Prep1 (pKnox1)-deficiency leads to spontaneous tumor development in mice and accelerates EmuMyc lymphomagenesis: a tumor suppressor role for Prep1

Abstract

The Prep1 homeodomain transcription factor is essential for embryonic development. 25% of hypomorphic Prep1(i/i) embryos, expressing the gene at 2% of the normal levels, survive pregnancy and live a normal-length life. Later in life, however, these mice develop spontaneous pre-tumoral lesions or solid tumors (lymphomas and carcinomas). In addition, transplantation of E14.5 fetal liver (FL) Prep1(i/i) cells into lethally irradiated mice induces lymphomas. In agreement with the above data, haploinsufficiency of a different Prep1-deficient (null) allele accelerates EmuMyc lymphoma growth. Therefore Prep1 has a tumor suppressor function in mice. Immunohistochemistry on tissue micrroarrays (TMA) generated from three distinct human cohorts comprising a total of some 1000 human tumors revealed that 70% of the tumors express no or extremely low levels of Prep1, unlike normal tissues. Our data in mice are thus potentially relevant to human cancer.

Figure 1

Development of tumors and pre‐tumoral lesions in Prep1i/i mice. The graph shows the percentage of wt (blue) and Prep1i/i (green) mice that develop a tumor or a PTL. Mice were sacrificed within the indicated age windows (in months) and autopsied. n indicates the number of mice within each group.

Figure 2

Cytofluorimetry, genotyping and immunoblotting of the TT1 tumor. A: Flow cytometry analysis of tumor TT1, a T‐cell lymphoma generated by transplantation of Prep1i/i embryo FL cells into wt mice. Thymus tumor cells (i/i) are compared with the cells from the same organ from a mouse transplanted with wt (+/+) FL cells. FSC and SSC parameters show an abnormal cell population in the hypomorphic thymus. B: PCR genotyping (see Methods) of the DNA extracted from tumor TT1. CTL is an heterozygous control thymus showing the wild type and the hypomorphic allele bands. The arrow identifies the hypomorphic band. WT2 cells are thymus cells from a mouse transplanted with wt littermate FL cells. Mock, is a reaction in the absence of reverse transcriptase. C: Immunoblotting analysis for Prep1. Extracts of thymus of two mice (wt1 and wt2) transplanted with wt FL cells are compared with those of the TT1 lymphoma. Immunoblotting of actin is used as loading control.

Figure 3

Prep1 haploinsufficiency accelerates the death of transgenic EμMyc mice. A: Kaplan Meyer curves for EμMyc C57BL/6 transgenic mice carrying two wild type (heavy line) or one wild type and one deleted (fine line) allele for Prep1. Median survival: 21 weeks for Prep1+/−EμMyc, 58 weeks for EμMyc mice (p‐value 0.0022). In the absence of the EμMyc transgene all the Prep1+/− mice survive and show no signs of disease for at least 25 weeks (not shown). B: Quantitative (real‐time) PCR analysis on CD19+splenic cells purified from pre‐tumoral (two months old) littermate mice of the indicated genotype.

Figure 4

Immunoblotting analysis of Pbx1, Pbx2 and Meis1 in wt or homozygous Prep1i/i MEFs. On top the immunoblotting with an anti‐Prep1 antibody is shown. In the middle, the immunoblots with specific anti Meis1, Pbx1 and Pbx2 antibodies; loading control is an anti‐actin antibody. At the bottom is the quantitation of the bands by densitometry.

Figure 5

Prep1 is absent or decreased in most human tumors. Human tissue microarrays (TMA) analyzed by immunohistochemistry for Prep1 (see Methods and Suppl. Figure S3). The scores of Prep1 expression in tumors was defined as: 0 = negative; 1 = low expression; 2 = moderate; 3 = high. A. Histogram reporting the data of the Table in panel B. B. The table reports the number of the different tumors analyzed and their subdivision according to their IHC score. C: Examples of Prep1 immunohistochemistry in normal human mammary gland and breast cancer, as well as in normal and cancerous larynx. Representative negative and positive images (score 0 and 2) of respectively breast and larynx tumors are shown. Prep1 level were high in normal breast and larynx epithelia. D: One example of a skin cancer, showing normal cells (thick arrow) expressing Prep1 in the nucleus and cancer cells (thin arrows) expressing no or low Prep1.

Figure 6

Summary of Prep1 levels determination in 445 human NSCLC. A cohort of lung cancer patients was analyzed by immunohistochemistry with the anti‐Prep1 antibody. The tumors were scored as described in the text and in the legend of Figure 5. In this group, 50% of the patients showed no Prep1 expression, and 20% a very low expression. Below the histogram the numbers of patients in each score group is indicated. ND: sections in which the score could not be determined.