In 1998, Fire and colleagues reported that double-stranded RNA (dsRNA) was substantially more effective in producing interference than either strand was individually [1]. Specifically, purified single strands were shown to have modest effects after injection into adult animals, whereas double-stranded mixtures caused potent and specific interference [1]. This phenomenon, known as RNA interference (RNAi), is characterized by the presence of small RNAs that are approximately 21 or 22 nucleotides (nt) in length and homologous to genes being suppressed [2–4]. Long dsRNAs are processed by Dicer into 21- and 22-nt short interfering RNAs (siRNAs) that serve as guide sequences to instruct a multicomponent nuclease, the RNA-induced silencing complex or RISC, to destroy homologous messenger RNAs (mRNAs) [3,5].

In addition to siRNA production, Dicer is also essential for the biogenesis of microRNAs (miRNAs). Primary miRNAs (pri-miRNAs), mostly transcribed by RNA polymerase II (RNAPII) [6,7], are processed into a stem-loop structure containing 60- to 70-nt precursor miRNAs (pre-miRNAs) by a microprocessor consisting of an RNAase III endonuclease, Drosha, and a dsRNA-binding protein, DGCR8 [8]. The pre-miRNAs are then transported out of the nucleus via exportin-5 and processed by Dicer into mature miRNAs of 21 or 22 nt in length. These mature miRNAs subsequently bind to the 3′-untranslated region (UTR) of mRNAs and either promote their degradation or inhibit their translation [6–8].

The RNAi machinery plays important roles in the regulation of heterochromatin structure and function [9–11]. Specifically, loss of Dicer2 has been shown to result in not only decondensation of heterochromatin but also accumulation of extrachromosomal circular repeated DNAs (eccDNA) [12]. Ligase IV, an essential regulator of nonhomologous end joining, perhaps along with other DNA damage repair machinery, have been suggested to participate in the eccDNA formation in Dicer2-deficient cells [12]. These findings imply that in addition to increased accessibility of DNA repair and recombination proteins to repeated DNA caused by heterochromatin decondensation, activation of DNA damage response (DDR) may also contribute to the eccDNA formation in Dicer2-mutant cells. Moreover, it has been reported that the temporal control of satellite DNA replication in mouse ES cells is sensitive to loss of Dicer [13]. Misregulation of the timing of DNA replication may cause stalled and collapsed replication forks, which can in turn elicit a DNA damage response [14]. Furthermore, the RNAi machinery is known to be essential for transposon silencing [15–19]; therefore, loss of key components in the pathway may activate transposons and consequently lead to DNA damage [20]. Based on these observations, we hypothesized that loss of Dicer can induce DNA damage [21,22]. To test this hypothesis, we knocked down Dicer in human cells. The immunostaining assays for the phosphorylated form of histone H2AX (γ-H2AX; a widely used marker for double-strand DNA breaks [22]) and the replication protein A 70 (RPA70; a protein that becomes phosphorylated and forms intranuclear foci upon exposure of cells to DNA damage [23]) demonstrated that a much higher percentage of Dicer-knockdown cells displayed intense γ-H2AX and RPA70 foci than the control [21]. Using a comet assay to directly assess DNA damage, we confirmed that Dicer knockdown resulted in the accumulation of DNA breaks [21]. Consistent with our results, Peng and Karpen showed that Drosophila mutants lacking Dicer2 displayed significantly elevated frequencies of spontaneous DNA damage in heterochromatin [24]. Mudhasani and colleagues also reported that loss of Dicer activated a DNA damage checkpoint, upregulated the p19(Arf)-p53 signaling, and induced senescence in primary cells [25]. Moreover, Teta and colleagues found that deletion of Drosha or Dicer in rapidly proliferating follicular matrix cells led to DNA damage [26]. Furthermore, 2 groups have reported that knockdown of Dicer and Ago2 resulted in hypersensitivity to UV or gamma irradiation [27,28].

Compared to normal tissues, tumor tissues exhibit a general downregulation of miRNAs [56]. Similarly, the Dicer mRNA and protein levels, although still controversial, have been frequently found to be downregulated in tumor tissues [57–59]. The analysis of human cancer genome copy number data has also revealed frequent deletion of Dicer [60]. In addition, Heravi-Moussavi and colleagues recently reported that mutations in the RNase IIIb domain of Dicer are frequently associated with nonepithelial ovarian tumors [61], in which the mutations are restricted to codons encoding metal-binding sites within the RNase IIIb catalytic centers that are critical for microRNA biogenesis [61]. Signs of DDR, including phosphorylation of histone H2AX and Chk2, accumulation of p53, and focal staining of p53-binding protein 1, have been widely observed in clinical specimens from different stages of human tumors and precancerous lesions, but not in normal tissues [62–65]. Since decreased Dicer expression elicits DNA damage [21,24,25], we have raised the following questions: Is there an association between DNA damage and Dicer downregulation in cancer tissues? If the answer is yes, what is the causal relationship between the two in the process of tumorigenesis [35]?

Kumar and colleagues have proposed that loss of Dicer promotes tumorigenesis [66]. They showed that Dicer-knockdown cancer cells had a more pronounced transformed phenotype in animals; that is, Dicer-knockdown cells formed more invasive tumors with accelerated growth than the control tumor cells. In addition, these authors demonstrated that conditional deletion of Dicer enhanced tumor development in a mouse model of K-Ras-induced lung cancer [66]. The same group also suggested that Dicer functions as a haploinsufficient tumor suppressor gene, because full loss of Dicer1 expression caused inhibition of tumorigenesis [60]. In contrast, Ravi and colleagues recently showed that full loss of Dicer1 did not preclude tumor formation [67]. They found that transformed or immortalized Dicer1-null somatic cells can be isolated readily in vitro and that these cells maintained the characteristics of Dicer1-expressing controls and remained stably proliferative. In addition, Dicer1-null cells from a sarcoma cell line, though depleted of miRNAs, were competent for tumor formation [67]. Similarly, Sekine and colleagues, by disrupting Dicer in hepatocytes using a conditional knockout mouse model, found that Dicer elimination induced hepatocyte proliferation and overwhelming apoptosis [68]. Unexpectedly, they further found that two-thirds of mutant mice spontaneously developed hepatocellular carcinomas (HCCs) at 1 year of age. Deletion of Dicer in thyroid follicular cells has also been shown to cause hypothyroidism with signs of neoplastic alterations [69]. The thyroid tissue in Dicer mutant mice showed marked proliferation of follicular cells as well as an ongoing de-differentiation in the center of the thyroid gland, with loss of Pax8, FoxE1, Nis, and Tpo expression. These observations all suggested that reduced Dicer expression may be a cause of tumorigenesis. Considering the facts that the majority of Dicer-deficient hepatocytes undergo apoptosis and that only a minor subset of them give rise to HCCs, Sekine and colleagues speculated that a “second hit” was required to promote hepatocarcinogenesis in Dicer-deficient hepatocytes [68]. Consistent with this speculation, Kim and colleagues recently reported that Dicer-Pten double-knockout mice universally developed early serous carcinomas in the fallopian tube [70].

Taken together, we have proposed a model to explain the role of Dicer in tumorigenesis [35], in which decreased Dicer expression induces DNA damage and in turn leads to either cell apoptosis or senescence. Alternatively, DNA damage may result in DNA mutations, and cells that contain oncogenic mutations may escape from apoptosis and senescence and eventually form cancers. Furthermore, decreased Dicer expression leads to a global downregulation of cellular miRNAs; since some miRNAs, such as miR-34a and miR-16, may function as tumor suppressors [48,49,71,72], downregulation of such tumor-suppressive miRNAs could also contribute to tumorigenesis.

The role of Dicer in DNA damage repair and cancer etiology has just begun to surface, and the molecular mechanisms by which loss of Dicer leads to DNA damage still need to be further investigated. While the current data implicate that the Dicer- and Drosha-dependent small RNAs (~21 nt) are involved in homologous recombination-mediated DSB repair [29,30], it is important to address the role of these small RNAs in nonhomologous end joining-mediated DSB repair. Moreover, in silico predications suggested that several miRNAs could target genes in the DNA damage repair pathways [71]; however, these predications should be further verified experimentally. Another important issue is to address the roles and detailed molecular mechanisms of Dicer and DNA damage responsive microRNAs in cancer development, which will serve as a starting point for developing novel diagnostic and therapeutic strategies for cancer treatment.