Critical domains for NACC2-NTRK2 fusion protein activation.

Neurotrophic receptor tyrosine kinases (NTRKs) belong to the receptor tyrosine kinase (RTK) family. NTRKs are responsible for the activation of multiple downstream signaling pathways that regulate cell growth, proliferation, differentiation, and apoptosis. NTRK-associated mutations often result in oncogenesis and lead to aberrant activation of downstream signaling pathways including MAPK, JAK/STAT, and PLCγ1. This study characterizes the NACC2-NTRK2 oncogenic fusion protein that leads to pilocytic astrocytoma and pediatric glioblastoma. This fusion joins the BTB domain (Broad-complex, Tramtrack, and Bric-a-brac) domain of NACC2 (Nucleus Accumbens-associated protein 2) with the transmembrane helix and tyrosine kinase domain of NTRK2. We focus on identifying critical domains for the biological activity of the fusion protein. Mutations were introduced in the charged pocket of the BTB domain or in the monomer core, based on a structural comparison of the NACC2 BTB domain with that of PLZF, another BTB-containing protein. Mutations were also introduced into the NTRK2-derived portion to allow comparison of two different breakpoints that have been clinically reported. We show that activation of the NTRK2 kinase domain relies on multimerization of the BTB domain in NACC2-NTRK2. Mutations which disrupt BTB-mediated multimerization significantly reduce kinase activity and downstream signaling. The ability of these mutations to abrogate biological activity suggests that BTB domain inhibition could be a potential treatment for NACC2-NTRK2-induced cancers. Removal of the transmembrane helix leads to enhanced stability of the fusion protein and increased activity of the NACC2-NTRK2 fusion, suggesting a mechanism for the oncogenicity of a distinct NACC2-NTRK2 isoform observed in pediatric glioblastoma.

Oncogenic driver FGFR3-TACC3 requires five coiled-coil heptads for activation and disulfide bond formation for stability.

FGFR3-TACC3 represents an oncogenic fusion protein frequently identified in glioblastoma, lung cancer, bladder cancer, oral cancer, head and neck squamous cell carcinoma, gallbladder cancer, and cervical cancer. Various exon breakpoints of FGFR3-TACC3 have been identified in cancers; these were analyzed to determine the minimum contribution of TACC3 for activation of the FGFR3-TACC3 fusion protein. While TACC3 exons 11 and 12 are dispensable for activity, our results show that FGFR3-TACC3 requires exons 13-16 for biological activity. A detailed analysis of exon 13, which consists of 8 heptads forming a coiled coil, further defined the minimal region for biological activity as consisting of 5 heptads from exon 13, in addition to exons 14-16. These conclusions were supported by transformation assays of biological activity, examination of MAPK pathway activation, analysis of disulfide-bonded FGFR3-TACC3, and by examination of the Endoglycosidase H-resistant portion of FGFR3-TACC3. These results demonstrate that clinically identified FGFR3-TACC3 fusion proteins differ in their biological activity, depending upon the specific breakpoint. This study further suggests the TACC3 dimerization domain of FGFR3-TACC3 as a novel target in treating FGFR translocation driven cancers.

Nefarious NTRK oncogenic fusions in pediatric sarcomas: Too many to Trk.

Neurotrophic Tyrosine Receptor Kinase (NTRK) genes undergo chromosomal translocations to create novel open reading frames coding for oncogenic fusion proteins; the N-terminal portion, donated by various partner genes, becomes fused to the tyrosine kinase domain of either NTRK1, NTRK2, or NTRK3. NTRK fusion proteins have been identified as driver oncogenes in a wide variety of tumors over the past three decades, including Pediatric Gliomas, Papillary Thyroid Carcinoma, Spitzoid Neoplasms, Glioblastoma, and additional tumors. Importantly, NTRK fusions function as drivers of pediatric sarcomas, accounting for approximately 15% of childhood cancers including Infantile Fibrosarcoma (IFS), a subset of pediatric soft tissue sarcoma (STS). While tyrosine kinase inhibitors (TKIs), such as larotrectinib and entrectinib, have demonstrated profound results against NTRK fusion-positive cancers, acquired resistance to these TKIs has resulted in the formation of gatekeeper, solvent-front, and compound mutations. We present a comprehensive compilation of oncogenic fusions involving NTRKs focusing specifically on pediatric STS, examining their biological signaling pathways and mechanisms of activation. The importance of an obligatory dimerization or multimerization domain, invariably donated by the N-terminal fusion partner, is discussed using characteristic fusions that occur in pediatric sarcomas. In addition, examples are presented of oncogenic fusion proteins in which the N-terminal partners may contribute additional biological activities beyond an oligomerization domain. Lastly, therapeutic approaches to the treatment of pediatric sarcoma will be presented, using first generation and second-generation agents such as selitrectinib and repotrectinib.

Proteomic analysis reveals dual requirement for Grb2 and PLCγ1 interactions for BCR-FGFR1-Driven 8p11 cell proliferation.

Translocation of Fibroblast Growth Factor Receptors (FGFRs) often leads to aberrant cell proliferation and cancer. The BCR-FGFR1 fusion protein, created by chromosomal translocation t(8;22)(p11;q11), contains Breakpoint Cluster Region (BCR) joined to Fibroblast Growth Factor Receptor 1 (FGFR1). BCR-FGFR1 represents a significant driver of 8p11 myeloproliferative syndrome, or stem cell leukemia/lymphoma, which progresses to acute myeloid leukemia or T-cell lymphoblastic leukemia/lymphoma. Mutations were introduced at Y177F, the binding site for adapter protein Grb2 within BCR; and at Y766F, the binding site for the membrane associated enzyme PLCγ1 within FGFR1. We examined anchorage-independent cell growth, overall cell proliferation using hematopoietic cells, and activation of downstream signaling pathways. BCR-FGFR1-induced changes in protein phosphorylation, binding partners, and signaling pathways were dissected using quantitative proteomics to interrogate the protein interactome, the phosphoproteome, and the interactome of BCR-FGFR1. The effects on BCR-FGFR1-stimulated cell proliferation were examined using the PLCγ1 inhibitor U73122, and the irreversible FGFR inhibitor futibatinib (TAS-120), both of which demonstrated efficacy. An absolute requirement is demonstrated for the dual binding partners Grb2 and PLCγ1 in BCR-FGFR1-driven cell proliferation, and new proteins such as ECSIT, USP15, GPR89, GAB1, and PTPN11 are identified as key effectors for hematopoietic transformation by BCR-FGFR1.

Fibroblast growth factor receptor 4 promotes glioblastoma progression: a central role of integrin-mediated cell invasiveness.

Glioblastoma (GBM) is characterized by a particularly invasive phenotype, supported by oncogenic signals from the fibroblast growth factor (FGF)/ FGF receptor (FGFR) network. However, a possible role of FGFR4 remained elusive so far. Several transcriptomic glioma datasets were analyzed. An extended panel of primary surgical specimen-derived and immortalized GBM (stem)cell models and original tumor tissues were screened for FGFR4 expression. GBM models engineered for wild-type and dominant-negative FGFR4 overexpression were investigated regarding aggressiveness and xenograft formation. Gene set enrichment analyses of FGFR4-modulated GBM models were compared to patient-derived datasets. Despite widely absent in adult brain, FGFR4 mRNA was distinctly expressed in embryonic neural stem cells and significantly upregulated in glioblastoma. Pronounced FGFR4 overexpression defined a distinct GBM patient subgroup with dismal prognosis. Expression levels of FGFR4 and its specific ligands FGF19/FGF23 correlated both in vitro and in vivo and were progressively upregulated in the vast majority of recurrent tumors. Based on overexpression/blockade experiments in respective GBM models, a central pro-oncogenic function of FGFR4 concerning viability, adhesion, migration, and clonogenicity was identified. Expression of dominant-negative FGFR4 resulted in diminished (subcutaneous) or blocked (orthotopic) GBM xenograft formation in the mouse and reduced invasiveness in zebrafish xenotransplantation models. In vitro and in vivo data consistently revealed distinct FGFR4 and integrin/extracellular matrix interactions. Accordingly, FGFR4 blockade profoundly sensitized FGFR4-overexpressing GBM models towards integrin/focal adhesion kinase inhibitors. Collectively, FGFR4 overexpression contributes to the malignant phenotype of a highly aggressive GBM subgroup and is associated with integrin-related therapeutic vulnerabilities.

Characterization of FGFR signaling in prostate cancer stem cells and inhibition via TKI treatment.

Metastatic castrate-resistant prostate cancer (CRPC) remains uncurable and novel therapies are needed to better treat patients. Aberrant Fibroblast Growth Factor Receptor (FGFR) signaling has been implicated in advanced prostate cancer (PCa), and FGFR1 is suggested to be a promising therapeutic target along with current androgen deprivation therapy. We established a novel in vitro 3D culture system to study endogenous FGFR signaling in a rare subpopulation of prostate cancer stem cells (CSCs) in the cell lines PC3, DU145, LNCaP, and the induced pluripotent iPS87 cell line. 3D-propagation of PCa cells generated spheroids with increased stemness markers ALDH7A1 and OCT4, while inhibition of FGFR signaling by BGJ398 or Dovitinib decreased cell survival and proliferation of 3D spheroids. The 3D spheroids exhibited altered expression of EMT markers associated with metastasis such as E-cadherin, vimentin and Snail, compared to 2D monolayer cells. TKI treatment did not result in significant changes of EMT markers, however, specific inhibition of FGFR signaling by BGJ398 showed more favorable molecular-level changes than treatment with the multi-RTK inhibitor Dovitinib. This study provides evidence for the first time that FGFR1 plays an essential role in the proliferation of PCa CSCs at a molecular and cellular level, and suggests that TKI targeting of FGFR signaling may be a promising strategy for AR-independent CRPC.

Typical achondroplasia secondary to a unique insertional variant of FGFR3 with in vitro demonstration of its effect on FGFR3 function.

We describe an individual in whom clinical and radiographic features are typical for achondroplasia, but in whom the common variants of FGFR3 that result in achondroplasia are absent. Whole exome sequencing demonstrated a novel, de novo 6 base pair tandem duplication in FGFR3 that results in the insertion of Ser-Phe after position Leu324. in vitro studies showed that this variant results in aberrant dimerization, excessive spontaneous phosphorylation of FGFR3 dimers and excessive, ligand-independent tyrosine kinase activity. Together, these data suggest that this variant leads to constitutive disulfide bond-mediated dimerization, and that this, surprisingly, occurs to an extent similar to the neonatal lethal thanatophoric dysplasia type I Ser249Cys variant.

Oncogenic fusion protein FGFR2-PPHLN1: Requirements for biological activation, and efficacy of inhibitors.

AIM OF STUDY: Chromosomal translocations such as t(10;12)(q26,q12) are associated with intrahepatic cholangiocarcinoma, a universally fatal category of liver cancer. This translocation creates the oncogenic fusion protein of Fibroblast Growth Factor Receptor 2 joined to Periphilin 1. The aims of this study were to identify significant features required for biological activation, analyze the activation of downstream signaling pathways, and examine the efficacy of the TKIs BGJ398 and TAS-120, and of the MEK inhibitor Trametinib. METHODS: These studies examined FGFR2-PPHLN1 proteins containing a kinase-dead, kinase-activated, or WT kinase domain in comparison with analogous FGFR2 proteins. Biological activity was assayed using soft agar colony formation in epithelial RIE-1 cells and focus assays in NIH3T3 cells. The MAPK/ERK, JAK/STAT3 and PI3K/AKT signaling pathways were examined for activation. Membrane association was analyzed by indirect immunofluorescence comparing proteins altered by deletion of the signal peptide, or by addition of a myristylation signal. RESULTS: Biological activity of FGFR2-PPHLN1 required an active FGFR2-derived tyrosine kinase domain, and a dimerization domain contributed by PPHLN1. Strong activation of canonical MAPK/ERK, JAK/STAT3 and PI3K/AKT signaling pathways was observed. The efficacy of the tyrosine kinase inhibitors BGJ398 and TAS-120 was examined individually and combinatorially with the MEK inhibitor Trametinib; heterogeneous responses were observed in a mutation-specific manner. A requirement for membrane localization of the fusion protein was also demonstrated. CONCLUDING STATEMENT: Our study collectively demonstrates the potent transforming potential of FGFR2-PPHLN1 in driving cellular proliferation. We discuss the importance of sequencing-based, mutation-specific personalized therapeutics in treating FGFR2 fusion-positive intrahepatic cholangiocarcinoma.

Characterization of iPS87, a prostate cancer stem cell-like cell line.

Prostate cancer affects hundreds of thousands of men and families throughout the world. Although chemotherapy, radiation, surgery, and androgen deprivation therapy are applied, these therapies do not cure metastatic prostate cancer. Patients treated by androgen deprivation often develop castration resistant prostate cancer which is incurable. Novel approaches of treatment are clearly necessary. We have previously shown that prostate cancer originates as a stem cell disease. A prostate cancer patient sample, #87, obtained from prostatectomy surgery, was collected and frozen as single cell suspension. Cancer stem cell cultures were grown, single cell-cloned, and shown to be tumorigenic in SCID mice. However, outside its natural niche, the cultured prostate cancer stem cells lost their tumor-inducing capability and stem cell marker expression after approximately 8 transfers at a 1:3 split ratio. Tumor-inducing activity could be restored by inducing the cells to pluripotency using the method of Yamanaka. Cultures of human prostate-derived normal epithelial cells acquired from commercial sources were similarly induced to pluripotency and these did not acquire a tumor phenotype in vivo. To characterize the iPS87 cell line, cells were stained with antibodies to various markers of stem cells including: ALDH7A1, LGR5, Oct4, Nanog, Sox2, Androgen Receptor, and Retinoid X Receptor. These markers were found to be expressed by iPS87 cells, and the high tumorigenicity in SCID mice of iPS87 was confirmed by histopathology. This research thus characterizes the iPS87 cell line as a cancer-inducing, stem cell-like cell line, which can be used in the development of novel treatments for prostate cancer.

Functions of FGFR2 corrupted by translocations in intrahepatic cholangiocarcinoma.

Cholangiocarcinoma, originating from the biliary duct, represents a subset of liver cancer. With about 8000 new cases of cholangiocarcinoma diagnosed annually in the U.S., these fall into three categories: intrahepatic, peri-hilar, and extrahepatic cholangiocarcinoma. Arising from the epithelium of the bile duct, intrahepatic cholangiocarcinoma (ICC) is a universally fatal malignancy with very few treatment options. The poor prognosis and lack of molecular targeted therapies highlights ICC as a critical unmet medical need. With advances in sequencing technology, numerous chromosomal translocations have been discovered as drivers in cancer initiation and progression. Particularly in ICC, chromosomal translocations involving Fibroblast Growth Factor Receptor 2 (FGFR2) have been frequently identified, resulting in the creation of oncogenic fusion proteins. At the N-terminus, these fusion proteins share a nearly-identical FGFR2 moiety retaining an intact kinase domain and, at the C-terminus, a dimerization/oligomerization domain provided by different partner genes, including: Periphilin 1 (PPHLN1), Bicaudal family RNA binding protein 1 (BICC1), Adenosylhomocysteinase Like 1 (AHCYL1), and Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3). A number of pre-clinical and clinical trials have shown the effectiveness of FGFR inhibitors in treating FGFR2 fusion-positive ICC patients. However, the efficacy of these inhibitors may be short-lived due to acquired resistance. In this review, we provide an overview of FGFR2 fusions, comparing their structures and mechanism of dimerization, examining the importance of FGFR2 as a partner gene, as well as highlighting the significance of alternative splicing of FGFR2 in these fusion proteins. In addition, we discuss various therapeutic options and their associated potencies in targeting these translocation-induced ICCs.

Oncogenic fusion protein BCR-FGFR1 requires the breakpoint cluster region-mediated oligomerization and chaperonin Hsp90 for activation.

Mutation and translocation of fibroblast growth factor receptors often lead to aberrant signaling and cancer. This work focuses on the t(8;22)(p11;q11) chromosomal translocation which creates the breakpoint cluster region (BCR) fibroblast growth factor receptor1 (FGFR1) (BCR-FGFR1) fusion protein. This fusion occurs in stem cell leukemia/lymphoma, which can progress to atypical chronic myeloid leukemia, acute myeloid leukemia, or B-cell lymphoma. This work focuses on the biochemical characterization of BCR-FGFR1 and identification of novel therapeutic targets. The tyrosine kinase activity of FGFR1 is required for biological activity as shown using transformation assays, interleukin-3 independent cell proliferation, and liquid chromatography/mass spectroscopy analyses. Furthermore, BCR contributes a coiled-coil oligomerization domain, also essential for oncogenic transformation by BCR-FGFR1. The importance of salt bridge formation within the coiled-coil domain is demonstrated, as disruption of three salt bridges abrogates cellular transforming ability. Lastly, BCR-FGFR1 acts as a client of the chaperonin heat shock protein 90 (Hsp90), suggesting that BCR-FGFR1 relies on Hsp90 complex to evade proteasomal degradation. Transformed cells expressing BCR-FGFR1 are sensitive to the Hsp90 inhibitor Ganetespib, and also respond to combined treatment with Ganetespib plus the FGFR inhibitor BGJ398. Collectively, these data suggest novel therapeutic approaches for future stem cell leukemia/lymphoma treatment: inhibition of BCR oligomerization by disruption of required salt bridges; and inhibition of the chaperonin Hsp90 complex.

BCR: a promiscuous fusion partner in hematopoietic disorders.

Considerable advances have been made in our understanding of the molecular basis of hematopoietic cancers. The discovery of the BCR-ABL fusion protein over 50 years ago has brought about a new era of therapeutic progress and overall improvement in patient care, mainly due to the development and use of personalized medicine and tyrosine kinase inhibitors (TKIs). However, since the detection of BCR-ABL, BCR has been identified as a commonly occurring fusion partner in hematopoietic disorders. BCR has been discovered fused to additional tyrosine kinases, including: Fibroblast Growth Factor Receptor 1 (FGFR1), Platelet-derived Growth Factor Receptor Alpha (PDGFRA), Ret Proto-Oncogene (RET), and Janus Kinase 2 (JAK2). While BCR translocations are infrequent in hematopoietic malignancies, clinical evidence suggests that patients who harbor these mutations benefit from TKIs and additional personalized therapies. The improvement of further methodologies for characterization of these fusions is crucial to determine a patient's treatment regimen, and optimal outcome. However, potential relapse and drug resistance among patients' highlights the need for additional treatment options and further understanding of these oncogenic fusion proteins. This review explores the mechanisms behind cancer progression of these BCR oncogenic fusion proteins, comparing their similarities and differences, examining the significance of BCR as a partner gene, and discussing current treatment options for these translocation-induced hematopoietic malignancies.

Oncogenic mutations in IKKß function through global changes induced by K63-linked ubiquitination and result in autocrine stimulation.

Mutations at position K171 in the kinase activation loop of Inhibitor of κB kinase beta (IKKß) occur in multiple myeloma, spleen marginal zone lymphoma and mantle cell lymphoma. Previously, we demonstrated that these result in constitutive kinase activation and stimulate Signal Transducer and Activator of Transcription 3 (STAT3). This work also identified K147 as a site of K63-linked regulatory ubiquitination required for activation of signaling pathways. We now present a more detailed analysis of ubiquitination sites together with a comprehensive examination of the signaling pathways activated by IKKß K171E mutants. Downstream activation of STAT3 is dependent upon the activity of: UBE2N, the E2 ubiquitin ligase involved in K63-linked ubiquitination; TAK1 (MAP3K7), or TGFß Activated Kinase, which forms a complex required for NFκB activation; JAK kinases, involved proximally in the phosphorylation of STAT transcription factors in response to inflammatory cytokines; and gp130, or IL-6 Receptor Subunit Beta which, upon binding IL-6 or other specific cytokines, undergoes homodimerization leading to activation of associated JAKs, resulting in STAT activation. We further demonstrate, using an IL-6-responsive cell line, that IKKß K171E mutants stimulate the release of IL-6 activity into conditioned media. These results show that IKKß K171E mutants trigger an autocrine loop in which IL-6 is secreted and binds to the IL-6 receptor complex gp130, resulting in JAK activation. Lastly, by examining the differential abundance of proteins associated with K63-only-ubiquitinated IKKß K171E, proteomic analysis demonstrates the global activation of proliferative responses. As cancers harboring K171-mutated IKKß are likely to also exhibit activated STAT3 and p44/42 MAPK (Erk1/2), this suggests the possibility of using MAPK (Erk1/2) and JAK inhibitors, or specific ubiquitination inhibitors. K63-linked ubiquitination occurs in other kinases at sites homologous to K147 in IKKß, including K578 in BRAF V600E, which serves as an oncogenic driver in melanoma and other cancers.

Oncogenic driver FGFR3-TACC3 is dependent on membrane trafficking and ERK signaling.

Fusion proteins resulting from chromosomal translocations have been identified as oncogenic drivers in many cancers, allowing them to serve as potential drug targets in clinical practice. The genes encoding FGFRs, Fibroblast Growth Factor Receptors, are commonly involved in such translocations, with the FGFR3-TACC3 fusion protein frequently identified in many cancers, including glioblastoma, cervical cancer, bladder cancer, nasopharyngeal carcinoma, and lung adenocarcinoma among others. FGFR3-TACC3 retains the entire extracellular domain and most of the kinase domain of FGFR3, with its C-terminal domain fused to TACC3. We examine here the effects of targeting FGFR3-TACC3 to different subcellular localizations by appending either a nuclear localization signal (NLS) or a myristylation signal, or by deletion of the normal signal sequence. We demonstrate that the oncogenic effects of FGFR3-TACC3 require either entrance to the secretory pathway or plasma membrane localization, leading to overactivation of canonical MAPK/ERK pathways. We also examined the effects of different translocation breakpoints in FGFR3-TACC3, comparing fusion at TACC3 exon 11 with fusion at exon 8. Transformation resulting from FGFR3-TACC3 was not affected by association with the canonical TACC3-interacting proteins Aurora-A, clathrin, and ch-TOG. We have shown that kinase inhibitors for MEK (Trametinib) and FGFR (BGJ398) are effective in blocking cell transformation and MAPK pathway upregulation. The development of personalized medicines will be essential in treating patients who harbor oncogenic drivers such as FGFR3-TACC3.

RAS at the Golgi antagonizes malignant transformation through PTPRκ-mediated inhibition of ERK activation.

RAS GTPases are frequently mutated in human cancer. H- and NRAS isoforms are distributed over both plasma-membrane and endomembranes, including the Golgi complex, but how this organizational context contributes to cellular transformation is unknown. Here we show that RAS at the Golgi is selectively activated by apoptogenic stimuli and antagonizes cell survival by suppressing ERK activity through the induction of PTPRκ, which targets CRAF for dephosphorylation. Consistently, in contrast to what occurs at the plasma-membrane, RAS at the Golgi cannot induce melanoma in zebrafish. Inactivation of PTPRκ, which occurs frequently in human melanoma, often coincident with TP53 inactivation, accelerates RAS-ERK pathway-driven melanomagenesis in zebrafish. Likewise, tp53 disruption in zebrafish facilitates oncogenesis driven by RAS from the Golgi complex. Thus, RAS oncogenic potential is strictly dependent on its sublocalization, with Golgi complex-located RAS antagonizing tumor development.

Receptor Tyrosine Kinases: Translocation Partners in Hematopoietic Disorders.

Receptor tyrosine kinases (RTKs) activate various signaling pathways and regulate cellular proliferation, survival, migration, and angiogenesis. Malignant neoplasms often circumvent or subjugate these pathways by promoting RTK overactivation through mutation or chromosomal translocation. RTK translocations create a fusion protein containing a dimerizing partner fused to an RTK kinase domain, resulting in constitutive kinase domain activation, altered RTK cellular localization, upregulation of downstream signaling, and novel pathway activation. While RTK translocations in hematological malignancies are relatively rare, clinical evidence suggests that patients with these genetic abnormalities benefit from RTK-targeted inhibitors. Here, we present a timely review of an exciting field by examining RTK chromosomal translocations in hematological cancers, such as Anaplastic Lymphoma Kinase (ALK), Fibroblast Growth Factor Receptor (FGFR), Platelet-Derived Growth Factor Receptor (PDGFR), REarranged during Transfection (RET), Colony Stimulating Factor 1 Receptor (CSF1R), and Neurotrophic Tyrosine Kinase Receptor Type 3 (NTRK3) fusions, and discuss current therapeutic options.

Visualization of early prostatic adenocarcinoma as a stem cell disease.

Prostate Cancer represents the second leading cause of cancer death among men in the United States, and the third leading cause of cancer death among men in Europe. We have previously shown that cells possessing Cancer Stem Cell (CSC) characteristics can be grown from human PrCa tissue harvested at the time of prostatectomy. However, the cellular origin of these CSCs was not previously known. In most cases, simple hematoxylin and eosin (H&E) stained sections are sufficient to make a definitive diagnosis of prostatic adenocarcinoma (PrCa) in needle biopsy samples. We utilized six different antibodies specific for stem cell antigens to examine paraffin sections of PrCa taken at the time of needle-biopsy diagnosis. These antisera were specific for CD44, CD133, ALDH7A1, LGR-5, Oct-4 and NANOG. We demonstrate specific staining of tumor cells with all six antisera specific for stem cell antigens. Some of these antibodies also react with cells of hyperplastic glands, but the patterns of reactivity differ from those of malignant glands. These findings demonstrate that at the time of diagnosis, PrCa consists of cells exhibiting properties of CSCs and consistent with the possibility that PrCa is a stem cell disease.

Oncogenic Gene Fusion FGFR3-TACC3 Is Regulated by Tyrosine Phosphorylation.

UNLABELLED: Fibroblast growth factor receptors (FGFR) are critical for cell proliferation and differentiation. Mutation and/or translocation of FGFRs lead to aberrant signaling that often results in developmental syndromes or cancer growth. As sequencing of human tumors becomes more frequent, so does the detection of FGFR translocations and fusion proteins. The research conducted in this article examines a frequently identified fusion protein between FGFR3 and transforming acidic coiled-coil containing protein 3 (TACC3), frequently identified in glioblastoma, lung cancer, bladder cancer, oral cancer, head and neck squamous cell carcinoma, gallbladder cancer, and cervical cancer. Using titanium dioxide-based phosphopeptide enrichment (TiO2)-liquid chromatography (LC)-high mass accuracy tandem mass spectrometry (MS/MS), it was demonstrated that the fused coiled-coil TACC3 domain results in constitutive phosphorylation of key activating FGFR3 tyrosine residues. The presence of the TACC coiled-coil domain leads to increased and altered levels of FGFR3 activation, fusion protein phosphorylation, MAPK pathway activation, nuclear localization, cellular transformation, and IL3-independent proliferation. Introduction of K508R FGFR3 kinase-dead mutation abrogates these effects, except for nuclear localization which is due solely to the TACC3 domain. IMPLICATIONS: These results demonstrate that FGFR3 kinase activity is essential for the oncogenic effects of the FGFR3-TACC3 fusion protein and could serve as a therapeutic target, but that phosphorylated tyrosine residues within the TACC3-derived portion are not critical for activity. Mol Cancer Res; 14(5); 458-69. ©2016 AACR.

Functions of Fibroblast Growth Factor Receptors in cancer defined by novel translocations and mutations.

The four receptor tyrosine kinases (RTKs) within the family of Fibroblast Growth Factor Receptors (FGFRs) are critical for normal development but also play an enormous role in oncogenesis. Mutations and/or abnormal expression often lead to constitutive dimerization and kinase activation of FGFRs, and represent the primary mechanism for aberrant signaling. Sequencing of human tumors has revealed a plethora of somatic mutations in FGFRs that are frequently identical to germline mutations in developmental syndromes, and has also identified novel FGFR fusion proteins arising from chromosomal rearrangements that contribute to malignancy. This review details approximately 200 specific point mutations in FGFRs and 40 different fusion proteins created by translocations involving FGFRs that have been identified in human cancer. This review discusses the effects of these genetic alterations on downstream signaling cascades, and the challenge of drug resistance in cancer treatment with antagonists of FGFRs.

Novel Lys63-linked ubiquitination of IKKß induces STAT3 signaling.

NFκB signaling plays a significant role in human disease, including breast and ovarian carcinoma, insulin resistance, embryonic lethality and liver degeneration, rheumatoid arthritis, aging and Multiple Myeloma (MM). Inhibitor of κB (IκB) kinase ß (IKKß) regulates canonical Nuclear Factor κB (NFκB) signaling in response to inflammation and cellular stresses. NFκB activation requires Lys63-linked (K63-linked) ubiquitination of upstream proteins such as NEMO or TAK1, forming molecular complexes with membrane-bound receptors. We demonstrate that IKKß itself undergoes K63-linked ubiquitination. Mutations in IKKß at Lys171, identified in Multiple Myeloma and other cancers, lead to a dramatic increase in kinase activation and K63-linked ubiquitination. These mutations also result in persistent activation of STAT3 signaling. Liquid chromatography (LC)-high mass accuracy tandem mass spectrometry (MS/MS) analysis identified Lys147, Lys418, Lys555 and Lys703 as predominant ubiquitination sites in IKKß. Specific inhibition of the UBC13-UEV1A complex responsible for K63-linked ubiquitination establishes Lys147 as the predominant site of K63-ubiquitin conjugation and responsible for STAT3 activation. Thus, IKKß activation leads to ubiquitination within the kinase domain and assemblage of a K63-ubiquitin conjugated signaling platform. These results are discussed with respect to the importance of upregulated NFκB signaling known to occur frequently in MM and other cancers.