Kim L. Mercer

Impaired microRNA processing enhances cellular transformation and tumorigenesis.

MicroRNAs (miRNAs) are a new class of small noncoding RNAs that post-transcriptionally regulate the expression of target mRNA transcripts. Many of these target mRNA transcripts are involved in proliferation, differentiation and apoptosis, processes commonly altered during tumorigenesis. Recent work has shown a global decrease of mature miRNA expression in human cancers. However, it is unclear whether this global repression of miRNAs reflects the undifferentiated state of tumors or causally contributes to the transformed phenotype. Here we show that global repression of miRNA maturation promotes cellular transformation and tumorigenesis. Cancer cells expressing short hairpin RNAs (shRNAs) targeting three different components of the miRNA processing machinery showed a substantial decrease in steady-state miRNA levels and a more pronounced transformed phenotype. In animals, miRNA processing–impaired cells formed tumors with accelerated kinetics. These tumors were more invasive than control tumors, suggesting that global miRNA loss enhances tumorigenesis. Furthermore, conditional deletion of Dicer1 enhanced tumor development in a K-Ras–induced mouse model of lung cancer. Overall, these studies indicate that abrogation of global miRNA processing promotes tumorigenesis.

Somatic activation of the K-ras oncogene causes early onset lung cancer in mice.

About 30% of human tumours carry ras gene mutations. Of the three genes in this family (composed of K-ras, N-ras and H-ras), K-ras is the most frequently mutated member in human tumours, including adenocarcinomas of the pancreas (∼70–90% incidence), colon (∼50%) and lung (∼25–50%). To constuct mouse tumour models involving K-ras, we used a new gene targeting procedure to create mouse strains carrying oncogenic alleles of K-ras that can be activated only on a spontaneous recombination event in the whole animal. Here we show that mice carrying these mutations were highly predisposed to a range of tumour types, predominantly early onset lung cancer. This model was further characterized by examining the effects of germline mutations in the tumour suppressor gene p53, which is known to be mutated along with K-ras in human tumours. This approach has several advantages over traditional transgenic strategies, including that it more closely recapitulates spontaneous oncogene activation as seen in human cancers.

Endogenous oncogenic K-rasG12D stimulates proliferation and widespread neoplastic and developmental defects

Activating mutations in the ras oncogene are not considered sufficient to induce abnormal cellular proliferation in the absence of cooperating oncogenes. We demonstrate that the conditional expression of an endogenous K-ras(G12D) allele in murine embryonic fibroblasts causes enhanced proliferation and partial transformation in the absence of further genetic abnormalities. Interestingly, K-ras(G12D)-expressing fibroblasts demonstrate attenuation and altered regulation of canonical Ras effector signaling pathways. Widespread expression of endogenous K-ras(G12D) is not tolerated during embryonic development, and directed expression in the lung and GI tract induces preneoplastic epithelial hyperplasias. Our results suggest that endogenous oncogenic ras is sufficient to initiate transformation by stimulating proliferation, while further genetic lesions may be necessary for progression to frank malignancy.

Targeted Deletion Reveals an Essential Function for the Telomere Length Regulator Trf1

ABSTRACT The human telomeric DNA binding factor TRF1 (hTRF1) and its interacting proteins TIN2, tankyrase 1 and 2, and PINX1 have been implicated in the regulation of telomerase-dependent telomere length maintenance. Here we show that targeted deletion of exon 1 of the mouse gene encoding Trf1 causes early (day 5 to 6 postcoitus) embryonic lethality. The absence of telomerase did not alter the Terf1ex1Δ/ex1Δ lethality, indicating that the phenotype was not due to inappropriate telomere elongation by telomerase. Terf1ex1Δ/ex1Δ blastocysts had a severe growth defect of the inner cell mass that was accompanied by apoptosis. However, no evidence was found for telomere uncapping causing this cell death; chromosome spreads of Terf1ex1Δ/ex1Δ blastocysts did not reveal chromosome end-to-end fusions, and p53 deficiency only briefly delayed Terf1ex1Δ/ex1Δ lethality. These data suggest that murine Trf1 has an essential function that is independent of telomere length regulation.

ARF mutation accelerates pituitary tumor development in Rb+/- mice

Mice heterozygous for the retinoblastoma (Rb) tumor suppressor gene develop pituitary and thyroid tumors with high penetrance. We demonstrate here that loss of the ARF tumor suppressor strongly accelerates intermediate lobe pituitary tumorigenesis in Rb heterozygous mice. These effects in the pituitary are greater than those conferred by p53 loss in that Rb+/−;ARF−/− mice display significantly more early atypical lesions than Rb+/−; p53−/− mice. Also, Rb+/−;ARF−/− compound mutants do not develop many of the novel tumors or precancerous lesions seen in Rb+/−;p53−/− compound mutants. Although complete loss of ARF expression is not obligatory for pituitary tumorigenesis in Rb+/− mice, alterations of the ARF locus are observed in tumors from Rb+/−;ARF+/− mice, consistent with a selective advantage of ARF inactivation in this context. We conclude that inactivation of ARF acts more broadly than that of p53 in connecting abrogation of the Rb pathway to tumorigenesis.