Knockout of MCT1 results in total absence of spermatozoa, sex hormones dysregulation, and morphological alterations in the testicular tissue.

Lactate is a key metabolite for the normal occurrence of spermatogenesis. In the testis, lactate is produced by the Sertoli cells and transported to germline cells. Monocarboxylate transporters (MCTs) are key players in that process. Among the family of MCTs, MCT1 is at least partly responsible for lactate uptake by the germ cells. We aimed to perform a first assessment of the role of MCT1 in male reproductive potential. Mct1 conditional knockout (cKO) mice were used for morphometric evaluation, testicular morphology, and sperm parameter assessment. Serum steroid hormones levels were also measured. cKO animals showed a decrease in gonadosomatic index, testis weight, and seminiferous tubular diameters. Deletion of MCT1 also causes morphological changes in the organization of the seminiferous tubules and on Sertoli cell morphology. These changes resulted in failure of spermatogenesis with depletion of germ cells and total absence of spermatozoa. MCT1 cKO animals presented also hormonal dysregulation, with a decrease in serum 17ß-estradiol levels. In conclusion, MCT1 is pivotal for male reproductive potential. Absence of MCT1 results in maintenance of undifferentiated spermatogonia pool and compromised sperm production.

Identification of functionally distinct TRAF proinflammatory and phosphatidylinositol 3-kinase/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (PI3K/MEK) transforming activities emanating from RET/PTC fusion oncoprotein.

Thyroid carcinomas that harbor RET/PTC oncogenes are well differentiated, relatively benign neoplasms compared with those expressing oncogenic RAS or BRAF mutations despite signaling through shared transforming pathways. A distinction, however, is that RET/PTCs induce immunostimulatory programs, suggesting that, in the case of this tumor type, the additional pro-inflammatory pathway reduces aggressiveness. Here, we demonstrate that pro-inflammatory programs are selectively activated by TRAF2 and TRAF6 association with RET/PTC oncoproteins. Eliminating this mechanism reduces pro-inflammatory cytokine production without decreasing transformation efficiency. Conversely, ablating MEK/ERK or PI3K/AKT signaling eliminates transformation but not pro-inflammatory cytokine secretion. Functional uncoupling of the two pathways demonstrates that intrinsic pro-inflammatory pathways are not required for cellular transformation and suggests a need for further investigation into the role inflammation plays in thyroid tumor progression.

Oncoprotein signaling mediates tumor-specific inflammation and enhances tumor progression.

The RET/PTC3 (RP3) fusion protein is an oncogene expressed during the development of thyroid cancer and in thyroid epithelial cells of patients with Hashimoto's thyroiditis. RP3 has two immunological properties: 1) it encodes a chimeric protein including peptides that may be targets of antitumor immune responses and 2) it is a tyrosine kinase that can activate NF-kappaB transcriptional programs, induce secretion of proinflammatory mediators, and stimulate innate immunity. To distinguish the antigenic properties of the RP3 oncoprotein from its signaling function, a transplantable tumor system was developed. Tumors expressing the functional, but not mutant, form of RP3 show enhanced infiltration of CD8(+) lymphocytes, myeloid-derived CD11b(+)Gr1(+) cells, and enhanced growth in immunocompetent mice. In contrast, RP3 signaling mutant-expressing tumors maintained enhanced infiltration of CD8(+) lymphocytes did not enhance recruitment of CD11b(+)Gr1(+) cells and showed a decreased tumor incidence. These results implicate a role for RP3 function in enhancing a tumor-suppressive innate inflammatory response. These experiments support a mechanism whereby oncogenes can directly recruit and activate innate and adaptive immune cells, resulting in enhanced tumor progression.

Impairment of the p27kip1 function enhances thyroid carcinogenesis in TRK-T1 transgenic mice.

Impairment of the p27(kip1) function, caused by a drastic reduction of its expression or cytoplasmic mislocalization, has been frequently observed in thyroid carcinomas. To understand the role of p27(kip1) impairment in thyroid carcinogenesis, we investigated the consequences of the loss of p27(kip1) expression in the context of a mouse modeling of papillary thyroid cancer, expressing the TRK-T1 oncogene under the transcriptional control of thyroglobulin promoter. We found that double mutant mice homozygous for a p27(kip1) null allele (TRK-T1/p27(-/-)) display a higher incidence of papillary thyroid carcinomas, with a shorter latency period and increased proliferation index, compared with p27(kip1) wild-type compounds (TRK-T1/p27(+/+)). Consistently, double mutant mice heterozygous for a p27(kip1) null allele (TRK-T1/p27(+/-)) show an incidence of thyroid carcinomas that is intermediate between TRK-T1/p27(-/-) and TRK-T1/p27(+/+) mice. Therefore, our findings suggest a dose-dependent role of p27(kip1) function in papillary thyroid cancer development.

RET/papillary thyroid cancer rearrangement in nonneoplastic thyrocytes: follicular cells of Hashimoto's thyroiditis share low-level recombination events with a subset of papillary carcinoma.

CONTEXT: RET/papillary thyroid cancer (PTC) is a marker for papillary thyroid carcinoma, but its specificity has been questioned because of the disputed identification of RET/PTC in Hashimoto's thyroiditis (HT), oncocytic tumors, and other thyroid lesions. OBJECTIVE: The objective of this study was to determine 1) whether RET/PTC occurs in nonneoplastic follicular cells of HT, and 2) its recombination rate in thyroid tumors. DESIGN/PATIENTS: Forty-three samples from 31 cases of HT were examined using interphase fluorescence in situ hybridization (FISH) with RET probes spanning the breakpoint region; real-time RT-PCR to quantify RET/PTC1, RET/PTC3, and c-RET transcripts; and RT-PCR after laser capture microdissection to enrich samples for follicular cells. The results were compared with those similarly obtained in 34 papillary carcinomas, eight thyroid oncocytic tumors, and 21 normal thyroids. RESULTS: Normal samples showed no RET rearrangement. Sixty-eight percent (15 of 22) of HT were positive by FISH; in all thyroiditis, signals were localized to rare nonneoplastic follicular cells; low-level RET/PTC was identified in 17% (five of 29) of thyroiditis cases by real-time RT-PCR and in an additional six of 11 real-time negative cases after increasing sensitivity with laser capture microdissection. Low RET/PTC1 levels were detected in 26% (nine of 34) of papillary carcinomas with an expression pattern and proportion of FISH-positive cells similar to those of the thyroiditis. Forty-seven percent (16 of 34) of papillary carcinomas and one oncocytic carcinoma expressed high RET/PTC1 mRNA levels. CONCLUSIONS: Low-level RET/PTC recombination occurs in nonneoplastic follicular cells in HT and in a subset of papillary thyroid carcinomas. RET/PTC expression variability should be taken into account for the molecular diagnosis of thyroid lesions. Overlapping molecular mechanisms may govern early stages of tumor development and inflammation in the thyroid.

Oncogenic inflammation and autoimmune disease.

Many models exist to explain the induction and perpetuation of autoimmune diseases. Despite their validation in a variety of animal models, the basis for autoimmune disease in humans remains unknown. Here, we propose that an important aspect of autoimmune disease is the active participation of the target organ due to endogenously produced co-stimulatory factors that cause prolonged antigen presentation and lymphocyte activation. Evidence suggests that a major source of such endogenous signaling comes from newly transformed cells within the target organ that produce pro-inflammatory factors.

Interleukin 24 is induced by the RET/PTC3 oncoprotein and is an autocrine growth factor for epithelial cells.

Thyroid cancers, like hematological malignancies, are commonly associated with chromosomal translocations leading to the formation of fusion proteins. Through altered signaling by fusion proteins, cell death and survival pathways are disrupted and the physiological balance of cell-cell communication may be lost. A consequence of this disruption is the release of factors by stressed cells that alert the host. One type of host response is leukocytic infiltration that may develop into chronic inflammation or autoimmune disease. Although inflammation can be associated with neoplastic tissue, the mechanism driving this process is largely unknown. Therefore, to address the mechanism of cancer inflammation we investigated the effects of an oncogene in a murine model system. A comprehensive genetic analysis revealed several soluble factors that were induced by RET/papillary thyroid carcinoma (PTC)3 gene expression including several proinflammatory cytokines, chemokines and immunologically relevant costimulatory molecules. Following a large genetic screen using RP3-expressing thyroid cells, we identified a highly abundant transcript and later identified it as interleukin 24 (Il24), a cytokine with diverse tumor suppressor and inflammatory activities. We show that RET/PTC3 induces Il24 expression in rat thyrocytes and that this expression is dependent on the signaling properties of its tyrosine kinase. Likewise, RET/PTC3 induces large amounts of Il24 following expression in murine thyrocytes, but its expression is dramatically reduced in poorly differentiated carcinomas, a finding that parallels the loss of RET/PTC3 expression. Consistent with its behavior as a tumor suppressor, the loss of Il24 coincided with the loss of RET/PTC3 in poorly differentiated mouse tumors. A functional role of Il24 in the autocrine growth/survival of RET/PTC3-expressing thyroid cells was identified helping to support its role in cellular transformation. These data suggest that the induction of Il24 by oncogenes may support tumor growth at the early stages of cancer.

Tyrosine kinase oncoprotein, RET/PTC3, induces the secretion of myeloid growth and chemotactic factors.

Differentiated thyroid carcinomas are the most frequent endocrine neoplasms, but account for few cancer-related deaths. Although the indolent growth of these cancers correlates well with longevity, the biological basis for this good prognosis is not known. In contrast, two of the most frequent autoimmune diseases involve the thyroid suggesting a high propensity for this organ to invoke destructive immunity. Unfortunately, the mechanism linking malignancy and autoimmunity is not clear, although the expression of the oncogenic fusion protein RET/PTC3 (RP3) in both of these disorders may provide a clue. Interestingly, the signaling caused by activated RET kinase involves overlapping pathways and some common to the inflammatory response. Accordingly, we analyzed the function of RP3 and a mutant RP3 molecule to induce proinflammatory pathways in thyroid epithelial cells. Indeed, we find that RP3 alone causes increases in nuclear NF-kappaB activity and secretion of MCP-1 and GM-CSF. Finally, transfer of RP3-expressing thyrocytes into mice in vivo attracted dense macrophage infiltrates, which lead to rapid thyroid cell death. Further, cytokine synthesis and inflammation was largely abrogated by mutation of RP3 Tyr588; an important protein-binding site for downstream signaling. Together, these studies implicate oncogene-induced cytokine-signaling pathways in a new mechanism linking inflammation with cancer.

Functional expression of the CXCR4 chemokine receptor is induced by RET/PTC oncogenes and is a common event in human papillary thyroid carcinomas.

To identify genes involved in the transformation of thyroid follicular cells, we explored, using DNA oligonucleotide microarrays, the transcriptional response of PC Cl3 rat thyroid epithelial cells to the ectopic expression of the RET/PTC oncogenes. We found that RET/PTC was able to induce the expression of CXCR4, the receptor for the chemokine CXCL12/SDF-1alpha/beta. We observed that CXCR4 expression correlated with the transforming ability of the oncoprotein and depended on the integrity of the RET/PTC-RAS/ERK signaling pathway. We found that CXCR4 was expressed in RET/PTC-positive human thyroid cancer cell lines, but not in normal thyroid cells. Furthermore, we found CXCR4 expression in human thyroid carcinomas, but not in normal thyroid samples by immunohistochemistry. Since CXCR4 has been recently implicated in tumor proliferation, motility and invasiveness, we asked whether treatment with SDF-1alpha was able to induce a biological response in thyroid cells. We observed that SDF-1alpha induced S-phase entry and survival of thyroid cells. Invasion through a reconstituted extracellular matrix was also supported by SDF-1alpha and inhibited by a blocking antibody to CXCR4. Taken together, these results suggest that human thyroid cancers bearing RET/PTC rearrangements may use the CXCR4/SDF-1alpha receptor-ligand pathway to proliferate, survive and migrate.

Cyclooxygenase-2 expression in human thyroid carcinoma and Hashimoto's thyroiditis.

OBJECTIVES: Cyclooxygenases (COX) are enzymes that catalyze the conversion of arachidonic acid to prostaglandins. COX-2, unlike the constitutively expressed COX-1, is an inducible enzyme upregulated during cell proliferation and inflammation. More recently, COX-2 has been implicated in the development of numerous types of epithelial cancers. In addition, COX-2 is highly expressed in several inflammatory diseases. Because of its dual role in inflammation and cancer, we were interested in determining if COX-2 plays a role in the development of human thyroid carcinoma and Hashimoto's thyroiditis, an autoimmune condition frequently associated with thyroid malignancy. MATERIALS AND METHODS: Twenty paraffin-embedded human tissue specimens, including normal, inflammatory, and neoplastic thyroid sections, were analyzed by immunohistochemical staining for expression of human COX-2. In addition, COX-2 protein expression was verified by Western blot in two specimens. RESULTS: Immunohistochemical staining confirmed the presence of COX-2 in thyroid epithelial neoplasms, including papillary and follicular carcinomas. Moreover, COX-2 expression was observed in patients with Hashimoto's thyroiditis. COX-2 expression, however, was not observed in normal thyroid tissue, multinodular goiter, or anaplastic carcinoma. CONCLUSIONS: We have shown that cyclooxygenase-2 is expressed in thyroid carcinoma and thyroid epithelium from patients with Hashimoto's thyroiditis but not in normal thyroid. The expression of COX-2 in both of these thyroid pathologies may provide a basis for the relationship between carcinogenesis and autoimmunity.

Inhibition of oncogene-induced inflammatory chemokines using a farnesyltransferase inhibitor.

BACKGROUND: Farnesyltransferase inhibitors (FTI) are small molecule agents originally formulated to inhibit the oncogenic functions of Ras. Although subsequent analysis of FTI activity revealed wider effects on other pathways, the drug has been demonstrated to reduce Ras signaling by direct measurements. The purpose of the current study was to determine if FTI could be used to inhibit the inflammatory activities of a known Ras-activating human oncoprotein, RET/PTC3. RET/PTC3 is a fusion oncoprotein expressed in the thyroid epithelium of patients afflicted with thyroid autoimmune disease and/or differentiated thyroid carcinoma. Previous studies have demonstrated that RET/PTC3 signals through Ras and can provoke nuclear translocation of NFkappaB and the downstream release of pro-inflammatory mediators from thyroid follicular cells in vitro and in vivo, making it an ideal target for studies using FTI. METHODS: For the studies described here, an in vitro assay was developed to measure FTI inhibition of RET/PTC3 pro-inflammatory effects. Rat thyrocytes transfected with RET/PTC3 or vector control cDNA were co-cultured with FTI and examined for inhibition of chemokine expression and secretion measured by RT-PCR and ELISA. Immunoblot analysis was used to confirm the level at which FTI acts on RET/PTC3-expressing cells, and Annexin V/PI staining of cells was used to assess cell death in RET/PTC3-expressing cells co-cultured with FTI. RESULTS: These analyses revealed significant mRNA and protein inhibition of chemokines Ccl2 and Cxcl1 with nanomolar doses of FTI. Neither RET/PTC3 protein expression nor apoptosis were affected at any dose of FTI investigated. CONCLUSION: These data suggest that FTI may be applied as an effective inhibitor for RET/PTC3-oncogene induced pro-inflammatory mediators.

Guanylyl cyclase C-induced immunotherapeutic responses opposing tumor metastases without autoimmunity.

BACKGROUND: One of the greatest impediments to cancer immunotherapy is the paucity of antigens that are tumor specific, sufficiently immunogenic, and shared among patients. Mucosa-restricted antigens that are expressed by tumor cells represent a novel class of vaccine targets that are characterized by immunologic privilege, which limits systemic tolerance to those antigens, and immunologic partitioning, which shields mucosae from systemic autoimmune responses. Here we defined the immunogenicity and antitumor efficacy of guanylyl cyclase C (GCC), a protein that is normally restricted to intestinal mucosa and universally expressed by metastatic colorectal cancer. METHODS: BALB/c mice (n = 197) were immunized with recombinant GCC-expressing viral vectors before (prophylactic) or after (therapeutic) a lethal challenge of GCC-expressing mouse colon cancer cells, and antitumor efficacy was monitored by quantifying metastasis and survival. Induction of autoimmunity was monitored by histopathology. Induction of GCC-specific B-cell and CD4(+) and CD8(+) T-cell responses were determined by enzyme-linked immunosorbent assay and ELISpot, respectively. Tolerance to GCC was quantified by comparing responses in GCC-deficient (n = 45) and wild-type (n = 69) C57BL/6 mice. Statistical tests were two-sided. RESULTS: Immunization with GCC-expressing viral vectors reduced the formation of metastases to liver (control vs GCC: mean = 30.4 vs 3.55 nodules, difference = 26.9 nodules, 95% confidence interval [CI] = 8.47 to 45.3 nodules; P = .008) and lung (control vs GCC: mean = 263 vs 55.7 nodules, difference = 207, 95% CI = 163 to 251; P < .001) and extended the median survival of mice with established lung metastases following therapeutic immunization (control vs GCC: 29 vs 38 days, P = .024), without autoimmunity. Antitumor efficacy reflected asymmetrical tolerance that was characterized by CD8(+) T-cell, but not CD4(+) T-cell or antibody, responses. CONCLUSIONS: Immunologic partitioning together with immunologic privilege highlight the potential of mucosa-restricted antigens, particularly GCC, as therapeutic targets for metastatic cancer.

Mucosally restricted antigens as novel immunological targets for antitumor therapy.

Colorectal cancer is the third most common malignancy and the second most common cause of cancer-related mortality worldwide. While surgery remains the mainstay of therapy, approximately 50% of persons who undergo resection develop parenchymal metastatic disease. Unfortunately, current therapeutic regimens offer little improvement in survival. Using immunotherapy to fill this therapeutic gap has enjoyed limited success, reflecting a paucity of tumor-associated antigens. In that context, there is a significant unrealized opportunity to exploit structural and functional immune system compartmentalization to generate a therapeutic immune response against metastatic colorectal tumors employing biomarkers whose expression is normally confined to intestinal epithelial cells and their derivative malignancies. This novel class of biomarkers, here termed cancer mucosa antigens, may fill the unmet therapeutic need for colorectal cancer-associated immune targets. As a concrete example, guanylyl cyclase C is an intestinal mucosa-specific biomarker ideally suited to test this hypothesis and serve as the first cancer mucosa antigen for colorectal cancer immunotherapy. Here, we discuss colorectal cancer immunity, immune compartmentalization and preliminary results targeting guanylyl cyclase C in mouse models of colorectal cancer, as well as the potential paradigm shift to employing cancer mucosa antigens in immunotherapy of colorectal cancer.

Analysis of expansion of myeloid progenitors in mice to identify leukemic susceptibility genes.

The myeloid progenitor cell compartment (MPC) exhibits pronounced expansion in human myeloid leukemias. It is becoming more apparent that progression of myelodysplastic syndromes and myeloproliferative diseases to acute myelogenous leukemia is the result of defects in progenitor cell maturation. The MPC of bone marrow was analyzed in mice using a cell culture assay for measuring the relative frequency of proliferative myeloid progenitors. Response to the cytokines SCF, IL-3, and GM-CSF was determined by this assay for the leukemic mouse strain BXH-2 and ten other inbred mouse strains. Significant differences were found to exist among ten inbred mouse strains in the nature of their MPC in bone marrow, indicating the presence of genetic polymorphisms responsible for the divergence. The SWR/J and FVB/J strains show consistently low frequencies of myeloid progenitors, while the DBA/2J and SJL/J inbred strains show consistently high frequencies of myeloid progenitors within the bone marrow compartment. In addition, in silico linkage disequilibrium analysis was conducted to identify possible chromosomal regions responsible for the phenotypic variation. Given the importance of this cell compartment in leukemia progression and the soon to be released genomic sequence of 15 mouse strains, these differences may provide a valuable tool for research into leukemia.

RET/PTC fusion gene rearrangements in Japanese thyroid carcinomas.

The activation of RET proto-oncogene through chromosomal translocation is reported as being unique to papillary thyroid carcinomas. However, the reported prevalence of RET/PTC activation in papillary carcinoma was variable, and the clinical relevance of RET/PTC rearrangements in papillary carcinomas is still controversial. To investigate the roles of RET rearrangement in the carcinogenesis of papillary thyroid carcinoma, we have studied RET activation and p53 overexpression in various thyroid lesions of the Japanese population by immunohistochemical technique. RET activation and p53 overexpression were studied in 40 papillary carcinomas, 6 poorly differentiated carcinomas, 4 undifferentiated carcinomas, 2 medullary carcinomas, 2 follicular carcinomas and 19 follicular adenomas. RET activation was observed in 12 out of 40 papillary carcinomas, while no immunoreactivity of RET was detected in other lesions. P53 overexpression was observed in only 1 of 40 papillary carcinomas, but in 2 poorly differentiated carcinomas and 4 undifferentiated carcinomas. The prevalence of RET/PTC activation in papillary carcinoma among the Japanese population was higher than in previous reports. Immunohistochemical technique is proved to be a useful tool to detect RFT/PTC activation in thyroid tumors. RET rearrangements are restricted to a well-differentiated papillary carcinoma, suggesting that RET/PTC positive papillary carcinomas do not progress to undifferentiated carcinoma.

Proinflammatory mediators and genetic background in oncogene mediated tumor progression.

RET/PTC3 (RP3) is an oncogenic fusion protein which is frequently expressed in papillary thyroid carcinomas and has been detected in thyroid tissue from patients diagnosed with Hashimoto's thyroiditis. The constitutive activation of the tyrosine kinase domain in the carboxyl-terminal end of RP3 induces signaling pathways within thyrocytes and causes cellular transformation. One of the signaling pathways activated in RP3-expressing cells involves the activity of the transcription factor NF-kappaB and the production of downstream targets including GM-CSF and macrophage chemotactic protein 1. These factors are known to be immunostimulatory, making RP3 a molecular adjuvant and potentially promoting tissue-specific immunity. However compelling, these in vitro data do not reliably predict gene function in vivo or the cumulative effects of time-dependent processes such as angiogenesis, inflammation, or the influence of genetic background. To address these issues, we analyzed the production of proinflammatory mediators in mouse thyroid organs and demonstrate consistency with in vitro studies performed previously that Il1alpha, Il1beta, Il6, and Tnfalpha and the enzyme Cox2 are produced by RP3-transgenic thyroid tissue, but absent from nontransgenic thyroids. Furthermore, we find that that the genetic background of the host is important in the observed RP3-induced inflammation and tumor progression. These findings provide support for the notion that oncogene-induced cytokine secretion is important for the development and progression of thyroid carcinomas in genetically permissive hosts.

A thyroid tumor-specific antigen formed by the fusion of two self proteins.

Thyroid epithelial cells frequently express one or more members of the rearranged during transfection/papillary thyroid carcinoma (RET/PTC) fusion oncogene family during early stages of cancer, and fusion gene transcripts have been found in inflammatory conditions of the thyroid such as the autoimmune disease, Hashimoto's thyroiditis. Because these oncogenes encode chimeric proteins, novel RET/PTC epitopes may be targets of antitumor immune responses. We have been interested in the RET/PTC3 (RP3) fusion protein because this family member is more frequently expressed in radiation-induced and childhood papillary carcinomas than other members of the fusion oncogene family. We hypothesized that the activated kinase of c-RET, in the form of RP3, when expressed in patients with thyroid disease, presents an unusual altered self target for T cell recognition. Interestingly, we find that immunization with mouse RP3 protein can induce a strongly immunogenic response to RP3, although this response is not directed against the peptide comprising the unique fusion region. Rather, the responses are specific for the carboxyl-terminal portion of RP3 that is derived from the self protein c-RET. Furthermore, transplantation of RP3-expressing thyroid tumors into naive mice resulted in leukocytic infiltration, tumor rejection, and induction of RP3-specific T cells. Thus, the somatic fusion of two unrelated self proteins results in the development of a uniquely immunogenic response directed against self epitopes within RP3. These studies may better define the mechanisms controlling the initiation of thyroid-specific immune responses and provide insight into the design of novel molecules for invoking tumor-specific immunity.

A novel transgenic line of mice exhibiting autosomal recessive male-specific lethality and non-alcoholic fatty liver disease.

We have isolated a Meis1a transgenic mouse line exhibiting recessive male-specific lethality and non-alcoholic fatty liver disease (NAFLD), which coincides with pubescence and is androgen-dependent. The phenotype is due to disruption of an endogenous locus, since other Meis1a transgenic lines do not exhibit these phenotypes. Necropsy analysis revealed hepatic microvesicular steatosis in pubescent male homozygous mice, which is absent in transgenic females. The transgene insertion site was localized to chromosome 1 and further refined by cloning the flanking regions. Sequence analysis shows that the integration site disrupts a putative metallo-beta-lactamase gene with a 21.3 kb deletion encompassing exons 5-7.