Tumours displaying differentiation towards normal fat constitute the most common subgroup of soft tissue neoplasms. A series of such tumours was investigated by whole-exome sequencing followed by targeted ultra-deep sequencing. Eighty per cent of angiolipomas, but not any other tumour type, displayed mutations in the protein kinase D2 (PRKD2) gene, typically in the part encoding the catalytic domain. The absence of other aberrations at the chromosome or RNA level suggests that PRKD2 mutations are critical for angiolipoma development. Consistently, the mutated PRKD2 alleles were present at low (3-15%) frequencies, indicating that only a subset of the tumour cells is affected. Indeed, by sequencing mature fat cells and other cells separately, the former typically showed the highest mutation frequencies. Thus, we hypothesize that altered PRKD2 signalling in the adipocytic cells drives tumourigenesis and, in agreement with its pivotal role in angiogenesis, induces the vessel formation that is characteristic for angiolipoma. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

In Philadelphia-positive chronic myeloid leukemia (CML), imatinib resistance frequently emerges because of point mutations in the ABL1 kinase domain, but may also be the consequence of uncontrolled upstream signaling. Recently, the heteromeric transcription factor GA-binding protein (GABP) was found to promote CML-like myeloproliferative disease in mice. In a cohort of 70 CML patients, we found that expression of the GABP α subunit (GABPα) is positively correlated to the BCR-ABL1/ABL1 ratio. Moreover, significantly higher GABPα expression was detected in blast crisis than in chronic phase CML after performing data mining on 91 CML patients. In functional studies, imatinib sensitivity is enhanced after GABPα knockdown in tyrosine kinase inhibitors (TKI)-sensitive K-562, as well as by overexpression of a deletion mutant in TKI-resistant NALM-1 cells. Moreover, in K-562 cells, GABP-dependent expression variations of PRKD2 and RAC2, relevant signaling mediators in CML, were observed. Notably, protein kinase D2 (Prkd2) was reported to be a GABP target gene in mice. In line with this, we detected a positive correlation between GABPA and PRKD2 expression in primary human CML, indicating that the effects of GABP are mediated by PRKD2. These findings illustrate an important role for GABP in disease development and imatinib sensitivity in human CML.

Background and purpose: The peptide hormone vasopressin regulates water transport in the renal collecting duct largely via the V2 receptor, which triggers a cAMP-mediated activation of a protein kinase A (PKA)-dependent signaling network. The protein kinases downstream from PKA have not been fully identified or mapped to regulated phosphoproteins.

Experimental approach: We carried out systems-level analysis of large-scale phosphoproteomic data quantifying vasopressin-induced changes in phosphorylation in aquaporin-2-expressing cultured collecting duct cells (mpkCCD). Quantification was done using stable isotope labeling (SILAC method).

Key results: 9640 phosphopeptides were quantified. Stringent statistical analysis identified significant changes in response to vasopressin in 429 of these phosphopeptides. The corresponding phosphoproteins were mapped to known vasopressin-regulated cellular processes. The vasopressin-regulated sites were classified according to the sequences surrounding the phosphorylated amino acids giving 11 groups. Among the vasopressin-regulated phosphoproteins were 25 distinct protein kinases. Among these, six plus PKA appeared to account for phosphorylation of about 81% of the 313 vasopressin-regulated phosphorylation sites. The six downstream kinases were salt-inducible kinase 2 (Sik2), cyclin-dependent kinase 18 (Cdk18/PCTAIRE-3), calmodulin-dependent kinase kinase 2 (Camkk2), protein kinase D2 (Prkd2), mitogen-activated kinase 3 (Mapk3/ERK1), and myosin light chain kinase (Mylk).

Conclusion and implications: In V2 receptor-mediated signaling, PKA is at the head of a complex network that includes at least 6 downstream vasopressin-regulated protein kinases that are prime targets for future study. The extensive phosphoproteomic data reported in this study is provided as a web-based data resource for future studies of G-protein coupled receptors.

Epigenetic regulation has profound influence on stem cell fate during normal development in maintenance of physiologic tissue homeostasis. Here we report diminished ten-eleven translocation (TET) methylcytosine dioxygenase expression and loss of the DNA hydroxymethylation mark, 5-hydroxymethylcytosine (5-hmC), in keratinocyte stem cells (KSCs) and transit-amplifying cells (TACs) in human psoriasis and in imiquimod-induced murine psoriasis. Loss of 5-hmC was associated with dysregulated KSC kinetics, resulting in accumulation of nestin and FABP5-expressing TACs to produce classic psoriatic epidermal architecture. Moreover, 5-hmC loss was accompanied by diminished TET1 and TET2 mRNA expression. Genome-wide mapping of epidermal 5-hmC in murine psoriasis revealed loci-specific loss of 5-hmC in genes regulating stem cell homeostasis, including MBD1, RTN1, STRN4, PRKD2, AKT1, MAPKAP2, as well as those associated with RAR and Wnt/β-catenin signaling pathways. In vitro restoration of TET expression by ascorbic acid was accomplished in cultured human KSCs to show similar [Ca++]-induced differentiation, resulting in increased 5-hmC levels and reduced nestin expression. To our knowledge, an epigenetic deficiency in psoriasis with relevance to stem cell dysregulation has not been previously reported. This observation raises the possibility that epigenetic modifiers that impact on the TET-5-hmC pathway may be a relevant approach of heretofore unappreciated therapeutic utility.

Polymorphous adenocarcinoma (PAC) and cribriform adenocarcinoma of (minor) salivary gland (CASG) are salivary gland tumors with overlapping spectrum of morphology. Whether these represent distinct entities or a histologic spectrum of the same tumor remains contentious. PACs harbor recurrent PRKD1 E710D hotspot mutations in >70% of cases, whereas 80% of CASGs display rearrangements involving PRKD1, PRKD2, or PRKD3 (PRKD1/2/3). We studied the molecular and morphologic features of 37 PACs/CASGs, seeking to identify the associations among genotype, histologic phenotype, and classification. DNA was subjected to Sanger sequencing analysis of the PRKD1 hotspot locus. Fluorescence in situ hybridization (FISH) analysis for PRKD1/2/3 was performed using dual-color break-apart probes. Tumors were classified into four categories as described previously: PAC, CASG, tumor with indeterminate features (TIF), and tumor with a predominant papillary pattern (TPPP). PRKD1 E710D hotspot mutations were identified in 56%, 20%, 43% and 0% of PACs, CASGs, TIFs, and TPPPs, respectively. FISH demonstrated PRKD1/2/3 rearrangements in 13%, 78%, 36%, and 75% of PACs, CASGs, TIFs, and TPPPs, respectively. Histologically, fusion-positive tumors were associated with a high percentage of papillary growth, low percentage of single filing arrangement, a propensity of base of tongue location, and frequent (50%) lymph node metastasis, compared with the mutation-related tumors which had negligible nodal metastasis risk. Our results demonstrated that (1) PACs/CASGs are underpinned by genetic alterations affecting PRKD genes; (2) despite the associations between PAC and PRKD1 hotspot mutations and CASG and PRKD1/2/3 fusion, such distinction is not absolute; and (3) there is of a novel genotypic-phenotypic association whereby fusion-positive tumors are usually located in the base of the tongue, show papillary architecture and have a high risk of nodal metastasis. Genetic analysis of PRKD genes appears to be useful characterizing this spectrum of tumors, not only histologically but also clinically identifying those tumors with high risk of nodal metastasis.

Protein Kinase D2 belongs to a family of evolutionarily conserved enzymes regulating several biological processes. In a forward genetic screen for zebrafish cardiovascular mutants, we identified a mutation in the prkd2 gene. Homozygous mutant embryos develop as wild-type up to 36hours post-fertilization and initiate blood flow, but fail to maintain it, resulting in a complete outflow tract stenosis. We identified a mutation in the prkd2 gene that results in a T757A substitution at a conserved residue in the kinase domain activation loop (T714A in human PRKD2) that disrupts catalytic activity and drives this phenotype. Homozygous mutants survive without circulation for several days, allowing us to study the extreme phenotype of no intracardiac flow, in the background of a functional heart. We show dysregulation of atrioventricular and outflow tract markers in the mutants and higher sensitivity to the Calcineurin inhibitor, Cyclosporin A. Finally we identify TBX5 as a potential regulator of PRKD2. Our results implicate PRKD2 catalytic activity in outflow tract development in zebrafish.

Keywords: Cardiac valves; Cardiovascular development; Protein kinase D2; Zebrafish.

Background: Atopy, the overall tendency to become sensitized to an allergen, is heritable but seldom ascribed to mutations within specific genes. Atopic individuals develop abnormally elevated IgE responses to immunization with potential allergens. To gain insight into the genetic causes of atopy, we carried out a forward genetic screen for atopy in mice.

Methods: We screened mice carrying homozygous and heterozygous N-ethyl-N-nitrosourea (ENU)-induced germline mutations for aberrant antigen-specific IgE and IgG1 production in response to immunization with the model allergen papain. Candidate genes were validated by independent gene mutation.

Results: Of 31 candidate genes selected for investigation, the effects of mutations in 23 genes on papain-specific IgE or IgG1 were verified. Among the 20 verified genes influencing the IgE response, 8 were necessary for the response, while 12 repressed IgE. Nine genes were not previously implicated in the IgE response. Fifteen genes encoded proteins contributing to IgE class switch recombinationor B cell receptor signaling. The precise roles of the five remaining genes (Flcn, Map1lc3b, Me2, Prkd2, and Scarb2) remain to be determined. Loss-of-function mutations in nine of the 12 genes limiting the IgE response were dominant or semi-dominant for the IgE phenotype but did not cause immunodeficiency in the heterozygous state. Using damaging allele frequencies for the corresponding human genes and in silico simulations (Monte Carlo) of undiscovered atopy mutations, we estimated the percentage of humans with heterozygous atopy risk mutations.

Conclusions: Up to 37% of individuals may be heterozygous carriers for at least one dominant atopy risk mutation.

Keywords: IgE; IgG1; allergy; atopy; papain.

Background: The aim of this study is to determine whether Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) could be used as a biomarker for the diagnosis and treatment of B cell malignancies. With 4.3% of all new cancers diagnosed as Non-Hodgkin lymphoma, finding new biomarkers for the treatment of B cell cancers is an ongoing pursuit. HPRT is a nucleotide salvage pathway enzyme responsible for the synthesis of guanine and inosine throughout the cell cycle.

Methods: Raji cells were used for this analysis due to their high HPRT internal expression. Internal expression was evaluated utilizing western blotting and RNA sequencing. Surface localization was analyzed using flow cytometry, confocal microscopy, and membrane biotinylation. To determine the source of HPRT surface expression, a CRISPR knockdown of HPRT was generated and confirmed using western blotting. To determine clinical significance, patient blood samples were collected and analyzed for HPRT surface localization.

Results: We found surface localization of HPRT on both Raji cancer cells and in 77% of the malignant ALL samples analyzed and observed no significant expression in healthy cells. Surface expression was confirmed in Raji cells with confocal microscopy, where a direct overlap between HPRT specific antibodies and a membrane-specific dye was observed. HPRT was also detected in biotinylated membranes of Raji cells. Upon HPRT knockdown in Raji cells, we found a significant reduction in surface expression, which shows that the HPRT found on the surface originates from the cells themselves. Finally, we found that cells that had elevated levels of HPRT had a direct correlation to XRCC2, BRCA1, PIK3CA, MSH2, MSH6, WDYHV1, AK7, and BLMH expression and an inverse correlation to PRKD2, PTGS2, TCF7L2, CDH1, IL6R, MC1R, AMPD1, TLR6, and BAK1 expression. Of the 17 genes with significant correlation, 9 are involved in cellular proliferation and DNA synthesis, regulation, and repair.

Conclusions: As a surface biomarker that is found on malignant cells and not on healthy cells, HPRT could be used as a surface antigen for targeted immunotherapy. In addition, the gene correlations show that HPRT may have an additional role in regulation of cancer proliferation that has not been previously discovered.

Keywords: ALL; Acute Lymphoblastic Leukemia; Biomarker; Burkitt’s B cell lymphoma; Hypoxanthine Guanine Phosphoribosyltransferase (HPRT or HGPRT); Lymphocytes; Raji; Surface Localization.

Background: Angiolipoma (AL) is considered as a lipoma variant, characterized by the combination of mature adipocytes and capillary blood vessels diffusely distributed within the tumor. With the exception of recurrent PRKD2 mutations of uncertain pathogenetic significance, the genetic abnormalities of ALs are unknown, in absence of any of the specific chromosomal aberrations described in other lipoma variants.

Methods: Formalin-fixed and paraffin-embedded blocks of 13 conventional ALs and 5 cellular ALs from 17 individuals were retrieved and analyzed for mutations in exons 9 and 20 of PIK3CA by polymerase chain reaction and Sanger sequencing.

Results: Activating PIK3CA mutations were identified in 14 tumors (78%). All PIK3CA-mutated samples carried the same exon 9 mutation, c.1634A>C (p.E545A). No mutation was detected in exon 20 of PIK3CA. No significant difference between PIK3CA-mutated and wild-type samples appeared to exist based on age, gender, and location of the tumor. All 5 cellular ALs carried the p.E545A PIK3CA mutation.

Conclusions: The high frequency of the p.E545A PIK3CA mutation in both conventional and cellular ALs suggests that activation of the PI3K/AKT pathway plays a key role in AL pathogenesis, and reinforces the concept that cellular AL should be regarded as a variant of AL. This article is protected by copyright. All rights reserved.

Keywords: Angiolipoma; PIK3CA; hemangioma; lipoma; vascular malformation.

Polymorphous adenocarcinoma (PAC) shows histologic diversity with streaming and targetoid features whereas cribriform adenocarcinoma of salivary gland (CASG) demonstrates predominantly cribriform and solid patterns with glomeruloid structures and optically clear nuclei. Opinions diverge on whether CASG represents a separate entity or a variant of PAC. We aimed to assess the level of agreement among 25 expert Head and Neck pathologists in classifying these tumors. Digital slides of 48 cases were reviewed and classified as: PAC, CASG, tumors with ≥50% of papillary architecture (PAP), and tumors with indeterminate features (IND). The consensus diagnoses were correlated with a previously reported molecular alteration. The consensus diagnoses were PAC in 18/48, CASG in16/48, PAP in 3/48, and IND in 11/48. There was a fair interobserver agreement in classifying the tumors (κ=0.370). The full consensus was achieved in 3 (6%) cases, all of which were classified as PAC. A moderate agreement was reached for PAC (κ=0.504) and PAP (κ=0.561), and a fair agreement was reached for CASG (κ=0.390). IND had only slight diagnostic concordance (κ=0.091). PAC predominantly harbored PRKD1 hotspot mutation, whereas CASG was associated with fusion involving PRKD1, PRKD2, or PRKD3. However, such molecular events were not exclusive as 7% of PAC had fusion and 13% of CASG had mutation. In conclusion, a fair to moderate interobserver agreement can be achieved in classifying PAC and CASG. However, a subset (23%) showed indeterminate features and was difficult to place along the morphologic spectrum of PAC/CASG among expert pathologists. This may explain the controversy in classifying these tumors.

Background: Glioblastoma (GBM) is the most prevalent malignant tumor of the central nervous system (CNS). However, current GBM treatments are ineffective, signifying the great importance of exploring new therapeutic targets. Curcumin has been found to be a natural compound with an anticancer potential. However, its targets and mechanisms in GBM are still unclear.

Methods: Differentially expressed genes (DEGs) were screened from the GBM dataset in the GEO database and intersected with the target genes of curcumin to select potential target genes. Subsequently, survival analysis was performed with the GEPIA database to confirm the effect of target genes on the prognosis of GBM, and functional enrichment analysis was performed using the DAVID database. In vitro, CCK-8 assay was used to screen the appropriate concentration of curcumin; scratch and transwell invasion assays were used to evaluate the effect of curcumin on the migration and invasion abilities of GBM cells. Furthermore, RT-qPCR and Western blotting were used to detect changes in target genes and flow cytometry was used to assess the apoptosis level.

Results: A total of 16 target genes of curcumin and GBM were obtained, among which ENO1, MMP2, and PRKD2 significantly affected the prognosis (P < 0.05). We further selected ENO1 for functional enrichment analysis and found that it was enriched in the glycolytic pathway. Meanwhile, in vitro experiments showed that curcumin could inhibit the migration and invasion of U251 cells and promote apoptosis (P < 0.05).

Conclusion: ENO1 could be a possible target for curcumin in the suppression of GBM cells.

Keywords: ENO1; apoptosis; computational biology; curcumin; glioblastoma; migration.

Lipids are the most energy-dense components of the diet, and their overconsumption promotes obesity and diabetes. Dietary fat content has been linked to the lipid processing activity by the intestine and its overall capacity to absorb triglycerides (TG). However, the signaling cascades driving intestinal lipid absorption in response to elevated dietary fat are largely unknown. Here, we describe an unexpected role of the protein kinase D2 (PKD2) in lipid homeostasis. We demonstrate that PKD2 activity promotes chylomicron-mediated TG transfer in enterocytes. PKD2 increases chylomicron size to enhance the TG secretion on the basolateral side of the mouse and human enterocytes, which is associated with decreased abundance of APOA4. PKD2 activation in intestine also correlates positively with circulating TG in obese human patients. Importantly, deletion, inactivation, or inhibition of PKD2 ameliorates high-fat diet-induced obesity and diabetes and improves gut microbiota profile in mice. Taken together, our findings suggest that PKD2 represents a key signaling node promoting dietary fat absorption and may serve as an attractive target for the treatment of obesity.

Keywords: chylomicron; fat absorption; intestine; obesity; protein kinase D2/PKD2/PRKD2.

Background: The cell-surface enzyme carbonic anhydrase IX (CAIX/CA9) promotes tumor growth, survival, invasion, and metastasis, mainly via its pH-regulating functions. Owing to its tumor-specific expression, CAIX-targeting antibodies/chemicals are utilized for therapeutic and diagnostic purposes. However, mechanisms of CAIX trafficking, which affects such CAIX-targeting modalities remain unclear. In this study, roles of the AMAP1-PRKD2 pathway, which mediates integrin recycling of invasive cancer cells, in CAIX trafficking were investigated.

Methods: Using highly invasive MDA-MB-231 breast cancer cells, the physical association and colocalization of endogenous proteins were analyzed by immunoprecipitation and immunofluorescence, protein/mRNA levels were quantified by western blotting/qPCR, and cell-surface transport and intracellular/extracellular pH regulation were measured by biotin-labeling and fluorescent dye-based assays, respectively. The correlation between mRNA levels and patients' prognoses was analyzed using a TCGA breast cancer dataset.

Results: AMAP1 associated with the CAIX protein complex, and they colocalized at the plasma membrane and tubulovesicular structures. AMAP1 knockdown reduced total/surface CAIX, induced its lysosomal accumulation and degradation, and affected intracellular/extracellular pH. PRKD2 knockdown excluded AMAP1 from the CAIX complex and reduced total CAIX in a lysosome-dependent manner. Unexpectedly, AMAP1 knockdown also reduced CAIX mRNA. AMAP1 interacted with PIAS3, which stabilizes HIF-1α, a transcriptional regulator of CA9. AMAP1 knockdown inhibited the PIAS3-HIF-1α interaction and destabilized the HIF-1α protein. High-ASAP1 (AMAP1-encoding gene) together with high-PIAS3 correlated with high-CA9 and an unfavorable prognosis in breast cancer.

Conclusion: The AMAP1-PRKD2 pathway regulates CAIX trafficking, and modulates its total/surface expression. The AMAP1-PIAS3 interaction augments CA9 transcription by stabilizing HIF-1α, presumably contributing to an unfavorable prognosis.

Keywords: ASAP1; CA9; HIF1A; PIAS3; Protein kinase D2.