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[Cancer Research 65, 787-796, February 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

Suppression of Melanotroph Carcinogenesis Leads to Accelerated Progression of Pituitary Anterior Lobe Tumors and Medullary Thyroid Carcinomas in Rb+/– Mice

Zongxiang Zhou1, Andrea Flesken-Nikitin1, Corinna G. Levine1, Elena N. Shmidt1, Jessica P. Eng1, Ekaterina Yu. Nikitina2, David M. Spencer2 and Alexander Yu. Nikitin1

1 Department of Biomedical Sciences, Cornell University, Ithaca, New York and 2 Department of Immunology, Baylor College of Medicine, Houston, Texas

Requests for reprints: Alexander Yu. Nikitin, Department of Biomedical Sciences, Cornell University, T2 014A Veterinary Research Tower, Ithaca, NY 14853-6401. Phone: 607-253-4347; Fax: 607-253-4212; E-mail: an58{at}cornell.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mice with a single copy of the retinoblastoma gene (Rb+/–) develop a syndrome of multiple neuroendocrine neoplasia. They usually succumb to fast-growing, Rb-deficient melanotroph tumors of the pituitary intermediate lobe, which are extremely rare in humans. Thus, full assessment of Rb role in other, more relevant to human pathology, neoplasms is complicated. To prevent melanotroph neoplasia while preserving spontaneous carcinogenesis in other types of cells, we have prepared transgenic mice in which 770-bp fragment of pro-opiomelanocortin promoter directs expression of the human RB gene to melanotrophs (TgPOMC-RB). In three independent lines, transgenic mice crossed to Rb+/– background are devoid of melanotroph tumors but develop the usual spectrum of other neoplasms. Interestingly, abrogation of melanotroph carcinogenesis results in accelerated progression of pituitary anterior lobe tumors and medullary thyroid carcinomas. A combination of immunologic tests, cell culture studies, and tumorigenicity assays indicates that {alpha}-melanocyte–stimulating hormone, which is overproduced by melanotroph tumors, attenuates neoplastic progression by decreasing cell proliferation and inducing apoptosis. Taken together, we show that cell lineage–specific complementation of Rb function can be successfully used for refining available models of stochastic carcinogenesis and identify {alpha}-melanocyte–stimulating hormone as a potential attenuating factor during progression of neuroendocrine neoplasms.

Key Words: endocrine effects • mouse models of cancer • multiple neuroendocrine neoplasia syndrome • Rb tumor suppressor • stochastic carcinogenesis


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The retinoblastoma gene (Rb) occupies special status because its discovery confirmed the concept of tumor suppressor genes and thereby became a milestone in understanding cancer as a genetic disease (reviewed in ref. 1). In addition to retinoblastoma, a childhood eye tumor, inactivation of the gene occurs in 95% of small cell lung carcinomas and, to a lesser extent, in mammary and prostate carcinomas, soft tissue sarcomas, and leukemias (reviewed in ref. 2). Intriguingly, besides small cell lung carcinoma, Rb deficiency or loss of heterozygosity for the Rb locus was described in several human tumors with neuroendocrine differentiation, including parathyroid carcinomas, medullary C-cell thyroid carcinomas, and pituitary anterior lobe tumors (3, 4).

Numerous upstream regulators and downstream effectors of Rb protein are oncogenes and tumor suppressor genes. Thus, defects in Rb-associated pathways are considered to be among the most common mechanisms contributing to cancer (reviewed in refs. 1, 5).

Like humans with germ line mutations, mice with a single copy of the intact Rb gene (Rb+/–) develop Rb-deficient tumors with nearly complete penetrance (6–8). However, the predominant tumors identified in Rb+/– mice derive from melanotroph precursor cells in the intermediate lobe of the pituitary gland rather than from retinoblasts (9, 10). Due to the high frequency and stochastic character of initiation, relatively synchronous development of melanotroph neoplasms, and the small size of the mouse pituitary intermediate lobe (~2 x 105 cells), melanotroph carcinogenesis has become an essential model for understanding carcinogenesis associated with Rb inactivation, for assessment of modifying factors, and for testing diagnostic and therapeutic approaches (10–22).

Albeit useful, this model of melanotroph carcinogenesis has several limitations. In humans, the intermediate lobe of the pituitary gland is rudimentary and melanotroph tumors are very rare. Furthermore, few cell types are regulated to proliferate or differentiate by immediate contacts with inhibitory nerve terminals similar to melanotrophs. Thus, extrapolating experimental results to other mouse neoplasms and comparative assessment of human pathology may be difficult. Fortunately, subsequent studies showed that Rb+/– mice develop neoplasms more commonly observed in human pathology, such as medullary thyroid carcinomas (10, 18, 20), hyperplasia of the adrenal medulla, pheochromocytomas (18, 20), {alpha}-glycoprotein subunit ({alpha}-GSU)–containing pituitary anterior lobe tumors (14, 18), parathyroid tumors (18), hyperplasia of pancreatic Langerhans islets (18), and pulmonary neuroendocrine cells (10, 18). These findings allowed us to conclude that Rb+/– mice develop a syndrome of multiple neuroendocrine neoplasia (18).

In spite of synchronous initiation of carcinogenesis in multiple neuroendocrine cell lineages, most Rb+/– mice succumb to fast-growing melanotroph tumors by ages 12 to 14 months (18). Thus, comprehensive evaluation of other neoplasias associated with Rb deficiency remains difficult. Furthermore, for some cell types, the role of Rb in carcinogenesis may be missed completely, because relevant neoplasms can be detected only in aging mice. To address these issues, we attempted to refine the Rb+/– model of spontaneous carcinogenesis by selective complementation of Rb function in melanotrophs.

Adequate functional activity of the human RB cDNA transgene was shown by rescue of tumor and embryonic lethal phenotypes in Rb+/– and Rb–/– mice, respectively (18, 21, 23, 24) . These results provide the premise that specific expression of additional RB in melanotrophs may prevent their tumor formation. To express RB in melanotrophs, we have chosen the pro-opiomelanocortin (POMC) promoter. POMC is a precursor for {alpha}-melanocyte-stimulating hormone ({alpha}-MSH), the hormone specifically expressed in mouse melanotroph cell lineage from gestational day 14.5 onward (25). POMC is expressed in adrenocorticotrophs of the pituitary gland and in a subset of hypothalamic and hindbrain neurons. However, earlier studies showed that the 770-bp fragment containing POMC sequence between –706 and +64 provides adequate and cell type–specific expression of the transgenes limited to the mouse pituitary gland (26, 27). Particularly, it has been shown that expression of SV40 large T antigen under control of the POMC promoter results in formation of melanotroph tumors only (26). Exclusively melanotroph carcinogenesis was also observed after Rb inactivation by Flp recombinase under the control of POMC promoter in Rbfrt/frt mice (27).

Thus, after intercrossing TgPOMC-RB transgenic mice with Rb+/– mice, it was expected that this newly generated strain will lack melanotroph tumors and therefore will live long enough to develop other tumors similar to their human counterparts. Rb+/–, TgPOMC-RB mice maintain the stochastic character of carcinogenesis initiation and therefore should allow modeling the entire process in the most faithful fashion. Using this model, we describe acceleration of neuroendocrine carcinogenesis in mice lacking melanotroph tumors and identify {alpha}-MSH as a likely tumor-attenuating endocrine factor overproduced by neoplastic melanotrophs. This observation may explain the unequal progression rate of multiple neuroendocrine neoplasms in Rb+/– mice and lead to a closer look at potential therapeutic applications of {alpha}-MSH and its synthetic agonists.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of the POMC-RB Transgene and Generation of Transgenic Mice. To generate the POMC-RB ß-globin pA plasmid, a SmaI-HindIII DNA fragment containing the 2.8-kb human RB cDNA and the 1.6-kb human ß-globin polyadenylation site (23, 24) was directionally cloned into SmaI-HindIII restricted pGEM7Z-POMCp containing the 770-bp rat POMC promoter (28). The 5.2-kb ApaI-HindIII POMC-RB transgene was isolated with GELase (Epicentre Technologies, Madison, WI) and injected into pronuclei of fertilized oocytes of superovulated C57BL/6 mice at a concentration of 2 ng/µL as described previously (24). Newborn transgenic pups were identified by PCR-based screening followed by verification of transgene structure and copy number with Southern blotting of DNA isolated from a 1 cm tail fragment. Selected transgenic lines were crossed with Rb+/– mice (B6.129S2-Rb1tm1Tyj/J, The Jackson Laboratory, Bar Harbor, ME), which were produced by backcrossing the original Rb1tm1Tyj mice (7) to C57BL/6 strain for 10 generations. All mice were maintained identically following the recommendations of the Institutional Laboratory Animal Use and Care Committee. The health of mice was monitored daily.

Genotyping of Mice. At postnatal days (PND) 7 to 10, TgPOMC-RB pups were identified by PCR genotyping using primers corresponding to sequence of RB/Rb exons 15 and 16, RB15,5'/Rb15,5' and RB16,3'/Rb16,3' (15). PCR amplification of transgenic RB cDNA and genomic Rb gene sequences results in 116- and 196-bp fragments, respectively. Rb+/– mice were identified by primers Rbint3fMU5' (5'-TAAGTGCACCATGTGCAATGCTTGA-3'), RI3M3' (5'-TTCAGGTGCCCATGTTCGGTCCCTA-3'), and RbpAM3' (5'-AGAACGAGATCAGCAGCCTCTGTTC-3'). These primers discriminate between mutant and wild-type Rb alleles resulting in 175 - and 122-bp fragments, respectively. DNA isolation and PCR conditions are as described previously (11). The PCR temperature profile was 94°C for 30 seconds, 60°C for 1 minute, and 72°C for 2 minutes with extension of the last cycle for 10 minutes at 72°C. PCR products were resolved on 3% agarose gel.

Protein Analyses. Immunoprecipitations were done as described previously (24). Supernatants were immunoprecipitated with either monoclonal antibody (mAb) 1F8 (Zymed, San Francisco, CA), which is specific for human RB, or rabbit polyclonal Ig fraction C-15 (Santa Cruz Biotechnology, Santa Cruz, CA) followed by protein-Sepharose CL-4B (Amersham Biosciences, Piscataway, NJ). Western immunoblottings were done with mAb 245 (BD Biosciences/PharMingen, San Diego, CA). C-15 and 245 antibody recognize both human RB (110 kDa) and mouse Rb (105 kDa).

Histologic Analyses. Moribund mice as well as those sacrificed according to schedule were anesthetized with avertin and, if necessary, subjected to cardiac perfusion at 90 mm Hg with PBS followed by phosphate-buffered 4% paraformaldehyde. After macroscopic evaluation during necropsy, tissues were embedded in paraffin, sectioned at 4 µm thickness, and stained with H&E (Mayer's hemalum). Serial sectioning and three-dimensional reconstruction of specimens were done as described previously (11, 18).

Immunohistochemical Analyses. A modified avidin-biotin peroxidase technique was used for immunohistochemical stainings on paraffin sections of paraformaldehyde-fixed tissues essentially as described previously (11, 18). See Supplementary Table 1 for details specific for each antibody.

Laser Microdissection-PCR. Paraffin sections (4 µm) were placed on plastic foil attached to glass slides, stained with H&E, and evaluated under a microscope. Tumor cells or surrounding normal tissue were microdissected using a blue laser (Laser Microdissection System, Leica AS, Heidelberg, Germany) and collected into caps of 0.6 mL Eppendorf tubes filled with lysis buffer, digested in proteinase K, and used for subsequent PCR amplification prepared as described previously (11).

Adrenocorticotropic Hormone Measurement and Body Weight Analyses. All mice were anesthetized with avertin at the same time of the day, and blood samples from the retrorbital venous plexus were collected to heparinized tubes. After centrifugation at 2,000 x g for 10 minutes at 4°C, plasma samples were collected and stored at –80°C. RIAs for adrenocorticotropic hormone (ACTH) were done by the diagnostic laboratory of Cornell University. Body weight analyses were done essentially as described in ref. 24.

Enzyme-Linked Immunospot Assay for Murine IFN-{gamma} and Estimation of Lymphocyte Subpopulations by Flow Cytometry. MultiScreen-HA plates (96-well, Millipore, Billerica, MA) were coated with 10 µg/mL purified rat anti-mouse IFN-{gamma} mAb (clone R4-6A2, BD Biosciences/PharMingen) and incubated with 2 x 105 splenocytes, RPMI 1640 supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamate, 20 mmol/L HEPES, 100 units/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B, and 5 ng/mL recombinant murine interleukin-2 (RDI Research Diagnostics, Flanders, NJ) for 24 hours at 37°C in a CO2 incubator after addition of pokeweed mitogen (10-100 µg/mL, Sigma, St. Louis, MO). After washing with PBS containing 0.1% Tween 20, plates were incubated overnight at 4°C with 5 µg/mL biotinylated rat anti-mouse IFN-{gamma} mAb (clone XMG1.2, BD Biosciences/PharMingen) followed by a 2-hour incubation with avidin alkaline phosphatase conjugate (1.25 µg/mL, Sigma) at room temperature. Spots were visualized with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium alkaline phosphatase substrate (Sigma), counted per triplicate well using a stereomicroscope, and normalized per 105 cells.

CD4+, CD8+, CD3{varepsilon}+, and B lymphocytes were detected by flow cytometry (FACSCalibur, Becton Dickinson, Mountain View, CA) of splenocytes using CD4-FITC, CD8-PE, CD3{varepsilon}-FITC, and B220-FITC antibodies (all from BD Biosciences), respectively, in accordance with standard procedures.

Cell Culture and Treatment with {alpha}-MSH. Cell line CTN3 was established from medullary thyroid carcinoma of 373-day-old Rb+/– Tg(POMC-RB)109Ayn male mouse using the approach described for establishing melanotroph tumor cell line MT-4 (11). Its Rb–/– status was verified by PCR-based genotyping. Cells were cultured in DMEM at 37°C in a humidified incubator with 5% CO2. For {alpha}-MSH (Sigma) treatment, the cells were collected by centrifugation, resuspended in DMEM, seeded into six-well dishes containing the desired concentration of {alpha}-MSH medium per well, and incubated at 37°C for 48 hours. Because full-length {alpha}-MSH and its synthetic analogue [Nle4,D-Phe7]-{alpha}-MSH NDP-{alpha}-MSH; (ref. 29) showed similar results in pilot experiments, the more stable NDP-{alpha}-MSH was used in the subsequent studies.

Proliferation and Apoptosis Assays on CTN3 Cells. Because CTN3 cells grow in floating and loosely attached aggregates, they were detached by gentle shaking cell culture dish, collected to 1.5 mL Eppendorf tube by centrifugation, and snap frozen in liquid nitrogen after being covered by a drop of OCT compound (Tissue-Tek, Sakura Finetek, Torrance, CA). Frozen sections of cell pellets were placed on SuperFrost Plus charged slides (Fisher Scientific, Pittsburgh, PA), fixed in 4% paraformaldehyde for 15 minutes followed by PBS wash, and processed for immunostaining. For the proliferation assay, CTN3 cells were incubated with 3 µg/mL bromodeoxyuridine (BrdUrd, Sigma) for 2 hours at 37°C, and BrdUrd was detected by staining with anti-BrdUrd antibody (1:50, Becton Dickinson) as described previously (11). For the apoptosis assay, cells were stained with rabbit polyclonal antibody recognizing activated cysteinyl aspartic acid protease-3 (cleaved caspase-3, Cell Signaling Technologies, Beverly, MA). Our pilot experiments using identification of apoptotic cells morphologically and by TUNEL assay determined that cleaved caspase-3 staining is an accurate marker of apoptosis in neuroendocrine cells.3

For estimation of apoptotic and BrdUrd indices, sections were collected with SPOT-RT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI) using 100x oil immersion objective and under the Zeiss Axioskop 2 Plus microscope. Ten digital images were collected for each slide and transferred to Photoshop 6.0 for manual counting of all cells (150 cells on average per field) and BrdUrd-labeled or apoptotic cells after overlaying a grid. All cell culture staining was done in duplicate, and each pellet, and subsequent sections, was evaluated independently.

Tumorigenicity Assays. Severe combined immunodeficient mice (4 to 5 weeks old females; The Jackson Laboratory) were injected with 5 x 106 CTN3 cells and were monitored for tumor formation daily. Forming tumors were measured in three dimensions with a caliper, and the volume was calculated using the formula: V = {pi}/ 6 (L x W x H). After tumors reached volume 0.04 cm3, NDP-{alpha}-MSH (1 µg/20 µL/g body weight in PBS) was given by i.p. injection daily. The experiments were terminated after tumors reached volume 0.4 cm3.

Statistical Analyses. All statistical analyses in this study were done with InStat 3.03 and Prism 4.02 software (GraphPad, Inc., San Diego, CA). Survival fractions were calculated using the Kaplan-Meier method, and survival curves were compared by log-rank Mantel-Haenszel tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of POMC-RB Transgenic Mice. A 5.2-kb transgene construct containing 770-bp rat POMC promoter, 2.8-kb human RB cDNA, and 1.6-kb human ß-globin polyadenylation site Fig. 1A) was used for preparing transgenic mice on C57BL/6 background. According to germ line transmission in subsequent crosses, five transgenic mice founded the transgenic lines. Because even 5% to 10% expression of exogenous RB can suppress tumor formation (21), which might be difficult to detect immunohistochemically, all five generated transgenic lines were screened for melanotroph neoplasia suppression by crosses to Rb+/– mice (Fig. 1B). All melanotroph neoplasms are likely to be initiated between gestational day 14 and PND 35, and early neoplastic lesions [foci of early atypical proliferation (EAP)] are detected in all Rb+/– mice by PND 120 (11). Thus, pituitary glands (at least six per group) of Rb+/–, TgPOMC-RB mice as well as those of their Rb+/– littermates have been examined for absence of EAP foci by morphologic evaluation of serial sections on PND 120 to 160. Although all Rb+/– mice had EAP foci and/or early invasive tumors in their intermediate lobe (Fig. 1C, top), no such lesions were observed in three transgenic lines [Tg(POMC-RB)109Ayn, Tg(POMC-RB)110Ayn, and Tg(POMC-RB)111Ayn]. Two other lines had somewhat reduced number of EAfP foci (1-2 versus 2-4 in Rb+/– littermates). All three transgenic lines devoid of melanotroph neoplasia were subjected to further characterization. According to Southern blotting, these lines contain unique single sites of transgene integration and carry 1 (line 111), 2 (line 110), or 12 (line 109) copies of the transgene. Because mice in these lines have an identical phenotype, they will be described as TgPOMC-RB unless otherwise indicated.



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  		Figure 1. Generation of TgPOMC-RB mice. A, structure and detection of POMC-RB transgene. POMC-RB transgene consists of the 0.8-kb rat POMC promoter followed by 2.8-kb human RB gene and 1.6-kb ß-globin polyadenylation site (top). Rb+/–, TgPOMC-RB transgenic mice are detected by PCR genotyping (bottom). Fragments (196 and 116 bp) are diagnostic for the mouse Rb gene and human RB cDNA, respectively. TgPOMC-RB transgenic mice (lanes 2 and 4-8). Fragments (175 and 122 bp) are diagnostic for mutant and wild-type mouse alleles, respectively. Rb+/– mice (lanes 1, 2, and 5-7). B, design for identification of TgPOMC-RB lines devoid of melanotroph tumors after cross with Rb+/– mice. At least six pituitary glands were collected on PND 120-160, serially sectioned, and examined for melanotroph EAP foci after staining with H&E. Mouse lines lacking EAP foci were subjected to further characterization, including long-term survival experiments with screening for multiple neuroendocrine neoplasia (see Results). C, melanotroph EAP foci are absent in the pituitary intermediate lobe of Rb+/–, Tg(POMC-RB)111Ayn mouse (Rb+/–, TgPOMC-RB) but easily detected in Rb+/– littermate (arrow, Rb+/–). HE, H&E. Human RB protein is detected in the nuclei of melanotroph cells (arrows) in the pituitary intermediate lobe of TgPOMC-RB mouse (TgPOMC-RB) but not wild-type C57BL/6 mouse (WT). Note that transgene expression is below detectable levels in some melanotroph cells. Immunohistochemical detection (IHC) with mAb 1F8, ABC Elite method, hematoxylin counterstaining. Bar, 50 µm for all images. AL, IL, and PL, anterior, intermediate, and posterior lobes of the pituitary gland, respectively. D, RB expression in the brain (lanes 1 and 5), lung (lanes 2 and 6), kidney (lanes 3 and 7), and spleen (lanes 4 and 8) tissues of 60-day-old mice containing either Tg(POMC-RB)110Ayn (lanes 1-4) or RTg (TgCMV*-1-RB and TgRB-tTA; lanes 5-8; ref. 24) mice. Immunoprecipitation (IP) with either mAb 1F8 or C-15 antiserum followed by Western blotting with mAb 245 antibodies. mAb 1F8 is specific for the human RB protein. mAb 245 and C-15 antiserum recognize both human and mouse RB. Bands (110 and 105 kDa) are specific for human RB and mouse Rb, respectively.

 
According to immunostaining, expression of human RB was detected only in melanotrophs of the intermediate lobe and few cells of the anterior lobe of the pituitary gland. Notably, not all melanotrophs expressed detectable amount of human RB (Fig. 1C, bottom), reaffirming our initial strategy of immediate screening for suppression of melanotroph neoplasms. No transgene expression was detected by immunoprecipitation-Western blotting analyses in the brain, lung, kidney, and spleen (Fig. 1D). These results are in a good agreement with earlier reports indicating that transgene expression directed by the 770-bp POMC fragment is limited to melanotrophs and adrenocorticotrophs of the pituitary gland (26, 27).

TgPOMC-RB Mice Are Phenotypically Normal. Two year-long observation of TgPOMC-RB mice followed by extensive pathologic evaluation of >40 organs and tissues did not find any significant deviations in their health and phenotypical traits compared with their littermates and published reports on health and pathology of C57BL/6 mice (30). Additional quantitative assays also revealed that TgPOMC-RB transgenic mice had no significant differences from sex-matched, wild-type littermates in the size of the intermediate lobe, density of melanotrophs, plasma levels of ACTH, number of adrenocorticotrophs in the pituitary anterior lobe, body and individual organ weights, and body length (Supplementary Fig. 1). Thus, there are no phenotypical indications that POMC-directed expression of RB has any adverse biological effects on mouse development and biology in studied transgenic lines.

Rb+/–, TgPOMC-RB Mice Develop Usual Spectrum of Neuroendocrine Neoplasms but No Melanotroph Tumors. In agreement with our initial screening for EAP foci, histologic evaluation of pituitary glands found no melanotroph tumors in 43 Rb+/–, TgPOMC-RB mice within age range of 286 to 537 days old, including 23 mice observed until their conditions became moribund. At the same time, other neuroendocrine neoplasias associated with Rb deficiency, such as medullary thyroid carcinomas, pheochromocytomas, and pituitary anterior lobe tumors, were observed with at least the same frequency (Fig. 2A; Table 1).



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  		Figure 2. Spectrum of neuroendocrine neoplasms in Rb+/–, TgPOMC-RB mice. A, histology of neoplasms stained with H&E. Medullary carcinoma of the thyroid gland (TG) invading vessel (arrow) and pheochromocytoma (arrow) of the adrenal gland (AG) in Rb+/–, TgPOMC-RB mice. AC, adrenal cortex. Tumor of the pituitary anterior lobe (AL) in Rb+/–, TgPOMC-RB is characterized by pronounced vascularization (arrow) and is histologically distinct from monomorphic solid melanotroph tumor with central necroses (arrow) in the pituitary intermediate lobe (IL) of Rb+/– mouse. Immunohistochemical detection of {alpha}-GSU (brown) but not ACTH in pituitary anterior lobe tumor (arrow) of Rb+/–, TgPOMC-RB mice. ABC Elite method, hematoxylin counterstaining. Bar, 50 µm for thyroid gland, anterior lobe, intermediate lobe, and {alpha}-GSU images and 100 µm for adrenal gland and ACTH images. See Table 2 for quantitative analysis. B, absence of the wild-type Rb allele (122-bp PCR product) in the pituitary anterior lobe tumor of Rb+/–, TgPOMC-RB mice. N, Rb+/– normal tissue (lane 1); T, tumor cells from pituitary anterior lobe of Rb+/–, TgPOMC-RB mice collected by laser microdissection (lanes 2-6); M, DNA marker. The 175-bp band corresponds to the mutant Rb allele.

 

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  		Table 2. Hormone expression in pituitary anterior lobe tumors (PALT) of Rb+/–, TgPOMC-RB mice and their Rb+/– littermates

 

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  		Table 1. Neoplasms in Rb+/–, TgPOMC-RB mice and their Rb+/– littermates on C57BL/6 background

 
To evaluate if the phenotype of pituitary anterior lobe tumors in Rb+/–, TgPOMC-RB mice is similar to that reported previously in Rb+/– mice (14, 18), nine anterior lobe tumors of Rb+/–, TgPOMC-RB mice were subjected to immunohistochemical analyses for content of pituitary hormones (Table 2). In agreement with earlier and current findings in Rb+/– mice, anterior lobe tumors of Rb+/–, TgPOMC-RB mice always contained {alpha}-GSU (9 of 9 animals, 50-100% positive cells; Fig. 2) and sometimes expressed growth hormone (3 of 9 animals, 5-40% positive cells) and ß-thyroid-stimulating hormone (5 of 9 animals, 5-90% positive cells). No expression of ACTH (Fig. 2A), follicle-stimulating hormone, and luteinizing hormone was detected in any tumor. The only variation from previously observed expression pattern (18) was detection of prolactin (7 of 9 animals, 5-80% positive cells). However, evaluation of anterior lobe tumors of Rb+/– mice on C57BL/6 background also revealed its expression. In agreement with our previous report (18), all five microdissected anterior lobe tumors from Rb+/–, TgPOMC-RB mice had no wild-type copy of mouse Rb (Fig. 2B). Taken together, the spectrum and phenotype of neuroendocrine neoplasms were not changed in mice Rb+/–, TgPOMC-RB mice devoid of melanotroph tumors.

Selective Suppression of Melanotroph Neoplasms Accelerates Progression of Pituitary Anterior Lobe Tumors and Medullary Thyroid Carcinomas. The major cause of death of Rb+/– mice is brainstem compression by melanotroph tumors (18). Therefore, it could be anticipated that absence of these neoplasm would increase mouse life span. Interestingly, no significant difference in survival rates between Rb+/– and Rb+/–, TgPOMC-RB mice was observed (Fig. 3).



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  		Figure 3. Survival of Rb+/–, TgPOMC-RB mice and their Rb+/– littermates. Median survivals are 382 and 368 days for Rb+/–, TgPOMC-RB (n = 30) and Rb+/– (n = 23) mice, respectively. P for log-rank comparisons of the survival curves is 0.387.

 
Thorough assessment of neuroendocrine carcinogenesis revealed that almost all Rb+/–, TgPOMC-RB mice developed large pituitary anterior lobe tumors (97%, 21 of 22 animals; Table 1), whereas only 62% (13 of 21 animals; P = 0.0093, Fisher's exact test) of Rb+/– mice had grossly detectable anterior lobe tumors in addition to pituitary melanotroph tumors (90%, 19 of 21 animals) by the time of their death. This difference is unlikely due to unequal initiation rate, because according to the evaluation of serial sections practically all Rb+/– mice (6 of 6 animals) carry early tumors and/or EAP foci at the same frequency (1-2 per gland) in the pituitary anterior lobe on PND 180 to 220. Similarly, both Rb+/–, TgPOMC-RB and Rb+/– mice developed thyroid C-cell tumors at the same frequency of 96% (Table 1). However, volume of Rb+/–, TgPOMC-RB mice (mean ± SE 0.3214 ± 0.004 cm3, n = 24) is larger than that of Rb+/– mice (0.1687 ± 0.009 cm3, n = 21; P < 0.0001, unpaired t test). Thus, prevention of melanotroph tumors accelerates progression of pituitary anterior lobe tumors and thyroid C-cell tumors formation in Rb+/–, TgPOMC-RB mice.

{alpha}-MSH Has Expected Immunosuppressive Effects in Rb+/– and Rb+/–, TgPOMC-RB Mice. Melanotroph tumors produce large amounts of functionally active {alpha}-MSH (9, 31). Because {alpha}-MSH is a known immunomodulator, we assessed its potential effects on immune system of Rb+/– and Rb+/–, TgPOMC-RB mice. In agreement with previous reports (reviewed in ref. 32), {alpha}-MSH administration reduced production of IFN-{gamma} by splenic lymphocytes (Supplementary Fig. 2A) and had little, if any, effect on proportions of lymphocyte subpopulations (Supplementary Fig. 2B). Given the consistency of these observations with previous reports, it would be difficult to explain the suppression of neuroendocrine carcinogenesis by systemic immunomodulatory effects of {alpha}-MSH.

{alpha}-MSH Decreases Proliferation, Induce Apoptosis, and Reduces Tumorigenicity of Medullary Thyroid Carcinoma. To test whether {alpha}-MSH has direct effects on neuroendocrine cells, we have established a new cell line CTN3 derived from medullary thyroid carcinoma developed in Rb+/–, Tg(POMC-RB)109Ayn mouse. Similar to primary tumors, this line expresses melanocortin receptor 1,4 which has the highest affinity for {alpha}-MSH and NDP-{alpha}-MSH among melanocortin receptors (33). According to the BrdUrd incorporation assay, NDP-{alpha}-MSH treatment resulted in dose-dependent decrease of CTN3 cell proliferation over the 10 and 1,000 nmol/L concentrations (Fig. 4A). Furthermore, based on detection of activated caspase-3, all concentrations of NDP-{alpha}-MSH (10 nmol/L to 1 µmol/L) caused an increase in the number of apoptotic cells, with the most prominent effect at 100 nmol/L NDP-{alpha}-MSH (Fig. 4B). The 1 µmol/L NDP-{alpha}-MSH concentration is considered supraphysiologic (34), which may explain its less pronounced effect on apoptosis.



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  		Figure 4. Effects of NDP-{alpha}-MSH on proliferation, apoptosis, and tumorigenicity of CTN3 cell line established from Rb-deficient medullary thyroid carcinoma. Proliferation (A) and apoptosis (B) of tumor cells detected by immunostaining (brown) with antibodies to BrdUrd (BrdU) and cleaved caspase-3 (Caspase-3), respectively, after treatment with either 100 nmol/L NDP-{alpha}-MSH (+) or saline (–), ABC Elite method, hematoxylin counterstaining. Bar, 25 µm for A (top) and B (top). For quantitative assessment, proliferation levels were measured as the percentage of BrdUrd-stained cells. A, lower left, BrdUrd index (BrdUI). Mean ± SE 18.72 ± 0.13 (0 nmol/L, control), 13.32 ± 0.13 (10 nmol/L), 11.18 ± 0.48 (100 nmol/L), and 8.80 ± 1.28 (1,000 nmol/L). All concentrations had significant two-tailed P values when compared with control: 0.0011 (10 nmol/L), 0.0042 (100 nmol/L), and 0.0164 (1,000 nmol/L). B, lower right, apoptotic index (AI) measured as the percentage of caspase-3-stained cells. Mean ± SE 5.21 ± 0.08 (0 nmol/L, control), 6.38 ± 0.02 (10 nmol/L), 9.82 ± 0.11 (100 nmol/L), and 7.85 ± 0.21 (1,000 nmol/L). All concentrations had significant two-tailed P values when compared with control: 0.0043 (10 nmol/L), 0.0008 (100 nmol/L), and 0.0068 (1,000 nmol/L). For estimation of BrdUrd index or apoptotic index, 10 images with area 10,384 µm2 each were analyzed in each sample. About 2,000 cells were analyzed in a single experiment. Experiments were done in triplicate. C, CTN3 cells (5 x 106) were injected to 4- to 5-week-old severe combined immunodeficient female mice. After tumors reached volume 0.04 cm3, NDP-{alpha}-MSH (1 µg/20 µL/g body weight in PBS) or PBS (20 µL/g body weight) was given by i.p. injection daily. Tumor dimensions were recorded daily, and the experiments were terminated after tumors reached volume 0.4 cm3. Median survivals are 10 and 9 days for Rb+/–, TgPOMC-RB mice treated with NDP-{alpha}-MSH (n = 14) and saline (n = 14), respectively. P for log-rank comparisons of the survival curves is 0.0384. D, effect of NDP-{alpha}-MSH on body weight of severe combined immunodeficient mice described in C. Body weights of PBS-treated group mice and NDP-{alpha}-MSH-treated group mice have no significant differences at any day (all Ps > 0.05). Normalized body weight relative unit (RU) is defined as percentage of values for the body weight of the control group mice just before treated with PBS.

 
To test the effects of {alpha}-MSH on tumorigenicity, CTN3 cells were injected under the skin of severe combined immunodeficient mice. Monitoring tumor formation after administration of NDP-{alpha}-MSH showed that {alpha}-MSH indeed attenuates tumor formation (Fig. 4C). At the concentration used, no effect on body weight was observed during the length of experiment (Fig. 4D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Studying Spontaneous Carcinogenesis by Selective Complementation of Gene Function. Evaluation of the genetic basis of cancer by conventional targeted mutagenesis, transgenesis, or genetic chimeras in the mouse is frequently complicated by embryonic lethality and/or early development and rapid progression of neoplasms with no or little resemblance to their human counterparts. Furthermore, mice with complete absence of a gene or carrying a large proportion of gene-deficient cells may adapt and compensate for missing gene functions during ontogenesis (reviewed in ref. 35). To circumvent these problems, several approaches aimed at somatic conditional inactivation of a tumor suppressor gene or activation of an oncogene have been developed during the past decade (reviewed in ref. 36). Such approaches rely on either cell type–specific expression Cre or Flp recombinase in cells carrying loxP or frt sites, respectively, or on reversible control of gene expression.

These methods have been crucial for advancing our understanding of cancer, addressing such topics as cell lineage specificity, acute effects of genetic alterations, genetic requirements for tumor progression and maintenance, and feasibility of gene therapeutic approaches. Unfortunately, all of these approaches rely on alteration of gene function in a large number of target cells. To date, the only exception is a mouse model carrying oncogenic alleles of K-ras that can be activated by a spontaneous recombination event in the whole animal (37). The relevance of such a process to natural mechanisms responsible for tumor initiation by spontaneous genetic mutation remains to be determined.

Carcinogenesis is usually considered to be a multistage process initiated by random inactivation of tumor suppressor genes or by activation of oncogenes and driven by sequential accumulation of genetic alterations responsible for expression of phenotypical traits beneficial for selection of the most autonomous and, by extension, most malignant cell clones (38, 39). Simultaneous alteration of a critical gene function in a large number of target cells may affect the natural course of cancer initiation resulting in biologically inaccurate host responses and/or formation of artificial microenvironments influencing the survival and growth of mutant cells. Therefore, modeling cell type–specific sporadic carcinogenesis from its initiating step remains a major unanswered challenge.

In an attempt to model naturally occurring spontaneous cancer initiation, we decided to test an approach of cell type–specific complementation of gene function. Using an extensive range of phenotypical characterization of transgene function and potential side effects, we show that the well-characterized model of neuroendocrine carcinogenesis in Rb+/– mice can be improved by preventing melanotroph carcinogenesis while preserving formation of other neoplasms associated with spontaneous Rb inactivation. Using such cell type–specific promoters as {alpha}-GSU and calcitonin/CGRP, it should be possible to prepare mice with even more restricted set of neoplasms associated with Rb deficiency.

In principle, a tissue-specific Cre transgene expression combined with heterozygosity for a conditional allele of Rb (RbloxP/+) would generate a situation in which stochastic loss of Rb could initiate carcinogenesis exclusively in a tissue of interest. In future studies, it would be of interest to test whether neoplasms require a "heterozygous" environment to develop. For example, it has been shown that loss of the neurofibromatosis 1 (Nf1) gene is sufficient to induce the development of schwannomas in mice only if the nonneoplastic cells are heterozygous for Nf1 (40).

Previous studies indicate pleiotropic functions of Rb in restriction of cell cycle progression, mediation of terminal differentiation, and control of cell survival (reviewed in refs. 1, 5). There is also an increasing body of evidence that Rb is involved in the control of genomic stability (41, 42). Although a critical role for the Rb gene in the initial stages of carcinogenesis is well established, the precise mechanisms by which loss of Rb occurs remain mostly uncharacterized. Only a subset of tumors, many of them with neuroendocrine phenotype, consistently exhibit Rb deficiency due to gene mutation. Therefore, spontaneous Rb inactivation and/or selection of mutant clones should be studied in the context of cell type specificity with the possibility of extrapolating observations to respective human neoplasms. Thus, we anticipate that "designer" Rb+/– mice with relevance to human pathology should be of particular interest for studying Rb role in spontaneous initiation.

Because requirements for progression of tumors can be defined by the size and composition of the pool of initiated cells, it will be of particular interest to compare gene expression profiles and genomic integrity of advanced spontaneous neuroendocrine tumors developing in our mice with those deriving as a consequence of Rb conditional inactivation and to establish their respective similarities with human neoplasms of the same type. Such studies may prove to be of additional value for rational design of pathway-based diagnostic and therapeutic approaches.

The ability to preserve the stochastic character of cancer initiation makes a selective complementation of tumor suppressor function a viable addition to current methods of tissue-specific gene alterations. Similar approaches should be applicable to other models with spontaneous inactivation of tumor susceptibility genes, such as p53, PTEN, MSH2, etc., which lead to a spectrum of neoplasms. Furthermore, because a wild-type copy of a proto-oncogene frequently has tumor-preventive effect (43, 44), this approach may be useful for modifying the spectrum of tumors in mice with spontaneous oncogene activation, such as those carrying latent oncogenic K-ras (37).

Endocrine (Paraneoplastic) Effects as Modifying Factors of Carcinogenesis. Sequential morphologic studies of pituitary anterior lobe and melanotroph tumors, thyroid C-cell carcinomas, and adrenal pheochromocytomas showed that early morphologically detectable stages of these neoplasms can be identified within a narrow developmental time period (PND 35-120), and loss of the wild-type Rb is critical in each case (18). However, clinically detectable neoplasms appear after a long latency period and progress at different rates in a cell type–dependent manner. Thus, it is likely that additional genetic and/or epigenetic changes must affect progression of Rb-deficient tumors in a cell type–dependent manner.

Several recent studies showed that neuroendocrine carcinogenesis in Rb+/– mice can be facilitated by inactivation of p53 (10), p27 (17), and Arf (45) and attenuated by loss of E2F1 (20), E2F3 (16), and E2F4 (19). Additional studies also indicated the importance of such factors as genomic imprinting (15), exposure to ethylnitrosourea (13), and genetic background (14). However, the role of possible endocrine effects on carcinogenesis in Rb+/– mice was not addressed.

Observation of accelerated carcinogenesis in mice lacking melanotroph tumors was not initially expected but is well corroborated by the most recent studies. It has been reported that the frequency of large melanotroph tumors is inversely correlated with those of anterior lobe tumors and thyroid carcinomas in mice with different genetic backgrounds (14). Similar correlation can be found by comparing distribution and frequency of tumors in our previous findings on Rb+/– mice bred on a mixed 129 x C57BL/6 background (18) with our present data using C57BL/6 mice. Observation of larger medullary thyroid carcinomas and pituitary anterior lobe tumors was also reported in E2f3–/– mice with attenuated formation of melanotroph tumors (16).

Endocrine effects are quite common in patients with multiple tumors. However, careful identification of cause and effect in humans is complicated because of challenges in identification of initiation and subsequent progression of independent tumors. Availability of Rb+/– mice lacking melanotroph neoplasms, as well as further modification of their tumor spectrum, should bring further insights into organism-wide effects of tumors associated with Rb deficiency in natural settings of stochastic carcinogenesis.

Attenuating Effects of {alpha}-MSH on Neuroendocrine Carcinogenesis. The acceleration of neuroendocrine carcinogenesis in mice lacking melanotroph tumors prompted us to examine a possibility that {alpha}-MSH has tumor-attenuating properties. Melanotroph tumors produce a large amount of {alpha}-MSH in Rb+/– mice (9, 11, 14, 31). The amount of circulating {alpha}-MSH increases with tumor progression and may be up to 50-fold greater than in control mice (9). Importantly, tumor-produced {alpha}-MSH is functionally active as evidenced by its effects on melanocyte pigmentation (31).5

{alpha}-MSH is a 13–amino acid peptide derived from the proteolytic processing of POMC. In addition to regulating pigmentation by stimulation of melanin synthesis, {alpha}-MSH has a well-established role in modulation of anti-inflammatory effects by regulating the production and actions of many proinflammatory cytokines, including IFN-{gamma}, and may act as an immunosuppressor (reviewed in refs. 32, 33). Our studies have confirmed that {alpha}-MSH indeed inhibits production of IFN-{gamma}. We could not find any unexpected effects of {alpha}-MSH on the immune system based on an assessment of lymphocyte subpopulations. Based on the immunosurveillance concept of cancer control, normalization of {alpha}-MSH production would likely lead to deceleration of carcinogenesis, in direct contradiction to our observations. Hence, the effects of {alpha}-MSH on tumor progression in our mouse model are unlikely to be due to immunomodulation.

{alpha}-MSH is also reported to have anorectic effect (reviewed in ref. 33). There have been several studies emphasizing that dietary restriction results in decreasing carcinogenesis (reviewed in ref. 46). However, dietary restriction lacked an appreciable effect on neuroendocrine carcinogenesis associated with Rb deficiency even on 45% to 50% reduction in dietary intake (47). Importantly, systemic administration of {alpha}-MSH did not reduce body weight of mice in our tumorigenicity studies, indicating that at concentrations used in our experiments its anorexic effect is minimal, if any.

Although we cannot exclude that the pleiotropic systemic effects of {alpha}-MSH may affect neuroendocrine carcinogenesis in Rb+/– mice, our current data suggest the more likely interpretation that {alpha}-MSH influences tumor growth directly. It has been extensively reported that {alpha}-MSH inhibits proliferation of melanoma cells and may affect some of their tumorigenic properties, such as motility, anchorage-independent growth, and invasion (48, 49). {alpha}-MSH receptors are present in many tissues (reviewed in ref. 33). However, potential effects of {alpha}-MSH on other tumor types have not been reported with the exception of its autocrine inhibitory influence on malignant pleural mesothelioma (50). As presented in our study, {alpha}-MSH reduces proliferation, increases apoptosis, and delays growth of medullary thyroid carcinoma in severe combined immunodeficient mice. Besides demonstrating tumor attenuation properties of {alpha}-MSH in our model, these findings raise an intriguing possibility that {alpha}-MSH may have similar effects on other tumors, particularly those with neuroendocrine differentiation. Because the effects of {alpha}-MSH on tumor growth observed in our experiments are significant but not dramatic, one could envision its application as a part of combinatorial therapeutic approaches.

Taken together, using the approach of selective complementation of Rb function, we describe a new "humanized" model for studying neuroendocrine carcinogenesis associated with spontaneous Rb deficiency. As illustrated by the identification of tumor-attenuating effects of {alpha}-MSH, this approach can be also useful for identification and characterization of endocrine effects in the context of multiple neoplasms.


   Acknowledgments
 
Grant support: NIH grants R01 CA96823 and National Center for Research Resources, NIH Midcareer Award in Mouse Pathobiology K26 RR017595 (A.Yu. Nikitin).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Malcolm J. Low for the kind gift of rPOMC promoter; Dr. Albert F. Parlow for providing NHPP antibodies; Dr. David MacPherson for sharing sequence information of Rb KO cassette; Dr. David W. Goodrich for critical reading of the article; Lori A. Cesario and David C. Corney for expert technical assistance; Ryo Hayama for ACTH immunohistochemical-positive cell counting; Dr. Stephen V. Lamb for help with measurements of plasma ACTH; and Kyung-Chul Choi, Daniel Kuprienko, Vasanth Sriram, Matthew Baron, and Alexander Urban for their histotechnological support.


   Footnotes
 
Note: Supplementary data for this article can be found at Cancer Research Online (http://cancerres.aacrjournals.org).

3 C.G. Levine and Nikitin, unpublished observations. Back

4 Our unpublished observations. Back

5 Our unpublished observations. Back

Received 9/27/04. Revised 11/19/04. Accepted 11/29/04.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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