Oncolytic viruses are designed to replicate in and lyse cancer cells on the basis of genetic changes present in tumor but not normal cells. A variety of such agents were developed. For example, H101, a variant of ONYX-015, has been approved for the use in head-and-neck cancer by the Chinese authorities.23
Theoretically, this therapeutic platform has the advantage of great flexibility. For example, oncolytic adenoviruses can be modified (‘armed') to express therapeutic genes or changed in their cell tropism.2, 24
These approaches rely on viral replication, the capacity of the virus to self-amplify and spread in the tumor. Our data show that loss of p21WAF1
promotes adenovirus replication and more effective cell killing compared with cells expressing p21WAF1
. We took advantage of a derivative of the human HCT116 colon cancer cell line harboring a homozygous deletion of p21−/−, and found that wild-type adenovirus and ONYX-015 replicate more efficiently in tumor cells lacking p21WAF1
expression compared with wild-type cells (). This finding was confirmed through our experiments using small-interfering RNAs directed against p21WAF1
, which increased virus-mediated cell killing and production of viable viral particles in HCT116 cells expressing wild-type p53 ( and ). In agreement with our findings, Hoti et al.
recently showed that valproic acid upregulates p21WAF1
expression resulting in inhibition of adenovirus replication. The exact mechanism by which CDK inhibitors mediate virus replication remains elusive. However, it is known that p21WAF1
interacts with SET (also called template activating factor TAF1) and proliferating cell nuclear antigen (PCNA). SET stimulates the replication of the adenovirus DNA and PCNA increases DNA replication and repair. Thus, binding of p21WAF1
to SET and PCNA may inhibit viral DNA replication.25, 26
We examined the effects of CDK inhibitors on the oncolytic adenoviruses ONYX-015 and Delta-24, which have been developed as therapeutics for the use in humans. These viruses differ in deletions within the adenovirus E1
-gene region. ONYX-015 is characterized by a deletion of E1B-55
K, whereas Delta-24 carries a deletion in the E1A region. Our results using p21WAF1
siRNAs in HCT116-WT cells show that both viruses behave similarly in regard to cell killing and virus production ( and ). HCT116 cells lacking p53 function, showed no difference in cell viability in the presence or absence of p21WAF1
siRNAs (). However, replication of adenoviruses was more productive in these cells when p21WAF1
expression was suppressed by siRNA before infection. We expanded our studies using two different cell lines with non-functioning p53, H1299 and DU-145, and we found that again virus production but not cell-killing effect were enhanced after p21 knockdown in cells infected with oncolytic adenoviruses, ONYX-015 or Delta-24. This suggest that p53 plays a role in cell killing or virus production when its downstream, p21, is silenced. Earlier reports have shown the requirement of p53 in adenovirus-induced cell death. Geoerger et al.27
reported that wild-type p53 was associated with increased anti-tumor activity of ONYX-015 in human malignant glioma xenografts. Given the complexities of virus replication and cancer cell environment, understanding the mechanism that increase oncolytic virus replication and cell killing is under investigation to provide information relevant to cancer therapy.
We found that TE7, an esophageal carcinoma cell line harboring wild-type p53, also showed enhanced viral cell killing when p21WAF1 expression was suppressed by siRNA before infection. The pancreatic cancer cell line, MIA PaCa-2, does not express p21WAF1 and was very susceptible to infection and cell killing by adenoviruses in the presence or absence of p21WAF1 siRNA.
Delta-24 carries a deletion within the CR2 region of E1A
, a region necessary for interaction with and inactivation of RB. Fueyo et al.28
showed that D54MG and U-251MG cells treated with replication-deficient adenoviral vectors expressing either Rb or p21WAF1
and infected the cells with Delta-24-RGD had less cell death. This finding also confirms that expression of p21WAF1
decreases the cytophatic effects of the Delta-24 adenovirus. Delta-24 has been extensively studied preclinically as a promising therapy for the treatment of gliomas and ovarian cancer. Recently, a phase I trial of Delta-24RGD have been approved (Dr J Fueyo, personal communication).
It is interesting to note that p21WAF1
has shown to regulate replication of other viruses. For example, p21WAF1
was found to restrict human immunodeficiency virus type 1 (HIV-1) infection of hematopoietic stem cells. Zhang et al.29
showed that p21WAF1
blocked viral infection by complexing with HIV-1 integrase and aborting chromosomal integration.
Taken together, our data illustrate an important role for p21WAF1 as modulator of replication of oncolytic adenoviruses with potential implications for the design of future clinical trials and improved oncolytic adenoviruses. p27KIP1 also reduced killing of HCT116 cells by the oncolytic adenoviruses used, particularly by WtD and Delta-24.
expression may represent a predictive marker for efficacy of virus replication in human cancer which could be useful for selecting patients with the highest likelihood of tumor response. Furthermore, improved oncolytic adenoviruses expressing p21WAF1
siRNA under the control of tumor or tissue-specific promoters or liposomal delivery of p21WAF1
siRNAs might represent interesting new approaches. Interestingly, antisense oligonucleotides directed against human p21WAF1
have been described as a means of improving cancer radiotherapy.30
In summary, this study highlights the role of p21WAF1 as regulators of oncolytic adenovirus replication in cancer cells. Our findings are potentially relevant for patient selection as well as for the development of new therapeutic strategies that include suppression of p21WAF1.