Bone is a frequent target of lung cancer metastasis, which is associated with significant morbidity and a dismal prognosis. To identify and functionally characterize genes involved in the mechanisms of osseous metastasis, we developed a murine lung cancer model. Comparative transcriptomic analysis identified genes encoding signaling molecules (such as TCF4 and PRKD3) and cell anchorage-related proteins (MCAM and SUSD5), some of which were basally modulated by transforming growth factor-beta (TGF-beta) in tumor cells and in conditions mimicking tumor-stromal interactions. Triple gene combinations induced not only high osteoclastogenic activity but also a marked enhancement of global metalloproteolytic activities in vitro. These effects were strongly associated with robust bone colonization in vivo, whereas this gene subset was ineffective in promoting local tumor growth and cell homing activity to bone. Interestingly, global inhibition of metalloproteolytic activities and simultaneous TGF-beta blockade in vivo led to increased survival and a remarkable attenuation of bone tumor burden and osteolytic metastasis. Thus, this metastatic gene signature mediates bone matrix degradation by a dual mechanism of induction of TGF-beta-dependent osteoclastogenic bone resorption and enhancement of stroma-dependent metalloproteolytic activities. Our findings suggest the cooperative contribution of host-derived and cell autonomous effects directed by a small subset of genes in mediating aggressive osseous colonization.

Invasive ductal carcinomas (IDC) of the breast are associated with altered expression of hormone receptors (HR), amplification or overexpression of HER2, or a triple-negative phenotype. The most aggressive cases of IDC are characterized by a high proliferation rate, a great propensity to metastasize, and their ability to resist to standard chemotherapy, hormone therapy, or HER2-targeted therapy. Using progression tissue microarrays, we here demonstrate that the serine/threonine kinase protein kinase D3 (PKD3) is highly upregulated in estrogen receptor (ER)-negative (ER(-)) tumors. We identify direct binding of the ER to the PRKD3 gene promoter as a mechanism of inhibition of PKD3 expression. Loss of ER results in upregulation of PKD3, leading to all hallmarks of aggressive IDC, including increased cell proliferation, migration, and invasion. This identifies ER(-) breast cancers as ideal for treatment with the PKD inhibitor CRT0066101. We show that similar to a knockdown of PKD3, treatment with this inhibitor targets all tumorigenic processes in vitro and decreases growth of primary tumors and metastasis in vivo. Our data strongly support the development of PKD inhibitors for clinical use for ER(-) breast cancers, including the triple-negative phenotype.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease without a well-defined genetic alteration responsible for the onset of the disease. Several lines of evidence coincide in identifying stimulatory and growth signals delivered by B-cell receptor (BCR), and co-receptors together with NFkB pathway, as being the driving force in B-cell survival in CLL. However, the molecular mechanism responsible for this activation has not been identified. Based on the hypothesis that BCR activation may depend on somatic mutations of the BCR and related pathways we have performed a complete mutational screening of 301 selected genes associated with BCR signaling and related pathways using massive parallel sequencing technology in 10 CLL cases. Four mutated genes in coding regions (KRAS, SMARCA2, NFKBIE and PRKD3) have been confirmed by capillary sequencing. In conclusion, this study identifies new genes mutated in CLL, all of them in cases with progressive disease, and demonstrates that next-generation sequencing technologies applied to selected genes or pathways of interest are powerful tools for identifying novel mutational changes.

Rapidly accumulated evidence has shown that long non-coding RNA (lncRNAs) disregulation is involved in human tumorigenesis in many cancers, including prostate cancer (PCa). LncRNAs can regulate essential pathways that contribute to tumor initiation and progression with tissue specificity, which suggests that lncRNAs could be valuable biomarkers and therapeutic targets. Prostate cancer antigen 3 (PCA3), also known as differential display code 3 (DD3), is one such lncRNA that maps to chromosome 9q21-22. PCA3 expression is highly specific to PCa. In the present study, the level of PCA3 expression in prostate cancer cells was reduced by small interfering RNA (siRNA). Subsequently, the ability of LNCaP cell proliferation, invasion, and migration of PCa was compromised both in vivo and in vitro with the occurrence of cell autophagy. Recently, a novel regulatory mechanism has been proposed in which RNAs cross talk via competing with the shared microRNAs (miRNAs). In addition, lncRNAs can directly interact with RNA-binding proteins and then bind to the gene promoter region to further regulate gene expression. The proposed competitive endogenous RNAs mediate the bioavailability of miRNAs on their targets, thus imposing another level of post-transcriptional regulation. Here, we demonstrated that binding of Snail to the promoter region of PCA3 could activate the expression of PCA3. Down-regulation of PCA3 by silencing could increase the expression of the miRNA-1261, which then targeted at the PRKD3 gene (protein kinase D3) through competitive sponging. In summary, these results suggest that the transcription factor, Snail, activated the expression of lncRNA PCA3, which could inhibit the translation of PRKD3 protein via competitive miR-1261 sponging, and thus high expression of PRKD3 further promoted invasion and migration of prostate cancer.

Polymorphous low-grade adenocarcinoma (PLGA) and cribriform adenocarcinoma of minor salivary gland (CAMSG) are low-grade carcinomas arising most often in oral cavity and oropharynx, respectively. Controversy exists as to whether these tumors represent separate entities or variants of one spectrum, as they appear to have significant overlap, but also clinicopathologic differences. As many salivary carcinomas harbor recurrent translocations, paired-end RNA sequencing and FusionSeq data analysis was applied for novel fusion discovery on two CAMSGs and two PLGAs. Validated rearrangements were then screened by fluorescence in situ hybridization (FISH) in 60 cases. Histologic classification was performed without knowledge of fusion status and included: 21 CAMSG, 18 classic PLGA, and 21 with "mixed/indeterminate" features. The RNAseq of 2 CAMSGs showed ARID1A-PRKD1 and DDX3X-PRKD1 fusions, respectively, while no fusion candidates were identified in two PLGAs. FISH for PRKD1 rearrangements identified 11 additional cases (22%), two more showing ARID1A-PRKD1 fusions. As PRKD2 and PRKD3 share similar functions with PRKD1 in the diacylglycerol and protein kinase C signal transduction pathway, we expanded the investigation for these genes by FISH. Six additional cases each showed PRKD2 and PRKD3 rearrangements. Of the 26 (43%) fusion-positive tumors, there were 16 (80%) CAMSGs and 9 (45%) indeterminate cases. A PRKD2 rearrangement was detected in one PLGA (6%). We describe novel and recurrent gene rearrangements in PRKD1-3 primarily in CAMSG, suggesting a possible pathogenetic dichotomy from "classic" PLGA. However, the presence of similar genetic findings in half of the indeterminate cases and a single PLGA suggests a possible shared pathogenesis for these tumor types.

RAS mutations occur in more than 30% of all human cancers but efforts to directly target mutant RAS signaling as a cancer therapy have yet to succeed. As alternative strategies, RAF and MEK inhibitors have been developed to block oncogenic signaling downstream of RAS. As might be expected, studies of these inhibitors have indicated that tumors with RAS or BRAF mutations display resistance RAF or MEK inhibitors. In order to better understand the mechanistic basis for this resistance, we conducted a RNAi-based screen to identify genes that mediated chemoresistance to the RAF kinase inhibitor RAF265 in a BRAF (V600E) mutant melanoma cell line that is resistant to this drug. In this way, we found that knockdown of protein kinase D3 (PRKD3) could enhance cell killing of RAF and MEK inhibitors across multiple melanoma cell lines of various genotypes and sensitivities to RAF265. PRKD3 blockade cooperated with RAF265 to prevent reactivation of the MAPK signaling pathway, interrupt cell cycle progression, trigger apoptosis, and inhibit colony formation growth. Our findings offer initial proof-of-concept that PRKD3 is a valid target to overcome drug resistance being encountered widely in the clinic with RAF or MEK inhibitors.

Many catabolic stimuli, including interleukin-1 (IL-1) in combination with oncostatin M (OSM), promote cartilage breakdown via the induction of collagen-degrading collagenases such as matrix metalloproteinase 1 (MMP1) and MMP13 in human articular chondrocytes. Indeed, joint diseases with an inflammatory component are characterised by excessive extracellular matrix (ECM) catabolism. Importantly, protein kinase C (PKC) signalling has a primary role in cytokine-induced MMP1/13 expression, and is known to regulate cellular functions associated with pathologies involving ECM remodelling. At present, substrates downstream of PKC remain undefined. Herein, we show that both IL-1- and OSM-induced phosphorylation of protein kinase D (PKD) in human chondrocytes is strongly associated with signalling via the atypical PKCι isoform. Consequently, inhibiting PKD activation with a pan-PKD inhibitor significantly reduced the expression of MMP1/13. Specific gene silencing of the PKD isoforms revealed that only PKD3 (PRKD3) depletion mirrored the observed MMP repression, indicative of the pharmacological inhibitor specifically affecting only this isoform. PRKD3 silencing was also shown to reduce serine phosphorylation of signal transducer and activator of transcription 3 (STAT3) as well as phosphorylation of all three mitogen-activated protein kinase groups. This altered signalling following PRKD3 silencing led to a significant reduction in the expression of the activator protein-1 (AP-1) genes FOS and JUN, critical for the induction of many MMPs including MMP1/13. Furthermore, the AP-1 factor activating transcription factor 3 (ATF3) was also reduced concomitant with the observed reduction in MMP13 expression. Taken together, we highlight an important role for PKD3 in the pro-inflammatory signalling that promotes cartilage destruction.

Protein Kinase D3 (PRKD3) functions as an important oncogenic driver in invasive breast cancer, which is the leading cause of women mortality. However, PRKD3 regulating network is largely unknown. In this study, we systematically explored PRKD3 regulating networks via investigating phosphoproteome, interactome and transcriptome to uncover the molecular mechanism of PRKD3 in invasive breast cancer. Using iTRAQ, 270 proteins were identified as PRKD3 regulated phosphoproteins from 4619 phosphosites matching 3666 phosphopeptides from 2016 phosphoproteins with p-value <0.005. Transcriptome analysis using affymetrix microarray identified 45 PRKD3 regulated genes, in which 20 genes were upregulated and 25 genes were downregulated with p-value <0.005 upon silencing PRKD3. Using Co-IP in combination of MS identification, 606 proteins were identified to be PRKD3 interacting proteins from 2659 peptides. Further network analysis of PRKD3 regulated phosphoproteins, interacting proteins and regulated genes, reveals 19 hub nodes, including ELAVL1, UBC and BRCA1. UBC was recognized as the most common hub node in PRKD3 regulating networks. The enriched pathway analysis reveals that PRKD3 regulates pathways contributing to multiple cancer related events, including cell cycle, migration and others. Enrichment of cell cycle and cell mobility related pathways across PRKD3 networks, explained the observations that depletion of oncogenic PRKD3 led to alternation of cell cycle and decrease of cell migration ability. Taken together, our current study provided valuable information on the roles as well as the molecular mechanisms of PRKD3 in invasive breast cancer.

Identifying reliable drug response biomarkers is a significant challenge in cancer research. We present computational analysis of resistance (CARE), a computational method focused on targeted therapies, to infer genome-wide transcriptomic signatures of drug efficacy from cell line compound screens. CARE outputs genome-scale scores to measure how the drug target gene interacts with other genes to affect the inhibitor efficacy in the compound screens. Such statistical interactions between drug targets and other genes were not considered in previous studies but are critical in identifying predictive biomarkers. When evaluated using transcriptome data from clinical studies, CARE can predict the therapy outcome better than signatures from other computational methods and genomics experiments. Moreover, the CARE signatures for the PLX4720 BRAF inhibitor are associated with an anti-programmed death 1 clinical response, suggesting a common efficacy signature between a targeted therapy and immunotherapy. When searching for genes related to lapatinib resistance, CARE identified PRKD3 as the top candidate. PRKD3 inhibition, by both small interfering RNA and compounds, significantly sensitized breast cancer cells to lapatinib. Thus, CARE should enable large-scale inference of response biomarkers and drug combinations for targeted therapies using compound screen data.

Genetic discoveries of Alzheimer's disease are the drivers of our understanding, and together with polygenetic risk stratification can contribute towards planning of feasible and efficient preventive and curative clinical trials. We first perform a large genetic association study by merging all available case-control datasets and by-proxy study results (discovery n = 409,435 and validation size n = 58,190). Here, we add six variants associated with Alzheimer's disease risk (near APP, CHRNE, PRKD3/NDUFAF7, PLCG2 and two exonic variants in the SHARPIN gene). Assessment of the polygenic risk score and stratifying by APOE reveal a 4 to 5.5 years difference in median age at onset of Alzheimer's disease patients in APOE ɛ4 carriers. Because of this study, the underlying mechanisms of APP can be studied to refine the amyloid cascade and the polygenic risk score provides a tool to select individuals at high risk of Alzheimer's disease.

Breast cancer is clinically classified into three main subtypes: estrogen receptor-positive (ER⁺) breast cancer, human epidermal growth factor receptor 2-positive (HER2⁺) breast cancer, and triple-negative breast cancer (TNBC). Without specific targeted therapies, patients with TNBC have poorer prognosis compared with those with ER⁺ and HER2⁺ breast cancer. Protein kinase D (PRKD) family members play crucial roles in cancer progression. CRT0066101, a PRKD inhibitor, has been reported to have anticancer activity in many cancer types. Nevertheless, the role and mechanism of CRT0066101 in TNBC have not been well investigated.

The expression level of PRKDs was analyzed in breast cancer samples and breast cancer cell lines. The effects of inhibiting PRKD activity with CRT0066101 on TNBC cell proliferation, cell cycle, apoptosis, and tumor growth were studied by Cell Counting Kit8 assay, cell cycle assay, propidium iodide/annexin-V assay, and a xenograft mouse model, respectively. To uncover the molecular mechanism of CRT0066101 in TNBC, comparative phosphoproteomic analysis using iTRAQ was employed.We found that PRKD2 and PRKD3 were preferentially expressed in breast cancers. Immunohistochemistry confirmed the overexpression of PRKD2 and PRKD3 in TNBC. CRT0066101, which inhibited the activity of PRKDs, dramatically inhibited proliferation, increased apoptosis and the G1-phase population of TNBC cells in vitro, and reduced breast tumor volume in vivo. Comparative phosphoproteomic analysis between breast cancer cells with and without CRT0066101 treatment revealed that the anti-breast cancer effects involved regulation of a complex network containing multiple enriched pathways and several hub-nodes contributing to multiple cancer-related processes, thus explaining the described effects of CRT0066101 on TNBC in vitro and in vivo. Finally, we validated several targets of PRKD inhibition by treatment with CRT0066101 and small interfering RNAs against PRKD2 and PRKD3 (siPRKD2 and siPRKD3), including p-MYC(T58/ S62), p-MAPK1/3(T202/Y204), p-AKT(S473), p-YAP(S127), and p-CDC2(T14).PRKD inhibitor CRT0066101 exhibits anti-TNBC effects via modulating a phosphor-signaling network and inhibiting the phosphorylation of many cancer-driving factors, including MYC, MAPK1/3, AKT, YAP, and CDC2, providing insight into the important roles as well as the molecular mechanism of CRT0066101 as an effective drug for TNBC.

OBJECTIVE

Polymorphous low-grade adenocarcinoma (PLGA) is the second most common intra-oral salivary gland malignancy. The vast majority of PLGAs harbour a PRKD1 E710D hot-spot somatic mutation or somatic rearrangements of PRKD1, PRKD2 or PRKD3. Given the kinase domain homology among PRKD1, PRKD2 and PRKD3, we sought to define whether PLGAs lacking PRKD1 somatic mutations or PRKD gene family rearrangements would be driven by somatic mutations affecting the kinase domains of PRKD2 or PRKD3.

RESULTS

DNA was extracted from eight microdissected PLGAs lacking PRKD1 somatic mutations or PRKD gene family rearrangements. Samples were thoroughly centrally reviewed, microdissected and subjected to Sanger sequencing of the kinase domains of the PRKD2 and PRKD3 genes. None of the PLGAs lacking PRKD1 somatic mutations or PRKD gene family rearrangements harboured somatic mutations in the kinase domains of the PRKD2 or PRKD3 genes.

CONCLUSIONS

PLGAs lacking PRKD1 somatic mutations or PRKD gene family rearrangements are unlikely to harbour somatic mutations in the kinase domains of PRKD2 or PRKD3. Further studies are warranted to define the driver genetic events in this subgroup of PLGAs.

Breast cancer is the second leading death cause of cancer death for all women. Previous study suggested that Protein Kinase D3 (PRKD3) was involved in breast cancer progression. In addition, the protein level of PRKD3 in triple-negative breast adenocarcinoma was higher than that in normal breast tissue. However, the oncogenic mechanisms of PRKD3 in breast cancer is not fully investigated. Multi-omic data showed that ERK1/c-MYC axis was identified as a major pivot in PRKD3-mediated downstream pathways. Our study provided the evidence to support that the PRKD3/ERK1/c-MYC pathway play an important role in breast cancer progression. We found that knocking out PRKD3 by performing CRISPR/Cas9 genome engineering technology suppressed phosphorylation of both ERK1 and c-MYC but did not down-regulate ERK1/2 expression or phosphorylation of ERK2. The inhibition of ERK1 and c-MYC phosphorylation further led to the lower protein level of c-MYC and then reduced the expression of the c-MYC target genes in breast cancer cells. We also found that loss of PRKD3 reduced the rate of the cell proliferation in vitro and tumour growth in vivo, whereas ectopic (over)expression of PRKD3, ERK1 or c-MYC in the PRKD3-knockout breast cells reverse the suppression of the cell proliferation and tumour growth. Collectively, our data strongly suggested that PRKD3 likely promote the cell proliferation in the breast cancer cells by activating ERK1-c-MYC axis.

Cribriform adenocarcinoma of the tongue and minor salivary glands (CAMSG) was first described 16 years ago. It typically presents as a mass at the base of the tongue with early spread to lymph nodes, but without potential for distant metastases. In the 2005 World Health Organization Classification of Tumors the entity was classified as a possible variant of polymorphous low-grade adenocarcinoma (PLGA). Since then, more than 40 cases have been described in the English literature. Recently, PRKD1-3 translocation was found in more than 80% of CAMSGs. In some of those cases ARID1A or DDX3X was the translocation partner. We reviewed 183 primary carcinomas of major and minor salivary glands, resected at the Medical University of Gdańsk, Poland, in the period 1992-2012, and identified only one case of CAMSG. A giant tumor developed at the base of the tongue in a 76-year-old man. The primary tumor was resected with multiple bilateral cervical lymph node metastases. The patient received radiotherapy but died 10 months after the surgery due to causes not related to the primary cancer. The tumor presented PRKD3 rearrangement as confirmed by FISH. As the tumor is extremely rare (it represented only 0.5% of salivary gland tumors in our series), the controversy on its nosological status is still unresolved. This is the first report in the world literature of a patient who died in the course of CAMSG.

Although serine/threonine-protein kinases are found to participate in a wide range of cancer progression, the involvement of protein kinase D3 (PRKD3) in gastric cancer has not been explored. Here, we investigated the role of PRKD3 in gastric cancer (GC) and its potential mechanisms. PRKD3 was over-expressed in gastric cancer tissues and cells. In vitro, PRKD3 ectopic expression accelerated the proliferation and growth of GES-1, SGC7901 and MKN-28 cells. By contrast, PRKD3 knockdown suppressed the proliferation of SGC7901 and MKN-28 GC cells. In vivo, xenograted tumorigenesis was blunted by PRKD3 silencing. Mechanistically, PRKD3 up-regulated 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and activated glycolysis as shown by increased glucose consumption and lactate production. Knockdown of PFKFB3 suppressed the glycolysis in gastric cancer cells with highly expressed PRKD3 but not in PRKD3 silenced cells. PRKD3 over-expression induced phosphorylation of p65 at serine 536 was critical for the up-regulation of glycolytic enzyme PFKFB3. Furthermore, PRKD and PFKFB3 inhibitor suppressed the viability of GC cells. Our results suggest that targeting PRKD3/p65/PFKFB3 cascade maybe a promising therapeutic strategy for gastric cancer.

RUNX2, a gene involved in skeletal development, has previously been shown to be potentially affected by positive selection during recent human evolution. Here we have used antibody-based proteomics to characterize potential differences in expression patterns of RUNX2 interacting partners during primate evolution. Tissue microarrays consisting of a large set of normal tissues from human and macaque were used for protein profiling of 50 RUNX2 partners with immunohistochemistry. Eleven proteins (AR, CREBBP, EP300, FGF2, HDAC3, JUN, PRKD3, RUNX1, SATB2, TCF3, and YAP1) showed differences in expression between humans and macaques. These proteins were further profiled in tissues from chimpanzee, gorilla, and orangutan, and the corresponding genes were analyzed with regard to genomic features. Moreover, protein expression data were compared with previously obtained RNA sequencing data from six different organs. One gene (TCF3) showed significant expression differences between human and macaque at both the protein and RNA level, with higher expression in a subset of germ cells in human testis compared with macaque. In conclusion, normal tissues from macaque and human showed differences in expression of some RUNX2 partners that could be mapped to various defined cell types. The applied strategy appears advantageous to characterize the consequences of altered genes selected during evolution.

Protein kinase D3 (PRKD3), a serine/threonine kinase, belongs to protein kinase D family, which contains three members: PRKD1, PRKD2, and PRKD3. PRKD3 is activated by many stimuli including phorbol esters, and G-protein-coupled receptor agonists. PRKD3 promotes cancer cell proliferation, growth, migration, and invasion in various tumor types including colorectal, gastric, hepatic, prostate, and breast cancer. Accumulating data supports that PRKD3 is a promising therapeutic target for treatment of cancer. This review discusses the functions and mechanisms of PRKD3 in promoting tumorigenesis and tumor progression of various tumor types as well as the latest developments of small-molecule inhibitors selection for PRKD/PRKD3.

Keywords: Cancer progression; Protein kinase D3.

Liver fibrosis is a central pathological process that occurs in most types of chronic liver diseases. Advanced liver fibrosis causes cirrhosis, hepatocellular carcinoma, and liver failure. However, the exact molecular mechanisms underlying the initiation and progression of liver fibrosis remain largely unknown. This study was designed to investigate the role of protein kinase D3 (PKD3, gene name Prkd3) in the regulation of liver homeostasis. We generated global Prkd3 knockout (Prkd3^-/- ) mice and myeloid cell-specific Prkd3 knockout (Prkd3^∆LysM ) mice, and we found that both Prkd3^-/- mice and Prkd3^∆LysM mice displayed spontaneous liver fibrosis. PKD3 deficiency also aggravated carbon tetrachloride (CCL4)-induced liver fibrosis. PKD3 is highly expressed in hepatic macrophages, and PKD3 deficiency skewed macrophage polarization toward a profibrotic phenotype. The activated profibrotic macrophages produced TGF-β that in turn activates hepatic stellate cells (HSCs) to become matrix-producing myofibroblasts. Moreover, PKD3 deficiency decreased the phosphatase activity of SHP1 (a bona fide PKD3 substrate) resulting in sustained STAT6 activation in macrophages. In addition, we observed that PKD3 expression in hepatic macrophages was downregulated in cirrhotic human liver tissues. Conclusion: PKD3 deletion in mice drives liver fibrosis through the profibrotic macrophage activation.

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.

Objective: We aimed to clarify the molecular mechanism of how PRKD3 promotes the malignant progression of oral squamous cell carcinoma (OSCC).

Patients and methods: 62 cases of OSCC tissues and normal adjacent ones which were further confirmed by a qualified pathologist were collected from patients in the Department of Pathology and Stomatology of our hospital. PRKD3 expression in the above tissue samples was studied by quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and its relationship with clinicopathological characteristics of these OSCC patients was analyzed. Meanwhile, a PRKD3 knockdown expression model was constructed in OSCC cell lines for cell functional experiments. The relationship between PRKD3 and KLF16 was elucidated through bioinformatics and Luciferase reporter gene experiments.

Results: Our data showed that PRKD3 expression in OSCC specimens was remarkably higher than that in adjacent ones. PRKD3 expression showed positive association with the incidence of distant metastasis, but not with other clinical indicators such as gender, age, tumor stage or lymph node metastasis incidence. Patients with high PRKD3 expression had lower overall survival compared to those with low expression. In addition, OSCC cells migration ability and invasiveness were remarkably attenuated after PRKD3 was knocked down. Bioinformatics and Luciferase assay revealed that PRKD3 could directly bind to KLF16 and Western blot suggested that KLF16 was upregulated after PRKD3 was knocked down. In addition, knocking down KLF16 reversed the inhibitory effect of PRKD3 knockdown on invasiveness and metastasis of OSCC cells.

Conclusions: The highly-expressed PRKD3, remarkably associated with metastasis incidence and poor prognosis of OSCC patients, may accelerate the malignant progression of OSCC through modulating KLF16 expression.

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.

Diagnosis of salivary gland neoplasms is often challenging due to their high morphological diversity and overlaps. Several recurrent molecular alterations have been described recently, which can serve as powerful diagnostic tools and potential therapeutic targets (e.g. NTRK or RET fusions). However, current sequential molecular testing can be expensive and time consuming. In order to facilitate the diagnosis of salivary gland neoplasms, we designed an all-in-one RNA-based next generation sequencing panel suitable for the detection of mutations, fusions and gene expression levels (including NR4A3) of 27 genes involved in salivary gland neoplasms. Here we present the validation of the "SalvGlandDx" panel on FFPE histological specimen including fine needle aspiration (FNA) cell block material, against the standard methods currently used at our institution. In a second part we describe selected unique cases in which the SalvGlandDx panel allowed proper diagnosis and new insights into special molecular characteristics of selected salivary gland tumors. We characterize a unique salivary gland adenocarcinoma harboring a ZCCHC7-NTRK2 fusion, a highly uncommon spindle cell and pseudoangiomatoid adenoid-cystic carcinoma with MYBL1-NFIB fusion, and a purely oncocytic mucoepidermoid carcinoma, whereas diagnosis could be made by detection of a CRTC3-MAML2 rearrangement on the cell block specimen of the FNA. Further, a rare case of a SS18-ZBTB7A rearranged low-grade adenocarcinoma previously described as potential spectrum of microsecretory adenocarcinoma, is reported. In addition, features of six cases within the spectrum of polymorphous adenocarcinoma / cribriform adenocarcinoma of salivary gland including PRKD1 p.E710D mutations and novel fusions involving PRKAR2A-PRKD1, SNX9-PRKD1 and ATL2-PRKD3, are described.

Keywords: Biopsy; Comprehensive; FNA; Molecular; Salivary gland neoplasm; Testing.

Triple negative breast cancer (TNBC), with its lack of targeted therapies, shows the worst mortality rate among all breast cancer subtypes. Clusterin (CLU) is implicated to play important oncogenic roles in cancer via promoting various downstream oncogenic pathways. Here, protein kinase D3 (PRKD3) is defined to be a key regulator of CLU in promoting TNBC tumor growth. Mechanically, PRKD3 with kinase activity binding to CLU is critical for CLU protein stability via inhibiting CLU's lysosomal distribution and degradation. CLU and PRKD3 protein level are significantly elevated and positively correlated in collected TNBC tumor samples. CLU silencer (OGX-011) and PRKDs inhibitor (CRT0066101) can both result in impressive tumor growth suppression in vitro and in vivo, suggesting targeting CLU and its key regulator-PRKD3 are promisingly efficient against TNBC. Finally, secreted CLU (sCLU) is found to be elevated in serums from TNBC patients and reduced in serum from TNBC murine models post OGX-011 and/or CRT0066101 treatment, suggesting serum sCLU is a promising blood-based biomarker for clinical management of TNBC. Taken together, this study provides a thorough molecular basis as well as preclinical evidences for targeting CLU pathway as a new promising strategy against TNBC via revealing PRKD3 as the key regulator of CLU in TNBC.

Keywords: clusterin; protein kinase D3; targeted therapies; triple‐negative breast cancer; tumor growth.