Adenoviruses Are Implicated in Lung Cancer

EXAMPLE: "Despite its spectacular achievements, the first successful form of gene therapy will for now be used only as a last resort due to the risk of cancer. And that's not the only bad news. The latest studies suggest that some other gene therapies using different viruses might also trigger cancer. The Necker hospital in Paris pioneered the use of gene therapy to cure the severe inherited immune disease called X-SCID. But two of the 11 boys treated have developed leukemia. Both these cases appear to be due to the corrective gene being inserted near another gene called Lmo2, which helps control cell growth and can contribute to cancer if turned on at the wrong time (New Scientist, 25 January, p 12). Calculations by Christof von Kalle of the University of Cincinnati College of Medicine in Ohio suggest that rather than being an extraordinary coincidence the engineered mouse retrovirus used to deliver the gene in the French study may insert it near Lmo2 about once per 100,000 insertions. Since millions of bone marrow cells are modified and returned to the boys, such insertions may crop up in most if not all of the patients." (Cancer risk clouds gene cures. New Scientist March 15, 2003. Gene therapy 'caused T-cell leukemia.' By Jo Lyford. The Scientist Oct. 20, 2003; a third child later developed leukemia: January 2005, third adverse event in Paris! By Sandro Rusconi. Swiss Gene Therapy Web Page.) Although adenovirus was not used in this therapy, the principle is the same. And, what they do not mention in these articles is that the same thing may happen in natural infections - but, despite foreboding evidence, there has been little or no investigation before the establishment rushed into gene therapy.

An online review of adenoviruses, which however expresses the "conventional wisdom" about the nononcogenicity of adenoviruses in humans: "In 1962, some adenoviruses were shown to cause tumors in rodents -- this caused a considerable panic (N.B. Adenovirus oncogenesis appears to be associated with abortive infections and has never been observed in humans.)" Mainly because there was never a serious effort to look.

The role of the Tobacco Industry Research Council and the National Institutes of Health in adenovirus cancer research in the 1960s:

In the 1960s and early 1970s, the National Cancer Institute funded some seroepidemiological studies of adenoviruses versus various types of cancer. They established nothing more than that exposure to adenoviruses was very common. Some were conducted by Maurice Green, RJ Huebner's leading contractor in the NCI's Cancer Virus Program, at St. Louis University, Huebner's alma mater, where Green was a professor. On the basis of these, Green and RV Gilden, of Huebner's Flow Laboratory, concluded that "Adenoviruses are not thought likely to be an important cause of human cancer" (Adenoviruses in human cancer. RM McAllister, RV Gilden, M Green. The Lancet 1972 Apr 15;1(7755):831-833; no abstract available). In the latter 1970s, a pitifully small sample of cancers were investigated for the presence of adenoviral DNA (the same four small cell lung cancers were used in the three 1979 studies), and the reported negative results were considered sufficient to dismiss the idea that adenoviruses could cause human cancer. Research that was directly relevant to human disease dried up, and the resurgence of research since 1994 is mainly concerned with the use of adenoviruses as vectors in gene therapy. And, in the meantime, the anti-smoking racketeers conspired to make it a thought crime to suggest that there could be any alternative cause for lung cancer other than smoking.

In 1969, Dr. Frank J. Rauscher, an associate scientific director of the National Cancer Institute and head of its cancer virus program, claimed that adenoviruses had been all but removed from suspicion of causing cancer, and killed off 380 monkeys in the NCI's research program who had just reached an age when cancer would be expected to develop.

Do highly oncogenic group A human adenoviruses cause human cancer? Analysis of human tumors for adenovirus 12 transforming DNA sequences. JK Mackey, PM Rigden, M Green. Proc Natl Acad Sci 1976 Dec;73(12):4657-4661. These included 17 squamous and 5 adenocarcinomas, but no small cell lung cancers.

Analysis of human cancer DNA for DNA sequences of human adenovirus type 4. JK Mackey, M Green, WSM Wold, P Rigden. J Natl Cancer Inst 1979 Jan;62(1):23-26.

Analysis of human cancer DNA's for DNA sequences of human adenovirus serotypes 3, 7, 11, 14, 16 and 21 in Group B. WSM Wold, JK Mackey, P Rigden, M Green. Cancer Res 1979 Sep;39(9):3479-3484.

Analysis of human tonsil and cancer DNAs and RNAs for DNA sequences of group C (serotypes 1, 2, 5, and 6) human adenoviruses. M Green, WSM Wold, JK Mackey, P Rigden. Proc Natl Acad Sci 1979 Dec;76(12):6606-6610.

Investigation resumes

Detection of group C adenovirus DNA in small-cell lung cancer with the nested polymerase chain reaction. K Kuwano, M Kawasaki, R Kunitake, N Hagimoto, Y Nomoto, T Matsuba, T Nakanishi, N Hara. J Cancer Res Clin Oncol 1997;123(7):377-382. "E1A target DNA was present in 11 (31%) of 35 cases of small-cell lung cancer but in none of the 40 cases of non-small-cell lung cancer (P<0.01). Of the 11 cases found positive by PCR, 8 were positive for adenovirus DNA by in situ hybridization."

The American Cancer Society says of small cell lung cancer: "About 20% of all lung cancers are of this type... Small cell lung cancer is almost always caused by smoking. It is very rare for someone who has never smoked to have small cell lung cancer. Other names for small cell lung cancer are oat cell carcinoma and small cell undifferentiated carcinoma" (http://www3.cancer.org/cancerinfo/load_cont.asp?ct=26&st=wi). In ACS News Today 1999 Sep 17, they call it "the type of lung cancer most strongly linked with exposure to tobacco... Small cell lung cancer accounts for about 25 percent of all lung cancers" (http://www2.cancer.org/zine/index.sfm?fn=001_09171999_0).

THE VOID SPEAKS FOR ITSELF

To the American Cancer Society and the National Cancer Institute, adenoviruses have historically been of interest only as gene therapy vectors. They are concerned enough about the possibility of inadvertently causing cancers in patients to remove the suspect genes from these viral vectors - at least the ones they know about. BUT THEY ARE STONEWALLING ABOUT CANCER CAUSED BY ALL THOSE NATURAL INFECTIONS WITH WILD-TYPE VIRUSES THAT HAVE ALL THEIR GENES. Just look at the nature of the research on adenoviruses. There's a flood of adenovirus vector research, versus virtually nothing on the role of natural adenoviruses in any cancer. The void speaks for itself.

Adenovirus binding to blood factors results in liver cell infection and hepatotoxicity. DM Shayakhmetov, A Gaggar, S Ni, Z-Y Li, A Lieber. J Virology 2005 Jun;79(12):7478-7491. Adenovirus vectors cause inflammation of the liver.

Mechanisms

Adenovirus proteins inhibit cell death and transform cells

Oncogenic potential of the adenovirus E4orf6 protein. M Moore, N Horikoshi, T Shenk. PNAS 1996 Oct;93(10):11295-11301. They note that "Although several early studies described the presence of E4-specific mRNAs in addition to the E1A and E1B species in adenovirus transformed rat and hamster cells (e.g., refs. 39-41; reviewed in ref. 42), a role for E4 in transformation was discounted because it was not always present in cells transformed by adenovirus types 2 and 5 and because cloned E1A and E1B genes were sufficient for transformation." The studies cited date from 1975, and about that time there was "The Big Push to Suppress Virus Work," and investigate only chemical carcinogens. Incidentally, "This work [the present paper] was supported by grants from the National Cancer Institute (CA41086) and the Cystic Fibrosis Foundation. T.S. is an American Cancer Society Professor and an Investigator of the Howard Hughes Medical Institute."

The cell death inhibitor Bcl-2 and its homologues influence control of cell cycle entry. LA O'Reilly, DC Huang, A Strasser. EMBO J 1996 Dec 16;15(24):6979-6790.

Bcl-2, Bcl-XL and adenovirus protein E1B19kD are functionally equivalent in their ability to inhibit cell death. DC Huang, S Cory, A Strasser. Oncogene 1997 Jan 30;14(4):405-414.

The adenovirus E4orf6 protein can promote E1A/E1B-induced focus formation by interfering with p53 tumor suppressor function. M Nevels, S Rubenwolf, T Spruss, H Wolf, T Dobner. PNAS 1997 Feb;94(2):1206-1211. "There is accumulating evidence that other viral proteins from different DNA tumor viruses -- i.e., human papillomavirus E6, hepatitis B virus Hbx, and Epstein-Barr virus BZLF-1 -- bind to the carboxyl region of p53. Given the importance of this domain in the regulation of the tumor-suppressor function of p53, these protein interactions may represent a general mechanism by which these viruses modulate p53 function and trigger oncogenic transformation."

Adenovirus E1B 55K represses p53 activation in vitro. MED Martin, AJ Berk. J Virol 1998 Apr;72(4):3146-3154. "The cellular phosphoprotein p53 acts as a tumor suppressor, and inactivation of this function is the most prevalent alteration found in human and animal tumors...."

Transforming potential of the adenovirus type 5 E4orf3 protein. M Nevels, B Tauber, E Kremmer, T Spruss, H Wolf, T Dobner. J Virol 1999 Feb;73(2):1591-1600. They observed that like E4orf6, E4orf3 disappeared from cell lines after transformation. "The absence of the viral DNA templates in these transformants implies that both E4 gene products can transform primary cells with E1A by a 'hit and run' mechanism." Nevels is co-author with 1988-98 CTR member PK Vogt of a chapter on "Cell transformation by viruses," in a 1996 book co-edited by 1982-86 CTR member Peter Howley, which is cited in the lead paragraph of the present paper. Howley was forced to resign from the CTR.  Needless to say, none of this has ever been brought up in a tobacco trial.

The adenovirus E4orf6 protein contributes to malignant transformation by antagonizing E1A-induced accumulation of the tumor suppressor protein p53. M Nevels, T Spruss, H Wolf, T Dobner. Oncogene 1999 Jan 7;18(1):9-17.

Transforming potential of the adenovirus type 5 E4orf3 protein. M Nevels, B Tauber, E Kremmer, T Spruss, H Wolf, T Dobner. J Virol 1999 Feb;73(2):1591-1600.

Adenovirus E4 34k and E4 11k inhibit double strand break repair and are physically associated with the cellular DNA-dependent protein kinase. J Boyer, K Rohleder, G Ketner. Virology 1999 Oct 25;263(2):307-312. "The adenovirus oncoproteins E4 34k and E4 11k, the products of E4 open reading frames 6 and 3, respectively, individually prevent the formation of concatemers of the linear viral genome in infected cells."

Two distinct activities contribute to the oncogenic potential of the adenovirus type 5 E4orf6 protein. M Nevels, S Rubenwolf, T Spruss, H Wolf, T Dobner. J Virol 2000 Jun;74(11):5168-5181.

Identification of three functions of the adenovirus E4orf6 protein that mediate p53 degradation by the E4orf6-E1B55K complex. E Querido, MR Morrison, Huan Chu-Pham-Dang, SW-L Thirlwell, D Boivin, PE Branton. J Virol 2001 Jan;75(2):699-709.

Degradation of p53 by adenovirus E4orf6 and E1B55K proteins occurs via a novel mechanism involving a Cullin-containing complex. Genes Dev 2001 Dec 1;15(23):3104-3117.

Regulation of the mitochondrial checkpoint in p53-mediated apoptosis confers resistance to cell death. H Henry, A Thomas, Y Shen, E White. Oncogene 2002 Jan 24;21(5):748-760.

Adenovirus and cell cycle control. H Ben-Israel, T Kleinberger. Front Biosci 2002 May 1;7:d1369-95. E1A proteins inactivate the pRb checkpoint, and E1B and E4orf6 inactivate p53.

E1A-based determinants of oncogenicity in human adenovirus groups A and C. JF Williams, Y Zhang, MA Williams, S Hou, D Kushner, RP Ricciardi. Curr Top Microbiol Immunol 2004;273:245-288. Review. "A broad spectrum of genetic and molecular investigations carried out with group C, Ad2 and Ad5, and with group A, Ad12, have shown that early region1 (E1) gene products are sufficient for complete transformation of rodent cells in vitro by these viruses.... In this chapter we review previous findings and present new evidence which demonstrates that Ad12 E1A possesses two or more independent functions enabling it to induce tumors. One of these functions lies in its capacity to repress transcription of MHC class I genes, allowing the tumor cells to avoid lysis by cytotoxic T lymphocytes.... In addition to mediating immune escape, E1A also determines the susceptibility of transformants to Natural Killer (NK) cell lysis, and in this case, also, Ad12 transformants are not susceptible."

The adenovirus E4orf6 protein inhibits DNA double strand break repair and radiosensitizes human tumor cells in an E1B-55K-independent manner. LS Hart, SM Yannone, C Naczki, JS Orlando, SB Waters, SA Akman, DJ Chen, D Ornelles, C Koumenis. J Biol Chem 2005 Jan 14;280(2):1474-1481.

E1A-based determinants of oncogenicity in human adenovirus groups A and C.

Adenovirus type 5 early region 1B 156R protein promotes cell transformation independent from repression of p53-stimulated transcription. T Sieber, T. Dobner. J Virol 2007 Jan;81(1):95-105. Two gene products of Early region 1B (E1B) of adenovirus type 5 (Ad5), E1B-19K and E1B-55K, are individually capable of cooperating with the Ad5 E1A proteins to completely transform rodent cells in culture. The remaining E1B proteins are E1B-156R, E1B-93R and E1B-84R. E1B-156R also enhances focal transformation of primary rat cells in cooperation with E1A. "Since E1B-156R seemed unable to relocalise p53 and inhibit its transactivating function, it must be assumed that it contributes to transformation independently of repression of p53-stimulated transcription. Furthermore, we discovered that E1B-156R contains a functional transcriptional repression domain and binds Ad5 E4orf6 and the cellular apoptosis regulator Daxx. While the ability to bind E4orf6 could indicate further biological functions of E1B-156R in viral infection, the interaction with Daxx might also be linked to its transforming potential. Taken together, these analyses introduce E1B-156R as a novel transformation-promoting E1B protein that acts without repressing p53-transactivation. Moreover, identification of the interaction partners E4orf6 and Daxx provide a first glance of E1B-156R's potential functions."

Adenovirus Integration

Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line. N Louis, C Evelegh, FL Graham. Virology 1997 Jul 7;233(2):423-429. "The Ad5 sequences... are located in the pregnancy-specific beta-1-glycoprotein 4 (PSG 4) gene. This maps the insertion of Ad5 DNA to human chromosome 19 (19q13.2)."

From the Online Mendelian Inheritance in Man (OMIM): "The human pregnancy-specific glycoproteins (PSGs) are a group of molecules that are mainly produced by the placental syncytiotrophoblasts during pregnancy...." They are believed to be essential for normal pregnancy. High levels are found in patients with choriocarcinoma and hydatidiform mole.

Chromosome damage

Chromosomal damage induced by human adenovirus type 12 requires expression of the E1B 55-kilodalton viral protein. S Schramayr, D Caporossi, I Mak, T Jelinek, S Bacchetti. J Virol 1990 May;64(5):2090-2095.

Adenovirus type 12-induced fragility of the human RNU2 locus requires p53 function. Z Li, A Yu, AM Weiner. J Virol 1998 May;72(5):4183-4191.

Coexpression of the adenovirus 12 E1B 55 kDa oncoprotein and cellular tumor suppressor p53 is sufficient to induce metaphase fragility of the human RNU2 locus. D Liao, A Yu, AM Weiner. Virology 1999 Feb 1;254(1):11-23.

RNA, U1 Small Nuclear; RNU1, located at 1p36.3

RNA, U2 Small Nuclear; RNU2, located at 17q21-q22. (OMIM).

Ribonucleic Acid, Ribosomal, 5S; 5S rRNA; RN5S; located at 1q42.11-q42.13.

"Hit-and-run" transformation

"According to classical concepts of viral oncogenesis, the persistence of virus-specific oncogenes is required to maintain the transformed cellular phenotype. In contrast, the "hit-and-run" hypothesis claims that viruses can mediate cellular transformation through an initial "hit," while maintenance of the transformed state is compatible with the loss ("run") of viral molecules." Cells that are transformed by adenovirus E1A and E1B permanently express these viral genes. However, a new mechanism of transformation has been found in which oncoproteins E4orf6 or E4orf3 cooperate with E1A in place of E1B. These cells do not express the E4 gene products, and only a subset contain E1A proteins. They conclude, "Our results strongly support the possibility that even tumors that lack any detectable virus-specific molecules can be of viral origin, which could have a significant impact on the use of adenoviral vectors for gene therapy." The health establishment is heavily invested in adenovirus-vector gene therapy; will they try to cover this up for this reason as well? "Hit-and-run" transformation by adenovirus oncogenes. M Nevels, B Tauber, T Spruss, H Wolf, T Dobner. J Virol 2001 Apr;75(7):3089-3094.

(AN EXAMPLE OF E4ORF6 IN GENE THERAPY RESEARCH): Improved adeno-associated virus vector production with transfection of a single helper adenovirus gene, E4orf6. JM Allen, CL Halbert, AD Miller. Mol Ther 2000 Jan;1(1):88-95.

Adenovirus interactions with other viruses

Human cytomegalovirus IE1 and IE2 proteins are mutagenic and mediate "hit-and-run" oncogenic transformation in cooperation with the adenovirus E1A proteins. Y Shen, H Zhu, T Shenk. Proc Natl Acad Sci USA 1997 Apr;94(7):3341-3345.

Human papilloma virus E6 and E7 proteins support DNA replication of adenoviruses deleted for the E1A and E1B genes. DS Steinwaerder, CA Carlson, A Lieber. Mol Ther 2001 Sep;4(3):211-216. The implications of co-infection in vivo with both HPV and Ad are not addressed.

Adenovirus and Epstein-Barr virus

Striking similarities are exhibited by two small Epstein-Barr virus-encoded ribonucleic acids and the adenovirus-associated ribonucleic acids VAI and VAII. MD Rosa, E Gottlieb, MR Lerner, JA Steitz. Mol Cell Biol 1981 Sep;1(9):785-796. "VAII RNA as well as VAI RNA and the EBERs exist in ribonucleoprotein complexes which are precipitable by anti-La antibodies associated with systemic lupus erythematosus."

Two small RNAs encoded by Epstein-Barr virus can functionally substitute for the virus-associated RNAs in the lytic growth of adenovirus 5. RA Bhat, B Thimmappaya. Proc Natl Acad Sci USA 1983 Aug;80(15):4789-4793. "To examine whether the EBRs can functionally substitute for the VA RNAs for the lytic growth of Ad5, we have constructed an Ad5 substitution mutant in which the two VA RNA genes have been deleted and replaced by an EBV DNA segment coding for the two EBERs. The resulting Ad5 mutant synthesizes large amounts of the EBERs and is viable."

Construction and analysis of additional adenovirus substitution mutants confirm the complementation of VAI RNA function by two small RNAs encoded by Epstein-Barr virus. RA Bhat, B Thimmappaya. J Virol 1985 Dec;56(3):750-756. "Adenovirus VAI RNA is essential for the efficient initiation of translation of viral mRNAs at late times after infection. Recently, by constructing an adenovirus type 5 substitution mutant, we showed that the Epstein-Barr virus encoded two small RNAs complemented for the VAI RNA function in the adenovirus type 5 lytic growth (Bhat and Thimmappaya, Proc. Natl. Acad. Sci. USA 80:4789-4793, 1983).... Our results convincingly demonstrated that the two Epstein-Barr virus-encoded RNAs can efficiently complement for the VAI RNA-mediated translational defect in adenovirus-infected cells."

Adenoviruses evade the immune system

E3/19K from adenovirus 2 is an immunosubversive protein that binds to a structural motif regulating the intracellular transport of major histocompatibility complex class I proteins. WA Jefferies, HG Burgert. J Exp Med 1990 Dec 1;172(6):1653-1664.

Adenovirus infection inhibits the phosphorylation of major histocompatibility complex class I proteins. R Lippe, E Luke, YT Kuah, C Lomas, WA Jefferies. J Exp Med 1991 Nov 1;174(5):1159-1166.

Specific inhibition of interferon signal transduction pathways by adenoviral infection. TD Joseph, DC Look. J Biol Chem 2001 Dec 14;276(50):47136-47142.

The adenovirus E3 RID Complex Protects Some Cultured Human T and B Lymphocytes from Fas-Induced Apoptosis. AL McNees, CT Garnett, LR Gooding. J Virol 2002 Oct 1;76(19):9716-9723.

New Strains of Adenovirus Emerge and Spread

"The emergence and apparent global spread of Ad7d2 are reminiscent of observations for another genome type of serotype 7, Ad7b. Originally described by Wadell and Varsanyi, Ad7b was associated with outbreaks of severe respiratory illness in Europe in the 1970s. Although first isolated in 1956 from a Paris orphanage outbreak, subsequent retrospective studies did not identify Ad7b in Europe again until 1969. Before then, the earliest documented occurrence of Ad7b was in China in 1958, where it was the predominant genome type circulating throught the early 1980s. With the exception of Paris, the first emergence of Ad7b outside China was on the US West Coast in 1962. By 1970, Ad7b was the predominant genome type circulating throughout the United States and eventually throughout many parts of the world." (Molecular epidemiology of adenovirus type 7 in the United States, 1966-2000. DD Erdman, W Xu, SI Gerber, GC Gray, D Schnurr, AE Kajon, LJ Anderson. Emerg Infect Dis 2002;8(3)./Medscape.)

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cast 12-11-07