  • 17
    Clifford SC, Astuti D, Hooper L, Maxwell PH, Ratcliffe PJ, Maher ER. The pVHL-associated SCF ubiquitin ligase complex: molecular genetic analysis of elongin B and C, Rbx1 and HIF-1alpha in renal cell carcinoma. Oncogene 2001;20:506774.
  • 18
    Tanimoto K, Yoshiga K, Eguchi H, Kaneyasu M, Ukon K, Kumazaki T, et al. Hypoxia-inducible factor-1alpha polymorphisms associated with enhanced transactivation capacity, implying clinical significance. Carcinogenesis 2003;24:177983.
  • 19
    Selak MA, Armour SM, MacKenzie ED, Boulahbel H, Watson DG, Mansfield KD, et al. Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase. Cancer Cell 2005;7:7785.
  • 20
    Isaacs JS, Jung YJ, Mole DR, Lee S, Torres-Cabala C, Chung YL, et al. HIF overexpression correlates with biallelic loss of fumarate hydratase in renal cancer: novel role of fumarate in regulation of HIF stability. Cancer Cell 2005;8:14353.
  • 21
    Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, Eshleman JR, Burt RW, et al. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 1998;58:524857.
  • 22
    Lee SH, Shin MS, Park WS, Kim SY, Kim HS, Han JY, et al. Alterations of Fas (Apo-1/CD95) gene in non-small cell lung cancer. Oncogene 1999;18:375460.
  • 23
    Lee JW, Soung YH, Kim SY, Lee HW, Park WS, Nam SW, et al. PIK3CA gene is frequently mutated in breast carcinomas and hepatocellular carcinomas. Oncogene 2005;24:147780.
  • 24
    Kipreos ET, Lander LE, Wing JP, He WW, Hedgecock EM. cul-1 is required for cell cycle exit in C. elegans and identifies a novel gene family. Cell 1996;85:82939.
  • 25
    Huh K, Zhou X, Hayakawa H, Cho JY, Libermann TA, Jin J, et al. Human papillomavirus type 16 E7 oncoprotein associates with the cullin 2 ubiquitin ligase complex, which contributes to degradation of the retinoblastoma tumor suppressor. J Virol 2007;81:973747.