Monoallelic but not biallelic loss of Dicer1 promotes tumorigenesis in vivo

Abstract

Human tumors are characterized by widespread reduction in microRNA (miRNA) expression, although it is unclear how such changes come about and whether they have an etiological role in the disease. Importantly, miRNA knockdown has been shown to enhance the tumorigenic potential of human lung adenocarcinoma cells. A defect in miRNA processing is one possible mechanism for global downregulation. To explore this possibility in more detail in vivo, we have manipulated Dicer1 gene dosage in a mouse model of retinoblastoma. We show that although monoallelic loss of Dicer1 does not affect normal retinal development, it dramatically accelerates tumor formation on a retinoblastoma-sensitized background. Importantly, these tumors retain one wild-type Dicer1 allele and exhibit only a partial decrease in miRNA processing. Accordingly, in silico analysis of human cancer genome data reveals frequent hemizygous, but not homozygous, deletions of DICER1. Strikingly, complete loss of Dicer1 function in mice did not accelerate retinoblastoma formation. miRNA profiling of these tumors identified members of the let-7 and miR-34 families as candidate tumor suppressors in retinoblastoma. We conclude that Dicer1 functions as a haploinsufficient tumor suppressor. This finding has implications for cancer etiology and cancer therapy.

Keywords: Dicer; haploinsufficiency; microRNA; retinoblastoma; tumor suppressor.

Figure 1. Dicer1 (Dic) heterozygosity enhances tumorigenesis on a retinoblastoma-sensitized background

(A) Kaplan-Meier curve showing the time to first observation of externally visible retinoblastoma. This time was respectively markedly decrease in Chx10Cre;Rb^lox/lox;p107^−/−;Dic^lox/+ mice and increased in Chx10Cre;Rb^lox/lox;p107^−/−;Dic^lox/lox mice (log rank test, P=0,0015 and P=0,0036) relative to Chx10Cre;Rb^lox/lox;p107^−/−;Dic^+/+ littermates. (B) A 3 months-old Chx10Cre;Rb^lox/lox;p107^−/−;Dic^lox/+ mouse with aggressive retinoblastoma. (C) Hematoxylin and eosin stain with the three retinal nuclear layers (GL: ganglion layer; INL: inner nuclear layer; ONL: outer nuclear layer) indicated. Early retinoblastoma lesions at P35 and invasive tumors seeding the vitreous at P60 are found in Chx10Cre;Rb^lox/lox;p107^−/−;Dic^lox/+ mice. P200 shows late stage retinoblastoma that had filled the vitreous and the anterior chamber (arrow). inset: a representative Homer-Wright rosette found in Chx10Cre;Rb^lox/lox;p107^−/−;Dic^lox/+ tumors. Scale bars in the top panels = 40µm and = 200µm in the lower panels.

Figure 2. Immunostaining of the Chx10Cre;Rb^lox/lox;p107^−/−;Dic^lox/+ retinoblastoma lesions

(A) Syntaxin, Chx10 and GFP immunostaining of Chx10Cre;Rb^lox/lox;p107^−/−;Dic^lox/+ (Dic^lox/+) and Chx10Cre;Rb^lox/lox;p107^−/−;Dic^+/+ (Dic^+/+) retinae at P45. (B) Proliferation and apoptosis in Chx10Cre;Rb^lox/lox;p107^−/−;Dic^lox/+ (Dic^lox/+) and Chx10Cre;Rb^lox/lox;p107^−/−;Dic^+/+ (Dic^+/+) at P14 and P21. BrdU incorporation, Ki67 and active Caspase3 immunostaining. Scale bars = 40µm. Quantification of BrdU and Caspase3 staining was performed on horizontal serial sections at the optic nerve level. The numbers (nb) of BrdU positive cells were normalized to the nb in Chx10Cre;Rb^lox/lox;p107^−/−;Dic^+/+(Dic^+/+), which was set 1 (top right panel). The numbers of immunoreactive cells to active caspase3 staining were normalized to the numbers of GFP-positive cells in serial consecutive sections. The data are presented relative to the observed ratio GFP/Caspase3 in Chx10Cre;Rb^lox/lox;p107^−/−;Dic^+/+ (Dic^+/+), which was set 1 (low right panel). Error bars represent standard deviation. P-values (Student’s t-test; N=3).

Figure 2. Immunostaining of the Chx10Cre;Rb^lox/lox;p107^−/−;Dic^lox/+ retinoblastoma lesions

(A) Syntaxin, Chx10 and GFP immunostaining of Chx10Cre;Rb^lox/lox;p107^−/−;Dic^lox/+ (Dic^lox/+) and Chx10Cre;Rb^lox/lox;p107^−/−;Dic^+/+ (Dic^+/+) retinae at P45. (B) Proliferation and apoptosis in Chx10Cre;Rb^lox/lox;p107^−/−;Dic^lox/+ (Dic^lox/+) and Chx10Cre;Rb^lox/lox;p107^−/−;Dic^+/+ (Dic^+/+) at P14 and P21. BrdU incorporation, Ki67 and active Caspase3 immunostaining. Scale bars = 40µm. Quantification of BrdU and Caspase3 staining was performed on horizontal serial sections at the optic nerve level. The numbers (nb) of BrdU positive cells were normalized to the nb in Chx10Cre;Rb^lox/lox;p107^−/−;Dic^+/+(Dic^+/+), which was set 1 (top right panel). The numbers of immunoreactive cells to active caspase3 staining were normalized to the numbers of GFP-positive cells in serial consecutive sections. The data are presented relative to the observed ratio GFP/Caspase3 in Chx10Cre;Rb^lox/lox;p107^−/−;Dic^+/+ (Dic^+/+), which was set 1 (low right panel). Error bars represent standard deviation. P-values (Student’s t-test; N=3).

Figure 3. Chx10Cre;Rb^lox/lox;p107^−/−;Dic^lox/+ retinoblastoma tumors retain a wild-type and functional Dic allele

(A) Schematic representation of the Dic wild-type, floxed and Cre-excised alleles. (B) DNA was prepared from two Chx10Cre;Rb^lox/lox;p107^−/−;Dic^lox/+ P20 retinae (Ctr Retina) and 10 isolated tumors (results from 4 are shown) and examined by PCR using the primers depicted in the top panel. (B) RT-qPCR analyses of mature miRNAs expression. Left panel: hierarchical clustering of miRNAs significantly down-regulated in five Chx10Cre;Rb^lox/lox;p107^−/−;Dic^lox/+ tumours (black, dKO; Dic^lox/+) compared to Chx10Cre;Rb^lox/lox;p107^−/−;Dic^+/+ P20 retinae (grey, dKO; Dic^+/+). Middle panel: heat map of all miRNAs expressed at higher levels than the mean in Chx10Cre;Rb^lox/lox;p107^−/−;Dic^lox/+ P20 retinae shows comparable expression levels in both Chx10Cre;Rb^lox/lox;p107^−/−;Dic^lox/+ P20 retinae and tumors. Right panel shows the mean cq-values for all miRNAs analyzed (522 different murine miRNAs) in both Chx10Cre;Rb^lox/lox;p107^−/−;Dic^lox/+ P20 retinae and tumors.

Figure 4. Search for putative tumor suppressor miRNAs in retinoblastoma

(A) Heatmap of selected differentially expressed miRNAs in Chx10Cre-negative mice (light grey, wild-type), Chx10Cre;Rb^lox/lox;p107^−/−;Dic^+/+ (dKO; Dic^+/+) and Chx10Cre;Rb^lox/lox;p107^−/−;Dic^lox/+ P20 retinae (black, dKO; Dic^lox/+). (B) Expression analysis by RT-qPCR of let-7 family members (let-7b, let-7c and let-7i) and miR-34c.

Figure 5. Monoallelic loss of Dicer enhances retinoblastoma formation

Our data support a model in which monoallelic loss of Dicer1 cooperates with Rb inactivation in the formation of aggressive and invasive retinoblastoma. In contrast, biallelic loss of Dicer1 leads to retinal degeneration on a retinoblastoma-sensitized background and inhibition of tumor formation.