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Genetic Analysis of Pten and Ink4a Arf Interactions in the Suppression of Tumorigenesis in Mice

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Genetic Analysis of Pten and Ink4a Arf Interactions in the Suppression of Tumorigenesis in Mice Genetic analysis of Pten and Ink4a͞Arf interactions in the suppression of tumorigenesis in mice Mingjian James You*†‡, Diego H. Castrillon*§, Boris C. Bastian¶,Ro´ na´ n C. O’Hagan*†‡, Marcus W. Bosenberg*§, Ramon Parsonsʈ, Lynda Chin*,**, and Ronald A. DePinho*†‡ †† *Departments of Adult Oncology, Dana–Farber Cancer Institute, Departments of †Medicine, §Pathology, and **Dermatology, Brigham and Women’s Hospital, Harvard Medical School, and ‡Department of Genetics, Harvard Medical School, Boston, MA 02115; ¶Departments of Dermatology and Pathology and the Comprehensive Cancer Center, University of California, San Francisco, CA 94143; and ʈInstitute of Cancer Genetics, College of Physicians and Surgeons, Columbia University, New York, NY 10031 Communicated by Lewis C. Cantley, Beth Israel Deaconess Medical Center, Boston, MA, November 28, 2001 (received for review September 26, 2001) Dual inactivation of PTEN and INK4a͞ARF tumor suppressor genes is phomas or sarcomas with median latency of Ϸ30 weeks (9). a common feature observed in a broad spectrum of human cancer Relative to wild-type (wt) control cultures, Ink4a͞ArfϪ/Ϫ mouse types. To validate functional collaboration between these genes in embryonic fibroblasts (MEFs) grow more rapidly, exhibit a high tumor suppression, we examined the biological consequences of Pten rate of colony formation and immortalization, and are suscep- and͞or Ink4a͞Arf deficiency in cells and mice. Relative to single tible to transformation by oncogenic H-Ras alone (9). mutant controls, Ink4a͞Arf؊/؊Pten؉/؊ mouse embryonic fibroblast The PTEN and INK4a͞ARF tumor suppressor genes are among cultures exhibited faster rates of growth in reduced serum, grew to the most frequently inactivated genes in human cancer (10, 11). higher saturation densities, produced more colonies upon low density Loss of PTEN function is common in glioblastoma, melanoma, seeding, and showed increased susceptibility to transformation by endometrial carcinoma, prostate adenocarcinoma, renal cell carci- oncogenic H-Ras. Ink4a͞Arf deficiency reduced tumor-free survival noma, and head and neck squamous carcinoma (10). PTEN mu- and shortened the latency of neoplasias associated with Pten het- tations have also been detected in sporadic cancers of the breast, erozygosity, specifically pheochromocytoma, prostatic intraepithelial thyroid, lung, stomach, and hematopoietic systems (10). In addition, neoplasia, and endometrial hyperplasia. Compound mutant mice also germ-line mutations of PTEN underlie three overlapping human exhibited an expanded spectrum of tumor types including melanoma autosomal-dominant hamartoma tumor syndromes: Cowden syn- and squamous cell carcinoma. Functional synergy between Ink4a͞Arf drome, Bannayan–Zonana syndrome, and Lhermitte–Duclos dis- and Pten manifested most prominently in the development of pheo- ease (2, 10). A wide spectrum of human cancer types also exhibit chromocytoma, prompting an analysis of genes and loci implicated in INK4a͞ARF inactivation by mutation, deletion, or epigenetic this rare human neoplasm. The classical pheochromocytoma genes silencing, particularly in malignant gliomas, melanoma, head and Ret, Vhl, and Nf-1 remained intact, a finding consistent with the neck squamous carcinoma, and lymphoblastic leukemia (8, 11, 12). intersection of these genes with pathways engaged by Pten and It is notable that dual inactivation of PTEN and INK4a͞ARF tumor ͞ Ink4a Arf. Notably, conventional and array-comparative genomic suppressor genes is encountered in several human cancer types, a GENETICS hybridization revealed frequent loss of distal mouse chromosome 4 in mutational profile implying functional collaboration between these a region syntenic to human chromosome 1p that is implicated in tumor suppressors. Indeed, this possible synergy is consistent with human pheochromocytoma. This study provides genetic evidence of RAS activation and Ink4a͞Arf loss in melanoma genesis in the collaboration between Pten and Ink4a͞Arf in constraining the growth mouse (13), coupled with the well established biochemical inter- and oncogenic transformation of cultured cells and in suppressing a actions between RAS and PTEN pathways (14). In this study, we wide spectrum of tumors in vivo. examined the potential collaborative interactions between these prominent tumor suppressors on the cellular and organismal levels. he PTEN (also known as MMAC1 and TEP1) gene encodes a Tphosphatase that negatively regulates the phosphatidylinositol Materials and Methods 3-kinase (PI3K) pathway activity (1, 2). PI3K activates a variety of Cellular Assays for Growth and Transformation. Cellular assays were key signaling proteins such as the Ser͞Thr kinase AKT. Activated performed as described previously (9, 15, 18). For growth curves AKT in turn phosphorylates and modulates the activity of a number and low density seeding assays, early passage (PD Յ 9) MEFs of important molecules governing cell cycle control and cell survival were cultured in DMEM containing 4% FCS. including forkhead transcription factors and BAD, among other substrates (1, 2). Consistent with the role of Pten as a signaling Mutant Mouse Tumor Studies. The production of Pten and Ink4a͞Arf antagonist of the PI3K pathway, PtenϪ/Ϫ embryonic stem cells mutant mice has been described elsewhere (7, 9) and maintained on ͞ ͞ ͞ exhibit an increased growth rate and an accelerated G1 S transition. a mixed FVB n C57BL 6 background. Mice heterozygous for Pten In addition, its reintroduction in PTEN-deficient tumor cells down- and Ink4a͞Arf mutant alleles were intercrossed to generate all of modulates AKT activity and induces cell cycle arrest and͞or the genotypes analyzed in this study. apoptosis (2). In the mouse, Pten nullizygosity leads to early embryonic lethality, whereas Pten heterozygotes survive and de- Histopathology and Immunohistochemistry. Normal and tumor tis- velop neoplasia in multiple tissues including lymphoid and epithe- sue samples were processed for immunohistochemical analysis lial hyperplasias and cancers of the prostate, endometrium, intes- by standard techniques. EPOS anti-neuron-specific enolase tine, thyroid, adrenal gland, and breast (3–7). (Dako) and with anti-chromagranin A antibody (DiaSorin, The Ink4a͞Arf gene encodes two distinct tumor suppressors, Stillwater, MN) were used according to the manufacturer’s p16INK4a and p19ARF, that function as regulators of the pRB and instructions. Diaminobenzadine was used as the chromogen and p53 pathways, respectively (8). p16INK4a and other members of the INK4 family inhibit G1 cyclin D-dependent kinases 4 and 6, ͞ Abbreviations: MEF, mouse embryonic fibroblast; CGH, comparative genomic hybridiza- thereby preventing CDK4 6-directed pRB hyperphosphoryla- tion; PI3K, phosphatidylinositol 3-kinase; wt, wild type; H&E, hematoxylin and eosin. ARF tion and blocking S phase entry. p19 inhibits MDM2- ††To whom reprint requests should be addressed. E-mail: [email protected]. mediated degradation of p53 and plays an important role in the The publication costs of this article were defrayed in part by page charge payment. This apoptotic elimination of aberrantly cycling cells (8). Mice doubly article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. null for p16INK4a and p19ARF are viable but succumb to lym- §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.022632099 PNAS ͉ February 5, 2002 ͉ vol. 99 ͉ no. 3 ͉ 1455–1460 Downloaded by guest on September 30, 2021 and oncogenic susceptibility of cultured cells (1, 2, 8, 9, 17). To assess potential cooperative interactions on the cellular level, we examined the compound effects of Pten heterozygosity and Ink4a͞ Arf nullizygosity. Independently derived early-passage Ink4a͞ ArfϪ/ϪPtenϩ/Ϫ and Ink4a͞ArfϪ/ϪPtenϩ/ϩ MEFs were found to be indistinguishable with respect to morphology, growth rates, and saturation densities under standard culture conditions (data not shown). However, when grown in reduced serum conditions (4% rather than 10%), Ink4a͞ArfϪ/Ϫ Ptenϩ/Ϫ MEFs proliferated more rapidly, attained higher saturation densities, and generated 2.5-fold more colonies following low density seeding (P Ͻ 0.001) relative to Ink4a͞ArfϪ/ϪPtenϩ/ϩ controls (Fig. 1 A and B). Under the same conditions, no differences in cellular growth rates and colony formation were detected between Ink4a͞Arfϩ/ϩPtenϩ/Ϫ and wt MEFs (Fig. 1A). Additionally, H-RAS (G12V) transduction gen- Fig. 1. Growth potential of Ink4a͞ArfϪ/ϪPtenϩ/ϩ and Ink͞Arf4aϪ/Ϫ Ptenϩ/Ϫ MEFs. (A) Growth curve of MEFs. MEFs were plated at 25,000 cells per erated a 2- to 3-fold increase in the number of transformed foci (foci well in triplicate wells of 12-well plates and cultured for the indicated number that emerged more rapidly and attained a larger size) in cultures of days. Cells were fixed and stained with crystal violet. Adsorption at OD 540 derived from Ink4a͞ArfϪ/ϪPtenϩ/Ϫ embryos relative to Ink4a͞ was measured for each time point for individual MEF lines and directly ArfϪ/ϪPtenϩ/ϩ controls (Fig. 1C and data not shown, P Ͻ 0.001). correlates with cell numbers (15). Each symbol represents the mean of tripli- In contrast, H-RAS (G12V) transduction failed to generate trans- cate readings of six lines. The standard errors are indicated. (B) Colony for- formed foci in Ink4a͞Arfϩ/ϩPtenϩ/Ϫ and Ink4a͞Arfϩ/ϩPtenϩ/ϩ mation in MEFs. MEFS were plated at 3,500 cells per 6-well plate, cultured for MEFs (data not shown). Together, these findings suggest that Pten 12 days, and fixed and stained
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