Role of the chromobox protein CBX7 in lymphomagenesis

Abstract

Chromobox 7 (CBX7) is a chromobox family protein and a component of the Polycomb repressive complex 1 (PRC1) that extends the lifespan of cultured epithelial cells and can act independently of BMI-1 to repress the INK4a/ARF tumor suppressor locus. To determine whether CBX7 might be oncogenic, we examined its expression pattern in a range of normal human tissues and tumor samples. CBX7 was expressed at high levels in germinal center lymphocytes and germinal center-derived follicular lymphomas, where elevated expression correlated with high c-Myc expression and a more advanced tumor grade. By targeting Cbx7 expression to the lymphoid compartment in mice, we showed that Cbx7 can initiate T cell lymphomagenesis and cooperate with c-Myc to produce highly aggressive B cell lymphomas. Furthermore, Cbx7 repressed transcription from the Ink4a/Arf locus and acted epistatically to the Arf-p53 pathway during tumorigenesis. These data identify CBX7 as a chromobox protein causally linked to cancer development and may help explain the low frequency of INK4a/ARF mutations observed in human follicular lymphoma.

Fig. 1.

Aberrant expression of CBX7 in human follicular lymphomas. (A) Expression of CBX7, BCL-2, and BMI-1 in germinal center (GC, green) and follicular lymphoma (FL, red), as reported in ref. . (B) FL samples were stained by IHC by using anti-CBX7 antibodies and the CBX7 levels evaluated and quantified as explained in Materials and Methods. (C) Representative pictures of FL samples stained with CBX7 antibodies are shown. (D) CBX7 expression in secondary GC of the tonsil. Arrows indicate high CBX7 expression in germinal center histiocytes.

Fig. 2.

Expression of Cbx7 in the murine lymphoid compartment causes mature T cell lymphomas. (A) Kaplan–Meier curve showing tumor-free survival. Mice reconstituted with HSCs infected as indicated were monitored for lymphoma onset and illness until they reached a terminal stage and were killed. The data are presented in a Kaplan–Meier format, showing the percentage of mouse survival at various times after reconstitution. (B) Characterization of Cbx7-overexpressing lymphomas. Representative pictures are shown. (C) Western blot of Cbx7 overexpressing lymphomas showing exogenously expressed Cbx7 and endogenous c-Myc levels. β-Actin is used as a loading control. MEFs of the indicated genotypes and a lymphoma arising in a vav-MYC transgenic mouse were used as controls.

Fig. 3.

Cbx7 accelerates the onset of lymphomas triggered by c-Myc. (A) Kaplan–Meier curve showing tumor-free survival in recipient animals reconstituted with Eμ-myc fetal liver cells infected as indicated. (B) Characterization of Eμ-myc-expressing lymphomas. Representative pictures are shown. (C) Immunophenotyping by flow cytometry.

Fig. 4.

The Ink4a/Arf locus and p53 mediate Cbx7 effects in lymphomagenesis. (A) TUNEL analysis. Number of TUNEL-positive cells per 400× field is shown. (B) Quantitative RT-PCR analysis of transcript levels for p19^Arf and p16^Ink4a for lymphomas of the indicated genotypes. (C) Expression of Cbx7 confers a growth advantage on fetal liver cells obtained from WT or Eμ-myc embryos but not on those derived from Ink4a/Arf^−/− embryos, as measured by the increase in GFP-positive cells after 7 days of in vitro culture. (D) Fetal liver cells derived from embryos of genotypes as indicated were infected with the named constructs coexpressing GFP and injected into irradiated mice. Resulting lymphomas were analyzed for GFP expression. (E) Eμ-myc/p53^+/− fetal liver cells were infected with the indicated constructs coexpressing GFP and injected into irradiated mice. Loss of heterozygosity of p53 was analyzed by allele-specific PCR in the resulting lymphomas.