Genomic binding of PAX8-PPARG fusion protein regulates cancer-related pathways and alters the immune landscape of thyroid cancer.

PAX8-PPARG fusion protein (PPFP) results from a t(2;3)(q13;p25) chromosomal translocation, is found in 30% of follicular thyroid carcinomas, and demonstrates oncogenic capacity in transgenic mice. A PPARG ligand, pioglitazone, is highly therapeutic in mice with PPFP thyroid cancer. However, only limited data exist to characterize the binding sites and oncogenic function of PPFP, or to explain the observed therapeutic effect of pioglitazone. Here we used our previously characterized transgenic mouse model of PPFP follicular thyroid carcinoma to identify PPFP binding sites in vivo using ChIP-seq, and to distinguish genes and pathways regulated directly or indirectly by PPFP with and without pioglitazone treatment via integration with RNA-seq data. PPFP bound to DNA regions containing the PAX8 and/or the PPARG motif, near genes involved in lipid metabolism, the cell cycle, apoptosis, and cell motility; the binding site distribution was highly concordant with our previous study in a rat PCCL3 cell line. Most strikingly, pioglitazone induced an immune cell infiltration including macrophages and T cells only in the presence of PPFP, which may be central to its therapeutic effect.

Identification of the Genomic Insertion Site of the Thyroid Peroxidase Promoter-Cre Recombinase Transgene Using a Novel, Efficient, Next-Generation DNA Sequencing Method.

BACKGROUND: It can be useful to know the transgene insertion site in transgenic mice for a variety of reasons, but determining the insertion site generally is a time consuming, expensive, and laborious task. METHODS: A simple method is presented to determine transgene insertion sites that combines the enrichment of a sequencing library by polymerase chain reaction (PCR) for sequences containing the transgene, followed by next-generation sequencing of the enriched library. This method was applied to determine the site of integration of the thyroid peroxidase promoter-Cre recombinase mouse transgene that is commonly used to create thyroid-specific gene deletions. RESULTS: The insertion site was found to be between bp 12,372,316 and 12,372,324 on mouse chromosome 9, with the nearest characterized genes being Cntn5 and Jrkl, ∼1.5 and 0.9 Mbp from the transgene, respectively. One advantage of knowing a transgene insertion site is that it facilitates distinguishing hemizygous from homozygous transgenic mice. Although this can be accomplished by real-time quantitative PCR, the expected Ct difference is only one cycle, which is challenging to assess accurately. Therefore, the transgene insertion site information was used to develop a 3-primer qualitative PCR assay that readily distinguishes wild type, hemizygous, and homozygous TPO-Cre mice based upon size differences of the wild type and transgenic allele PCR products. CONCLUSIONS: Identification of the genomic insertion site of the thyroid peroxidase promoter-Cre mouse transgene should facilitate the use of these mice in studies of thyroid biology.

The thyroid cancer PAX8-PPARG fusion protein activates Wnt/TCF-responsive cells that have a transformed phenotype.

A chromosomal translocation results in the production of a paired box 8-peroxisome proliferator-activated receptor gamma (PAX8-PPARG) fusion protein (PPFP) in ∼35% of follicular thyroid carcinomas. To examine the role of PPFP in thyroid oncogenesis, the fusion protein was stably expressed in the non-transformed rat thyroid cell line PCCL3. PPFP conferred on PCCL3 cells the ability to invade through Matrigel and to form colonies in anchorage-independent conditions. PPFP also increased the fraction of cells with Wnt/TCF-responsive green fluorescent protein reporter gene expression. This Wnt/TCF-activated population was enriched for colony-forming and invading cells. These actions of PPFP required a functional PPARG DNA binding domain (DBD) within PPFP and were further stimulated by PPARG agonists. These data indicate that PPFP, through its PPARG DBD, induces Wnt/TCF pathway activation in a subpopulation of cells, and these cells have properties of cellular transformation including increased invasiveness and anchorage-independent growth.

Pioglitazone induces a proadipogenic antitumor response in mice with PAX8-PPARgamma fusion protein thyroid carcinoma.

Approximately 35% of follicular thyroid carcinomas harbor a chromosomal translocation that results in expression of a paired box gene 8-peroxisome proliferator-activated receptor γ gene (PPARγ) fusion protein (PPFP). To better understand the oncogenic role of PPFP and its relationship to endogenous PPARγ, we generated a transgenic mouse model that combines Cre-dependent PPFP expression (PPFP;Cre) with homozygous deletion of floxed Pten (PtenFF;Cre), both thyroid specific. Although neither PPFP;Cre nor PtenFF;Cre mice develop thyroid tumors, the combined PPFP;PtenFF;Cre mice develop metastatic thyroid cancer, consistent with patient data that PPFP is occasionally found in benign thyroid adenomas and that PPFP carcinomas have increased phosphorylated AKT/protein kinase B. We then tested the effects of the PPARγ agonist pioglitazone in our mouse model. Pioglitazone had no effect on PtenFF;Cre mouse thyroids. However, the thyroids in pioglitazone-fed PPFP;PtenFF;Cre mice decreased 7-fold in size, and metastatic disease was prevented. Remarkably, pioglitazone caused an adipogenic response in the PPFP;PtenFF;Cre thyroids characterized by lipid accumulation and the induction of a broad array of adipocyte PPARγ target genes. These data indicate that, in the presence of pioglitazone, PPFP has PPARγ-like activity that results in trans-differentiation of thyroid carcinoma cells into adipocyte-like cells. Furthermore, the data predict that pioglitazone will be therapeutic in patients with PPFP-positive carcinomas.

Direct role of nucleotide metabolism in C-MYC-dependent proliferation of melanoma cells.

To identify C-MYC targets rate-limiting for proliferation of malignant melanoma, we stably inhibited C-MYC in several human metastatic melanoma lines via lentivirus-based shRNAs approximately to the levels detected in normal melanocytes. C-MYC depletion did not significantly affect levels of E2F1 protein reported to regulate expression of many S-phase specific genes, but resulted in the repression of several genes encoding enzymes rate-limiting for dNTP metabolism. These included thymidylate synthase (TS), inosine monophosphate dehydrogenase 2 (IMPDH2) and phosphoribosyl pyrophosphate synthetase 2 (PRPS2). C-MYC depletion also resulted in reduction in the amounts of deoxyribonucleoside triphosphates (dNTPs) and inhibition of proliferation. shRNA-mediated suppression of TS, IMPDH2 or PRPS2 resulted in the decrease of dNTP pools and retardation of the cell cycle progression of melanoma cells in a manner similar to that of C-MYC-depletion in those cells. Reciprocally, concurrent overexpression of cDNAs for TS, IMPDH2 and PRPS2 delayed proliferative arrest caused by inhibition of C-MYC in melanoma cells. Overexpression of C-MYC in normal melanocytes enhanced expression of the above enzymes and increased individual dNTP pools. Analysis of in vivo C-MYC interactions with TS, IMPDH2 and PRPS2 genes confirmed that they are direct C-MYC targets. Moreover, all three proteins express at higher levels in cells from several metastatic melanoma lines compared to normal melanocytes. Our data establish a novel functional link between C-MYC and dNTP metabolism and identify its role in proliferation of tumor cells.

Pathological responses to oncogenic Hedgehog signaling in skin are dependent on canonical Wnt/beta3-catenin signaling.

Constitutive Hedgehog (Hh) signaling underlies several human tumors, including basal cell carcinoma (BCC) and basaloid follicular hamartoma in skin. Intriguingly, superficial BCCs arise as de novo epithelial buds resembling embryonic hair germs, collections of epidermal cells whose development is regulated by canonical Wnt/beta-catenin signaling. Similar to embryonic hair germs, human BCC buds showed increased levels of cytoplasmic and nuclear beta-catenin and expressed early hair follicle lineage markers. We also detected canonical Wnt/ beta-catenin signaling in epithelial buds and hamartomas from mice expressing an oncogene, M2SMO, leading to constitutive Hh signaling in skin. Conditional overexpression of the Wnt pathway antagonist Dkk1 in M2SMO-expressing mice potently inhibited epithelial bud and hamartoma development without affecting Hh signaling. Our findings uncover a hitherto unknown requirement for ligand-driven, canonical Wnt/ beta-catenin signaling for Hh pathway-driven tumorigenesis, identify a new pharmacological target for these neoplasms and establish the molecular basis for the well-known similarity between early superficial BCCs and embryonic hair germs.

Receptor-type protein-tyrosine phosphatase-kappa regulates epidermal growth factor receptor function.

Epidermal growth factor receptor (EGFR), the prototypic receptor protein tyrosine kinase, is a major regulator of growth and survival for many epithelial cell types. We report here that receptor-type protein-tyrosine phosphatase-kappa (RPTP-kappa) dephosphorylates EGFR and thereby regulates its function in human keratinocytes. Protein-tyrosine phosphatase (PTP) inhibitors induced EGFR tyrosine phosphorylation in intact primary human keratinocytes and cell-free membrane preparations. Five highly expressed RPTPs (RPTP-beta, delta, kappa, mu, and xi) were functionally analyzed in a Chinese hamster ovary (CHO) cell-based expression system. Full-length human EGFR expressed in CHO cells, which lack endogenous EGFR, displayed high basal (i.e. in the absence of ligand) tyrosine phosphorylation. Co-expression of RPTP-kappa, but not other RPTPs, specifically reduced basal EGFR tyrosine phosphorylation. RPTP-kappa also reduced epidermal growth factor-dependent EGFR tyrosine phosphorylation in CHO cells. Purified RPTP-kappa preferentially dephosphorylated EGFR tyrosines 1068 and 1173 in vitro. Overexpression of wild-type or catalytically inactive RPTP-kappa reduced or enhanced, respectively, basal and EGF-induced EGFR tyrosine phosphorylation in human keratinocytes. Furthermore, siRNA-mediated knockdown of RPTP-kappa increased basal and EGF-stimulated EGFR tyrosine phosphorylation and augmented downstream Erk activation in human keratinocytes. RPTP-kappa levels increased in keratinocytes as cells reached confluency, and overexpression of RPTP-kappa in subconfluent keratinocytes reduced keratinocyte proliferation. Taken together, the above data indicate that RPTP-kappa is a key regulator of EGFR tyrosine phosphorylation and function in human keratinocytes.

Hedgehog signaling regulates sebaceous gland development.

Epithelial progenitor cells in skin give rise to multiple lineages, comprising the hair follicle, an associated sebaceous gland, and overlying epidermis; however, the signals that regulate sebocyte development are poorly understood. We tested the potential involvement of the Hedgehog pathway in sebaceous gland development using transgenes designed to either block or stimulate Hedgehog signaling in cutaneous keratinocytes in vivo. Whereas inhibition of the Hedgehog pathway selectively suppressed sebocyte development, Hedgehog pathway activation led to a striking increase both in size and number of sebaceous glands. Remarkably, ectopic Hedgehog signaling also triggered the formation of sebaceous glands from footpad epidermis, in regions normally devoid of hair follicles and associated structures. These ectopic sebaceous glands expressed molecular markers of sebocyte differentiation and were functional, secreting their contents directly onto the skin's surface instead of into a hair canal. The Hedgehog pathway thus plays a key role in sebocyte cell fate decisions and is a potential target for treatment of skin disorders linked to abnormal sebaceous gland function, such as acne.

The magnitude of hedgehog signaling activity defines skin tumor phenotype.

Gain-of-function mutations in SMO have been implicated in constitutive activation of the hedgehog signaling pathway in human basal cell carcinomas (BCCs). We used a truncated keratin 5 (DeltaK5) promoter to assess the potential role of the human M2SMO mutant in BCC development in adult transgenic mice. DeltaK5-M2SMO mouse epidermis is hyperproliferative, ex presses BCC protein markers and gives rise to numerous epithelial downgrowths invading the underlying dermis. Lesions strikingly similar to human basaloid follicular hamartomas develop, but BCCs do not arise even in elderly mice. Hedgehog target gene transcripts were only modestly upregulated in mouse and human follicular hamartomas, in contrast to the high levels detected in BCCs. Cyclins D1 and D2 were selectively upregulated in mouse BCCs. Our data suggest that the levels of hedgehog pathway activation and G(1) cyclins are major determinants of tumor phenotype in skin, and strongly implicate deregulated hedgehog signaling in the genesis of human basaloid follicular hamartomas. Expression of an activated SMO mutant in keratinocytes appears to be insufficient for the development and/or maintenance of full-blown BCCs.