The murine homologue of the TFEC was cloned as part of an analysis of the expression of the microphthalmia-TFE (MiT) subfamily of transcription factors in macrophages. TFEC, which most likely acts as a transcriptional repressor in heterodimers with other MiT family members, was identified in cells of the mononuclear phagocyte lineage, coexpressed with all other known MiT subfamily members (Mitf, TFE3, TFEB). Northern blot analysis of several different cell lineages indicated that the expression of murine TFEC (mTFEC) was restricted to macrophages. A 600-bp fragment of the TATA-less putative proximal promoter of TFEC shares features with many known macrophage-specific promoters and preferentially directs luciferase expression in the RAW264.7 macrophage cell line in transient transfection assays. Five of six putative Ets motifs identified in the TFEC promoter bind the macrophage-restricted transcription factor PU.1 under in vitro conditions and in transfected 3T3 fibroblasts; the minimal luciferase activity of the TFEC promoter could be induced by coexpression of PU.1 or the related transcription factor Ets-2. The functional importance of the tissue-restricted expression of TFEC and a possible role in macrophage-specific gene regulation require further investigation, but are likely to be linked to the role of the other MiT family members in this lineage.

Activation of transforming growth factor-beta (TGF-beta) receptors triggers phosphorylation of Smad2 and Smad3. After binding to Smad4, the complex enters the nucleus and interacts with other transcription factors to activate gene transcription. Unlike other Smads, Smad7 inhibits phosphorylation of Smad2 and Smad3, and its transcription is induced by TGF-beta, suggesting a negative feedback loop. Here, we show that TFE3 and Smad3 synergistically mediate TGF-beta-induced transcription from the Smad7 promoter by binding to an E-box and two adjacent Smad binding elements (SBEs), respectively. A precise 3-base pair spacer between one SBE and the E-box is essential. Previously, we showed that a similar arrangement between a SBE and an E-box of an element is essential for TGF-beta-dependent transcription of the plasminogen activator inhibitor-1 gene (PAI-1) and that TGF-beta-induced phosphorylation of Smad3 triggers its association with TFE3. Thus, TFE3-Smad3 response elements may represent a common target for TGF-beta-induced gene expression.

Binding sites for three families of sequence-specific DNA-binding proteins, microE3, C/EBP, and OCT, are found in both the promoters and the intronic enhancer of the immunoglobulin heavy-chain gene. We have used a cotransfection system to investigate how proteins binding these sites may participate in enhancer-promoter interactions. Basic helix-loop-helix-zipper (BHLHZIP) proteins TFE3 and TFEB activate from a distance in this assay, but the basic zipper (BZIP) protein NF-IL6 and endogenous OCT-binding proteins do not. Our results suggest that remotely bound TFE3 is recruited to the initiation site by association with proximally bound TFE3; this interaction is mediated by the BHLHZIP domain and not by activation domains of TFE3. The BZIP domain of Ig/EBP lacks this activity, revealing an important functional difference between these structurally related dimerization domains. We also show that TFE3 can exist as a tetramer in solution and that tetramerization is determined by the HLHZIP domain. These data support a model in which protein-protein interactions between proximally and remotely bound TFE3 recruit TFE3 to the initiation site for activation. The IgH gene is the first example of a cellular gene in which proximal and distal binding sites are found for a protein capable of mediating enhancer-promoter interaction.

OBJECTIVE

Recent studies suggest that paediatric renal cell carcinoma (RCC) may represent a distinct group of tumours; however, its biological behaviour and classification remain poorly understood. The aim was to analyse 13 RCCs from patients < or =23 years of age to determine their clinicopathological, immunohistochemical and molecular characteristics.

RESULTS

The histological spectrum included: Xp11.2 translocation-associated (6/13 patients, 46%), clear cell (5/13 patients, 38%), papillary (1/13 patients) and unclassified (1/13 patients) types. The Xp11.2 translocation-associated RCCs had a wide morphological spectrum, with high nuclear grade cells with abundant cytoplasm ranging from clear to granular and architecture ranging from solid to papillary. These tumours lacked cytokeratin expression and were confirmed by nuclear reactivity for TFE3 protein. Most of these translocation-associated tumours presented at high stage and had an unfavourable outcome. Three clear cell RCCs had unusual features that have not been previously characterized, including solid and cystic architecture, cells with abundant eosinophilic cytoplasm yet low nuclear grade and focal cytoplasmic inclusions, resembling oncocytoma. Deletion of subtelomeric 3p25 was observed in two of these RCCs.

CONCLUSIONS

Xp11.2 translocation-associated RCC represents a predominant and aggressive subtype in the paediatric age group. Increased awareness of this subtype is important due to its heterogeneous morphology.

Since the hallmark dermatologic features of Birt-Hogg-Dubé (BHD) syndrome were first described by three Canadian physicians in 1977, the clinical manifestations of BHD have been expanded to include hamartomas of the hair follicle, lung cysts, increased risk for spontaneous pneumothorax and kidney neoplasia. Twenty-five years later the causative gene FLCN was identified, and the mutation spectrum has now been defined to include mainly protein truncating mutations, but also rare missense mutations and large gene deletions/duplication. Second "hit" FLCN mutations in BHD kidney tumors and loss of tumorigenic potential of the FLCN-null UOK257 tumor cell line when FLCN is re-expressed underscore a tumor suppressor role for FLCN. The identification of novel FLCN interacting proteins FNIP1 and FNIP2/L and their interaction with 5'-AMP activated protein kinase (AMPK) has provided a link between FLCN and the AMPK-mTOR axis and suggested molecular targets for therapeutic intervention to treat BHD kidney cancer and fibrofolliculomas. The generation of FLCN-null cell lines and in vivo animal models in which FLCN (or FNIP1) has been inactivated have provided critical reagents to facilitate mechanistic studies of FLCN function. Research efforts utilizing these critical FLCN-deficient cell lines and mice have begun to uncover important signaling pathways in which FLCN and its protein partners may play a role, including TGF-β signaling, TFE3 transcriptional regulation, PGC1-α driven mitochondrial biogenesis, apoptotic response to cell stress, and vesicular transport. As the mechanisms by which FLCN inactivation leads to BHD manifestations are clarified, we can begin to develop therapeutic agents that target the pathways dysregulated in FLCN-deficient fibrofolliculomas and kidney tumors, providing improved prognosis and quality of life for BHD patients.

OBJECTIVE

To evaluate the clinical outcomes and histological types of renal cell carcinoma (RCC) arising in patients with end-stage renal disease (ESRD), and to analyse the relationship of histopathological features with the duration of dialysis.

METHODS

Clinical characteristics and outcomes of 34 patients who had a radical nephrectomy for RCC arising in ESRD between November 1994 and June 2008 were investigated. Archive paraffin-embedded tissue specimens obtained from 27 patients were histochemically and immunohistochemically analysed to determine the histopathological type.

RESULTS

There was one death from cancer and one patient with local progression within a median observation period of 29.5 months. Acquired cystic disease (ACD)-associated RCC, clear cell-papillary RCC, mucinous tubular and spindle-cell carcinoma, and Xp11.2 translocation/TFE3 gene fusion were identified in eight, two, three and one patient, respectively. Conventional clear-cell RCC was the predominant histological type (nine of 15) in patients with a duration of dialysis of <10 years, while ACD-associated RCC was predominant (seven of 12) in those with dialysis for>> or =10 years. Sarcomatoid foci were identified in three patients with dialysis for>> or =10 years. Papillary adenoma was microscopically identified as a satellite tumour in 10 patients.

CONCLUSIONS

The spectrum of histological types of RCCs arising in ESRD is distinct from that of sporadic RCCs. Patients with a longer duration of dialysis should have particular attention for progression and metastasis. Immunohistochemical profiling is efficient in the histological classification of RCCs arising in ESRD, although knowledge about genetic changes remains to be accumulated.

Renal cell carcinomas (RCCs) with an Xp11.2 translocation predominantly affect young patients, and can present at an advanced stage. However, more cases in older patients and incidentally detected cancers at earlier stages are also being identified. As the histology of Xp11.2 RCCs overlaps with clear cell and papillary RCCs, it is not infrequent that Xp11.2 RCCs are overlooked and misdiagnosed. The objective of this study was to validate the use of fluorescence in-situ hybridization (FISH) for identifying Xp11.2 RCCs. One hundred fifty-eight consecutive, unselected renal tumors were evaluated in tissue microarrays, including 109 clear cell RCCs, 20 papillary RCCs, 3 RCCs with mixed papillary and clear cell features, 1 Xp11.2 translocation RCC, 8 chromophobe RCCs, 10 oncocytomas, and 7 angiomyolipomas. FISH evaluation was performed blinded to karyotype data, available in about two-thirds of cases. Furthermore, conventional sections of 4 Xp11.2 RCCs, 4 RCCs with mixed papillary and clear cell features, and 4 cases of alveolar soft part sarcoma (the latter for control purposes) were also assessed by FISH. Break-apart signals were homogeneously identified throughout tumor cells in 2 cases from the tissue microarrays including 1 known Xp11.2 RCC and 1 misdiagnosed Xp11.2 RCC. All conventional sections from the Xp11.2 RCC and alveolar soft part sarcoma cases were positive for the TFE3 rearrangement by FISH. All remaining cases were negative. Our study shows the clinical application of FISH in formalin-fixed, paraffin-embedded tissue for detection of Xp11.2 translocation RCCs and other tumors with this genetic aberration.

OBJECTIVE

To report clinicopathological and genomic characteristics of (ccpRCC), a rare, recently characterized renal tumour entity.

RESULTS

Twenty-four renal tumours identified as ccpRCC were collected. Data from comparative genomic hybridization on microarrays (array-CGH) were obtained for seven of these. Most tumours (58%) occurred in the absence of renal disease. Mean patient age was 58.1 years. Tumours were small (mean size: 2.4 cm) and classified as pT1. Histological characteristics consisted of tubules and papillae lined by a single layer of small clear cells harbouring low-grade nuclei (Fuhrman grades 1 or 2). Architectural variations, with compact areas (41% of cases) and a micro- or macrocystic pattern (67% of cases) were observed frequently. Immunostaining demonstrated diffuse, strong expression of cytokeratin 7 and vimentin, whereas CD10, racemase, RCC antigen, translocation factor E3, TFE3 and translocation factor EB were consistently negative. In seven tumours, array-CGH detected no chromosomal imbalances.

CONCLUSIONS

Clear-cell papillary renal cell carcinoma (ccpRCC) were differentiated from other renal neoplasms by a specific constellation of histopathological and immunohistochemical features, without characteristic genomic imbalances. Clinical, histopathological and genomic data suggested that these tumours have a low potential for malignancy.

Perivascular epithelioid cell tumors (PEComas) are distinctive mesenchymal neoplasms that most often arise in the retroperitoneum, visceral organs, and abdominopelvic sites and usually show reactivity for melanocytic and smooth muscle markers. Fewer than 20 PEComas of the gastrointestinal (GI) tract have been reported, and behavior and criteria for malignancy are incompletely defined. The purpose of this study was to examine the clinicopathologic features of a series of GI PEComas and to evaluate prognostic parameters. A total of 35 PEComas of the GI tract were retrieved from consult and surgical files. Clinical and pathologic features were evaluated, and immunohistochemical analysis was performed. Clinical follow-up information was obtained from medical records and referring physicians. Nineteen patients were female and 16 male (median age 45 y; range, 7 to 70 y). One patient had tuberous sclerosis. Nineteen tumors arose in the colon, 12 in the small bowel, 2 in the stomach, and 1 each in gallbladder and omentum. Median tumor size was 6.2 cm (range, 0.8 to 22 cm). Three tumors were limited to the mucosa and submucosa, 8 extended to the muscularis propria, 15 to the subserosa/serosa, and 8 into the mesentery. The tumors were composed of nests and sheets of usually epithelioid cells with abundant granular eosinophilic to clear cytoplasm, surrounded by a delicate capillary vasculature. Thirteen tumors had mixed epithelioid and spindle cell components, and 2 were purely spindled. Sixteen tumors showed marked nuclear atypia. Seventeen tumors contained occasional pleomorphic cells, and 12 showed diffuse cellular pleomorphism. The median mitotic rate was 2/10 HPF (range, 0 to 36). Vascular invasion was present in 5 cases, and 16 tumors showed necrosis. By immunohistochemistry, 23/35 were positive for HMB45, 23/34 for melan-A, 15/25 for MiTF, 20/35 for smooth muscle actin, 26/35 for desmin, and 3/20 for TFE3. Focal cytoplasmic S100 protein was present in 5/27 cases, 2/25 cases were positive for KIT, and 1 case each was positive for EMA and keratin. Follow-up information was available for 31 patients (median 36 mo; range, 2 to 176 mo). Thirteen patients have developed metastases (10 liver, 3 peritoneum, 4 lymph node, 3 lung, 1 bone, 1 brain, and 1 adrenal). Thus far, 5 patients have died of disease. Metastases were significantly associated with marked atypia, diffuse pleomorphism, and mitoses ≥2/10 HPF. In summary, PEComas of the GI tract occur at similar frequency in female and male patients, most commonly involve the colon, and exhibit variable clinical behavior, ranging from benign lesions to aggressive, high-grade sarcomas. The presence of marked nuclear atypia, diffuse pleomorphism, and mitotic activity are the strongest predictors of malignant behavior.

Although histologic features enable an accurate diagnosis in most renal carcinomas, overlapping morphologic findings between some renal neoplasms make subclassification difficult. Some renal carcinomas show papillary architecture but are composed extensively of cells with clear cytoplasm, and it is unclear whether they should be classified as clear cell renal cell carcinomas or papillary renal cell carcinomas. We analyzed the immunohistochemical profiles and the cytogenetic patterns of 14 renal carcinomas showing papillary architecture in which there were variable amounts of cells with clear cytoplasm. The patients were 8 women and 6 men (mean age: 54 y). Immunohistochemistry and fluorescence in situ hybridization analysis distinguished 2 different groups. The first consisted of 10 renal cell carcinomas with strong immunoreactivity for alpha-methyl coenzyme A racemase, of which 9 also expressed cytokeratin 7. All of these neoplasms showed gains of chromosome 7 or 17 and chromosome Y was lost in all the male patients whereas 3p deletion was detected only in one case. In the other 4 renal cell carcinomas, cytokeratin 7 was not detected and alpha-methylacyl-CoA racemase was positive in only 1. In these neoplasms, no gain of chromosome 7 or 17 and no loss of chromosome Y were observed, whereas 3p deletion was detected in 3 of them. None of the 14 neoplasms showed immunoreactivity for TFE3. The combined use of immunohistochemistry and cytogenetics enabled us to provide a definitive diagnosis for 12 of 14 renal cell carcinomas with papillary architecture and clear cell components: 9 cases were confirmed to be papillary renal cell carcinomas and 3 cases were confirmed to be clear cell renal cell carcinomas. Despite these ancillary techniques, 2 cases remained unclassified. Our study establishes the utility of these procedures in accurately classifying the great majority of renal cell carcinomas with these findings.

Perivascular epithelioid cell tumor (PEComa) belongs to a family of tumors characterized by coexpression of melanocytic and muscle markers. Recent studies have shown that sporadic and tuberous sclerosis complex-associated PEComa may respond to mTOR inhibitors underscoring the importance of recognizing this tumor. However, its occurrence in the gynecologic tract continues to be disputed owing to its common misclassification as other types of uterine sarcoma and its controversial relationship with epithelioid smooth muscle tumors. To more fully characterize PEComa of the female genital tract, 16 cases of gynecologic PEComa were identified (1990 to 2012) and formed the basis of this study. Each case was analyzed for conventional morphologic and immunohistochemical characteristics established for PEComa of extrauterine sites; clinical outcome data were obtained for all cases. The 16 patients were aged 28 to 60 (mean 49; median 50) years, and 1 had a history of tuberous sclerosis complex. Thirteen cases were primary of the uterus, 2 of the adnexa, and 1 of the vagina. Tumor size ranged from 0.3 to 25.0 (mean 8.7) cm. Three patients died of disease, 6 were alive with disease, and 7 were alive without evidence of disease at last follow-up (1 mo to 13 y follow-up; mean 26 mo). All patients with an adverse outcome met established criteria for malignancy as proposed for extrauterine sites (ie, 2 or more features present: size ≥5 cm, high-grade nuclear features, infiltration, necrosis, lymphovascular invasion, or a mitotic rate ≥1/50 high-power fields). Of the melanocytic markers, HMB45 was most commonly expressed (16/16 positive, 100%), followed by microphthalmia transcription factor (11/12 positive, 92%), MelanA (14/16 positive, 88%), and S100 protein (2/10 positive, 20%). Of the smooth muscle markers, desmin was most commonly expressed (15/15 cases, 100%), followed by SMA (14/15 cases, 93%) and h-caldesmon (11/12 cases, 92%). TFE3 immunopositivity was identified in 5 of 13 cases; however, 3 tested cases were negative for a TFE3 rearrangement by fluorescence in situ hybridization. Current criteria for malignancy appear to be valid in the female genital tract, although modified criteria, as described herein, may be more specific. Awareness of the characteristic features of PEComa is important to help distinguish it from epithelioid smooth muscle tumors and other mimics as PEComa may respond to unique chemotherapeutic regimens.

TFE3 is a basic-helix-loop-helix-zipper (bHLHZIP) domain-containing protein that binds mu E3 sites in regulatory elements in the immunoglobulin heavy chain gene. The protein is a transcriptional activator that is expressed in vivo as two alternately spliced isoforms with different activating properties: TFE3L contains an N-terminal acidic activation domain; TFE3S lacks this activation domain and is a dominant negative inhibitor of TFE3L. We show that TFE3L and TFE3S contain a second, C-terminal activation domain rich in proline residues. This pro-rich activation domain has activity in a Gal4 fusion assay comparable to the N-terminal acidic activation domain present in TFE3L. The TFE3 pro-rich activation domain contains regions of strong homology with the related proteins microphthalmia and TFEB, suggesting that these regions are important for function. Using two different assays, we show that the N- and C-terminal activation domains of TFE3 act synergistically. This synergism explains in part the ability of TFE3S to act as a dominant negative. Our domain analysis of TFE3 is incorporated into a general structural model for the TFE3 protein that predicts that the activation domains of TFE3 will be widely separated in space.

The microphthalmia family (MITF, TFEB, TFE3, and TFEC) of transcription factors is emerging as global regulators of cancer cell survival and energy metabolism, both through the promotion of lysosomal genes as well as newly characterized targets, such as oxidative metabolism and the oxidative stress response. In addition, MiT/TFE factors can regulate lysosomal signaling, which includes the mTORC1 and Wnt/β-catenin pathways, which are both substantial contributors to oncogenic signaling. This review describes recent discoveries in MiT/TFE research and how they impact multiple cancer subtypes. Furthermore, the literature relating to TFE-fusion proteins in cancers and the potential mechanisms through which these genomic rearrangements promote tumorigenesis is reviewed. Likewise, the emerging function of the Folliculin (FLCN) tumor suppressor in negatively regulating the MiT/TFE family and how loss of this pathway promotes cancer is examined. Recent reports are also presented that relate to the role of MiT/TFE-driven lysosomal biogenesis in sustaining cancer cell metabolism and signaling in nutrient-limiting conditions. Finally, a discussion is provided on the future directions and unanswered questions in the field. In summary, the research surrounding the MiT/TFE family indicates that these transcription factors are promising therapeutic targets and biomarkers for cancers that thrive in stressful niches. Mol Cancer Res; 15(12); 1637-43. ©2017 AACR.

Noncanonical mechanistic target of rapamycin (mTOR) pathways remain poorly understood. Mutations in the tumor suppressor folliculin (FLCN) cause Birt-Hogg-Dubé syndrome, a hamartomatous disease marked by mitochondria-rich kidney tumors. FLCN functionally interacts with mTOR and is expressed in most tissues, but its role in fat has not been explored. We show here that FLCN regulates adipose tissue browning via mTOR and the transcription factor TFE3. Adipose-specific deletion of FLCN relieves mTOR-dependent cytoplasmic retention of TFE3, leading to direct induction of the PGC-1 transcriptional coactivators, drivers of mitochondrial biogenesis and the browning program. Cytoplasmic retention of TFE3 by mTOR is sensitive to ambient amino acids, is independent of growth factor and tuberous sclerosis complex (TSC) signaling, is driven by RagC/D, and is separable from canonical mTOR signaling to S6K. Codeletion of TFE3 in adipose-specific FLCN knockout animals rescues adipose tissue browning, as does codeletion of PGC-1β. Conversely, inducible expression of PGC-1β in white adipose tissue is sufficient to induce beige fat gene expression in vivo. These data thus unveil a novel FLCN-mTOR-TFE3-PGC-1β pathway-separate from the canonical TSC-mTOR-S6K pathway-that regulates browning of adipose tissue.

OBJECTIVE

Translocation renal cell carcinoma (tRCC) represents a rare subtype of kidney cancer associated with various TFE3, TFEB, or MITF gene fusions that are not responsive to standard treatments for RCC. Therefore, the identification of new therapeutic targets represents an unmet need for this disease.

METHODS

We have established and characterized a tRCC patient-derived xenograft, RP-R07, as a novel preclinical model for drug development by using next-generation sequencing and bioinformatics analysis. We then assessed the therapeutic potential of inhibiting the identified pathway using in vitro and in vivo models.

RESULTS

The presence of a SFPQ-TFE3 fusion [t(X;1) (p11.2; p34)] with chromosomal break-points was identified by RNA-seq and validated by RT-PCR. TFE3 chromatin immunoprecipitation followed by deep sequencing analysis indicated a strong enrichment for the PI3K/AKT/mTOR pathway. Consistently, miRNA microarray analysis also identified PI3K/AKT/mTOR as a highly enriched pathway in RP-R07. Upregulation of PI3/AKT/mTOR pathway in additional TFE3-tRCC models was confirmed by significantly higher expression of phospho-S6 (P < 0.0001) and phospho-4EBP1 (P < 0.0001) in established tRCC cell lines compared with clear cell RCC cells. Simultaneous vertical targeting of both PI3K/AKT and mTOR axis provided a greater antiproliferative effect both in vitro (P < 0.0001) and in vivo (P < 0.01) compared with single-node inhibition. Knockdown of TFE3 in RP-R07 resulted in decreased expression of IRS-1 and inhibited cell proliferation.

CONCLUSIONS

These results identify TFE3/IRS-1/PI3K/AKT/mTOR as a potential dysregulated pathway in TFE3-tRCC, and suggest a therapeutic potential of vertical inhibition of this axis by using a dual PI3K/mTOR inhibitor for patients with TFE3-tRCC.

Epithelioid hemangioendothelioma (EHE) is a malignant endothelial neoplasm characterized by recurrent translocations involving chromosomal regions 1p36.3 and 3q25, resulting in the formation of a WWTR1-CAMTA1 fusion gene in approximately 90% of cases; a small subset (<5%) have a YAP1-TFE3 fusion gene. The WWTR1-CAMTA1 fusion gene leads to overexpression of both genes. WWTR1 protein is expressed in many different cell types, whereas CAMTA1 expression is normally limited to the brain. A prior study using a polyclonal antibody directed against regions within the C-terminus of CAMTA1 reported widespread expression in both normal tissues and diverse tumor types. In contrast, a recent study using a different polyclonal antibody directed against the C-terminus of CAMTA1 suggested that this other antibody is a potentially useful diagnostic marker for EHE. Our study aimed to validate this finding in a large series of EHE cases and to determine whether CAMTA1 is expressed in other epithelioid mesenchymal tumors that may mimic EHE. Protein expression of CAMTA1 was evaluated in whole-tissue sections of 204 tumors using a polyclonal anti-CAMTA1 antibody: 59 EHE (48 conventional, 11 "malignant"; 4 with known TFE3 gene rearrangement); 70 other epithelioid vascular tumors; and 75 nonendothelial epithelioid mesenchymal neoplasms. In total, 51/59 cases (86%) of EHE showed diffuse nuclear staining for CAMTA1, including 44/48 cases (92%) with conventional histology and 7/11 cases (64%) with "malignant" histology. Of the 8 CAMTA1-negative tumors, 6 were positive for TFE3. With the exception of 1 case previously diagnosed as epithelioid angiosarcoma on core biopsy, all other tumor types examined were negative for CAMTA1. In conclusion, in keeping with the reported frequency of WWTR1-CAMTA1 in EHE, nuclear CAMTA1 expression is identified in the majority of EHE cases, whereas other epithelioid mesenchymal neoplasms are negative for CAMTA1. These findings support the diagnostic utility of immunohistochemistry for CAMTA1 in distinguishing EHE from histologic mimics, in particular benign epithelioid vascular tumors, epithelioid angiosarcoma, and epithelioid sarcoma, an important distinction given the differences in biological potential and clinical course.

OBJECTIVE

Recent studies have demonstrated that cathepsin K seems to be a powerful marker in identifying renal perivascular epithelioid cell neoplasms (PEComas). However, the expression in extrarenal PEComas has not been well characterized due to their rare incidence. Our aim was to investigate the expression of cathepsin K in a wide spectrum of extrarenal PEComas and evaluate its potential diagnostic usefulness in comparison with other commonly used markers.

RESULTS

Twenty-three cases of PEComa (liver, n = 9; lung, n = 1; broad ligament of uterus, n = 1; vertex subcutaneous soft tissue, n = 1; abdominal wall, n = 1; and kidney, n = 10) were selected for study. All displayed a high percentage of cells with moderately to strongly positive reactions for cathepsin K (mean 91%; range 80-100%). HMB45, Melan-A and smooth muscle actin (SMA) were expressed in 78, 87 and 87% of cases, respectively, with various percentages of positive cells (mean, 34, 40 and 38%; range 0-80, 0-90 and 0-90%). Transcription factor E3 (TFE3) was expressed strongly in only three cases; none exhibited evidence of TFE3 gene fusion or amplification.

CONCLUSIONS

Cathepsin K appears to be more powerful than other commonly used markers in diagnosing a wide spectrum of PEComas and distinguishing them from the majority of human cancers.

Germline mutations in the novel tumor suppressor gene FLCN are responsible for the autosomal dominant inherited disorder Birt-Hogg-Dubé (BHD) syndrome that predisposes to fibrofolliculomas, lung cysts and spontaneous pneumothorax, and an increased risk for developing kidney tumors. Although the encoded protein, folliculin (FLCN), has no sequence homology to known functional domains, x-ray crystallographic studies have shown that the C-terminus of FLCN has structural similarity to DENN (differentially expressed in normal cells and neoplasia) domain proteins that act as guanine nucleotide exchange factors (GEFs) for small Rab GTPases. FLCN forms a complex with folliculin interacting proteins 1 and 2 (FNIP1, FNIP2) and with 5' AMP-activated protein kinase (AMPK). This review summarizes FLCN functional studies which support a role for FLCN in diverse metabolic pathways and cellular processes that include modulation of the mTOR pathway, regulation of PGC1α and mitochondrial biogenesis, cell-cell adhesion and RhoA signaling, control of TFE3/TFEB transcriptional activity, amino acid-dependent activation of mTORC1 on lysosomes through Rag GTPases, and regulation of autophagy. Ongoing research efforts are focused on clarifying the primary FLCN-associated pathway(s) that drives the development of fibrofolliculomas, lung cysts and kidney tumors in BHD patients carrying germline FLCN mutations.

Lysosomes are becoming increasingly recognized as a hub that integrates diverse signals in order to control multiple aspects of cell physiology. This is illustrated by the discovery of a growing number of lysosome-localized proteins that respond to changes in growth factor and nutrient availability to regulate mTORC1 signaling as well as the identification of MiT/TFE transcription factors (MITF, TFEB and TFE3) as proteins that shuttle between lysosomes and the nucleus to elicit a transcriptional response to ongoing changes in lysosome status. These findings have been paralleled by advances in human genetics that connect mutations in genes involved in lysosomal signaling to a broad range of human illnesses ranging from cancer to neurological disease. This review summarizes these new discoveries at the interface between lysosome cell biology and human disease.

Receptor-independent G-protein regulators provide diverse mechanisms for signal input to G-protein-based signaling systems, revealing unexpected functional roles for G-proteins. As part of a broader effort to identify disease-specific regulators for heterotrimeric G-proteins, we screened for such proteins in cardiac hypertrophy using a yeast-based functional screen of mammalian cDNAs as a discovery platform. We report the identification of three transcription factors belonging to the same family, transcription factor E3 (TFE3), microphthalmia-associated transcription factor, and transcription factor EB, as novel receptor-independent activators of G-protein signaling selective for Gα(16). TFE3 and Gα(16) were both up-regulated in cardiac hypertrophy initiated by transverse aortic constriction. In protein interaction studies in vitro, TFE3 formed a complex with Gα(16) but not with Gα(i3) or Gα(s). Although increased expression of TFE3 in heterologous systems had no influence on receptor-mediated Gα(16) signaling at the plasma membrane, TFE3 actually translocated Gα(16) to the nucleus, leading to the induction of claudin 14 expression, a key component of membrane structure in cardiomyocytes. The induction of claudin 14 was dependent on both the accumulation and activation of Gα(16) by TFE3 in the nucleus. These findings indicate that TFE3 and Gα(16) are up-regulated under pathologic conditions and are involved in a novel mechanism of transcriptional regulation via the relocalization and activation of Gα(16).

To further study the characteristics of renal cell carcinoma (RCC) in young patients and better define their biological features, 46 RCCs of patients younger than 25 years were morphologically and immunohistochemically characterized with follow-up. Loss of heterozygosity (LOH) analysis of the von Hippel-Lindau (VHL) gene region and screening for VHL gene mutations were performed in all tumors. Applying the 2004 WHO classification for RCC, there were 19 Xp11.2 translocation RCCs, 9 clear cell RCCs, 17 papillary RCCs, and 1 unclassified RCC. All 19 Xp11.2 translocation RCCs showed moderate to strong immunoreactivity for TFE3. None had TFEB immunoreactivity. One Xp11.2 translocation RCC had an unreported morphology with empty or ground glass nuclei, occasional nuclear grooves, inconspicuous nucleoli and abundant mucinous material in stroma.VHL gene analysis revealed deletions at 3p25-26 in 1 clear cell RCC and 1 papillary type 2 RCC. The papillary type 2 RCC was also presented with a family history of VHL disease and found a germline mutation G → C on a splicing site at position 553+5. The present case widens the spectrum of microscopic features to be found in VHL-associated RCC. There were no VHL mutations in the remaining 45 RCCs. Statistical analysis of stage and outcome revealed that TFE+ pediatric RCCs were significantly more frequently associated with a higher pTNM pT3/pT4 stage and a poorer outcome than TFE-RCCs (P < .05). Owing to the already known aggressive behavior of these Xp11.2 translocation RCCs, patients with TFE+ pediatric RCCs should benefit from a stricter follow-up.

The papillary renal cell carcinoma-associated t(X;1)(p11;q21) leads to fusion of the transcription factor TFE3 gene on the X-chromosome to a novel gene, PRCC, on chromosome 1. As a result, two putative fusion proteins are formed: PRCCTFE3, which contains all known domains for DNA binding, dimerization, and transactivation of the TFE3 protein, and the reciprocal product TFE3PRCC. Upon transfection into COS cells, both wild type and fusion proteins were found to be located in the nucleus. When comparing the transactivating capacities of these (fusion) proteins, significant differences were noted. PRCCTFE3 acted as a threefold better transactivator than wild type TFE3 both in a TFE3-specific and in a general (Zebra) reporter assay. In addition, PRCC and the two fusion proteins were found to be potent transactivators in the Zebra reporter assay. We propose that, as a result of the (X;1) translocation, fusion of the N-terminal PRCC sequences to TFE3 alters the transactivation capacity of the transcription factor thus leading to aberrant gene regulation and, ultimately, tumor formation.

The retinal pigment epithelium (RPE) is crucial for the function and survival of retinal photoreceptors. VMD2 encodes bestrophin, an oligomeric chloride channel that is preferentially expressed in the RPE and, when mutated, causes Best macular dystrophy. Previously, we defined the VMD2 upstream region from -253 to +38 bp as being sufficient to direct RPE-specific expression in the eye, and we suggested microphthalmia-associated transcription factor (MITF) as a possible positive regulator. Here we show that in transgenic mice the -154 to +38 bp region is sufficient for RPE expression, and mutation of two E-boxes, 1 and 2, within this region leads to loss of promoter activity. A yeast one-hybrid screen using bait containing E-box 1 identified clones encoding MITF, TFE3, and TFEB, and chromatin immunoprecipitation with antibodies against these proteins enriched the VMD2 proximal promoter. Analysis using in vivo electroporation with constructs containing mutation of each E-box indicated that expression in native RPE requires both E-boxes, yet in vitro DNA binding studies suggested that MITF binds well to E-box 1 but only minimally to E-box 2. MITF knockdown by small interfering RNA (siRNA) in cell culture revealed a strong correlation between MITF and VMD2 mRNA levels. Sequential transfection of a luciferase construct with expression vectors following MITF siRNA revealed that TFE3 and TFEB can also transactivate the VMD2 promoter. Taken together, we suggest that VMD2 is regulated by the MITF-TFE family through two E-boxes, with E-box 1 required for a direct interaction of MITF-TFE factors and E-box 2 for binding of the as yet unidentified factor(s).

TFE3 translocation renal cell carcinoma (tRCC) is defined by chromosomal translocations involving the TFE3 transcription factor at chromosome Xp11.2. Genetically proven TFE3 tRCCs have a broad histologic spectrum with overlapping features to other renal tumor subtypes. In this study, we aimed for characterizing RCC with TFE3 protein expression. Using next-generation whole transcriptome sequencing (RNA-Seq) as a discovery tool, we analyzed fusion transcripts, gene expression profile, and somatic mutations in frozen tissue of one TFE3 tRCC. By applying a computational analysis developed to call chimeric RNA molecules from paired-end RNA-Seq data, we confirmed the known TFE3 translocation. Its fusion partner SFPQ has already been described as fusion partner in tRCCs. In addition, an RNA read-through chimera between TMED6 and COG8 as well as MET and KDR (VEGFR2) point mutations were identified. An EGFR mutation, but no chromosomal rearrangements, was identified in a control group of five clear cell RCCs (ccRCCs). The TFE3 tRCC could be clearly distinguished from the ccRCCs by RNA-Seq gene expression measurements using a previously reported tRCC gene signature. In validation experiments using reverse transcription-PCR, TMED6-COG8 chimera expression was significantly higher in nine TFE3 translocated and six TFE3-expressing/non-translocated RCCs than in 24 ccRCCs (P < .001) and 22 papillary RCCs (P < .05-.07). Immunohistochemical analysis of selected genes from the tRCC gene signature showed significantly higher eukaryotic translation elongation factor 1 alpha 2 (EEF1A2) and Contactin 3 (CNTN3) expression in 16 TFE3 translocated and six TFE3-expressing/non-translocated RCCs than in over 200 ccRCCs (P < .0001, both).