Therapeutically targeting cyclin D1 in primary tumors arising from loss of Ini1

Abstract

Rhabdoid tumors (RTs) are rare, highly aggressive pediatric malignancies with poor prognosis and with no standard or effective treatment strategies. RTs are characterized by biallelic inactivation of the INI1 tumor suppressor gene. INI1 directly represses CCND1 and activates cyclin-dependent kinase (cdk) inhibitors p16(Ink4a) and p21(CIP). RTs are exquisitely dependent on cyclin D1 for genesis and survival. To facilitate translation of unique therapeutic strategies, we have used genetically engineered, Ini1(+/-) mice for therapeutic testing. We found that PET can be used to noninvasively and accurately detect primary tumors in Ini1(+/-) mice. In a PET-guided longitudinal study, we found that treating Ini1(+/-) mice bearing primary tumors with the pan-cdk inhibitor flavopiridol resulted in complete and stable regression of some tumors. Other tumors showed resistance to flavopiridol, and one of the resistant tumors overexpressed cyclin D1, more than flavopiridol-sensitive cells. The concentration of flavopiridol used was not sufficient to down-modulate the high level of cyclin D1 and failed to induce cell death in the resistant cells. Furthermore, FISH and PCR analyses indicated that there is aneuploidy and increased CCND1 copy number in resistant cells. These studies indicate that resistance to flavopiridol may be correlated to elevated cyclin D1 levels. Our studies also indicate that Ini1(+/-) mice are valuable tools for testing unique therapeutic strategies and for understanding mechanisms of drug resistance in tumors that arise owing to loss of Ini1, which is essential for developing effective treatment strategies against these aggressive tumors.

Fig. 1.

PET analysis and flavopiridol-induced regression of a primary tumor in an Ini1^+/− mouse. (A) PET analysis of primary tumors arising in an Ini1^+/− mouse before treatment. (A1) Digital image of mouse D84, showing a neck mass; (A2) PET projection image of the same mouse, revealing increased ¹⁸F-FDG uptake, indicating the presence of multiple tumors within the neck and abdomen. (B) Digital and PET projection images of mouse D84 after round 1 of flavopiridol treatment. (B1) Digital image corresponding to the PET image in B2. Note the complete regression of all tumor masses in B2; the uptake seen above the heart is due to nonspecific muscle uptake. White arrows indicate the location of masses. H, heart; B, urinary bladder. (C) Graph showing SUVs of tumor masses. Liver is used as a measure of background uptake.

Fig. 2.

PET analysis of an Ini1^+/− mouse with a primary tumor resistant to flavopiridol. (A) Digital and PET images of Ini1^+/− mouse E7 treated with flavopiridol. (A1 and A4) Images before treatment (day 1); (A2 and A5) Images after 2 wk of treatment with flavopiridol; (A3 and A6) Images after 7 wk of treatment with flavopiridol. White arrows indicate the location of the tumor. E, eyes. (B) Graph showing SUVs of the tumor. Liver is used as a measure of background uptake.

Fig. 3.

Ultrastructural characterization of the flavopiridol-resistant tumor cells. Transmission electron micrographs of (A) MON and (B) E7 resistant tumor cells, showing (A1 and B1) an eccentric nucleus and prominent nucleolus and (A2 and B2) whorls of cytoplasmic filaments. (Magnification: A1 and B1, 5,000×; A2 and B2, 10,000×.)

Fig. 4.

Analysis of drug resistance and cyclin D1 expression in flavopiridol-resistant tumors and cells. (A and B) Percentage survival of MON, G401, and flavopiridol-resistant E7 cells in culture after treatment with flavopiridol (A) and fenretinide (B). (C) Immunoblot of INI1 and cyclin D1 in E7 cells compared with MON, G401, and 293T cells. (D) Immunoblot showing expression of cyclin D1 in MON and E7 cells untreated or treated with 0.188 μM flavopiridol. (E) Comparative qPCR analysis indicating the fold increase in CCND1 gene dosage compared with that of either PRKCC or RPS18 in normal liver cells and in flavopiridol-resistant E7 tumor cells. (F) Table summarizing the karyotype analysis and quantitation of CCND1 counts in metaphase cells from the E7 resistant tumor (primary data are shown in Fig. S7).