Nardilysin-regulated scission mechanism activates polo-like kinase 3 to suppress the development of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) develops through step-wise genetic and molecular alterations including Kras mutation and inactivation of various apoptotic pathways. Here, we find that development of apoptotic resistance and metastasis of KrasG12D-driven PDAC in mice is accelerated by deleting Plk3, explaining the often-reduced Plk3 expression in human PDAC. Importantly, a 41-kDa Plk3 (p41Plk3) that contains the entire kinase domain at the N-terminus (1-353 aa) is activated by scission of the precursor p72Plk3 at Arg354 by metalloendopeptidase nardilysin (NRDC), and the resulting p32Plk3 C-terminal Polo-box domain (PBD) is removed by proteasome degradation, preventing the inhibition of p41Plk3 by PBD. We find that p41Plk3 is the activated form of Plk3 that regulates a feed-forward mechanism to promote apoptosis and suppress PDAC and metastasis. p41Plk3 phosphorylates c-Fos on Thr164, which in turn induces expression of Plk3 and pro-apoptotic genes. These findings uncover an NRDC-regulated post-translational mechanism that activates Plk3, establishing a prototypic regulation by scission mechanism.

Revisiting the multisite phosphorylation that produces the M-phase supershift of key mitotic regulators.

The term M-phase supershift denotes the phosphorylation-dependent substantial increase in the apparent molecular weight of numerous proteins of varied biological functions during M-phase induction. Although the M-phase supershift of multiple key mitotic regulators has been attributed to the multisite phosphorylation catalyzed by the Cdk1/cyclin B/Cks complex, this view is challenged by multiple lines of paradoxical observations. To solve this problem, we reconstituted the M-phase supershift of Xenopus Cdc25C, Myt1, Wee1A, APC3, and Greatwall in Xenopus egg extracts and characterized the supershift-producing phosphorylations. Our results demonstrate that their M-phase supershifts are each due to simultaneous phosphorylation of a considerable portion of S/T/Y residues in a long intrinsically disordered region that is enriched in both S/T residues and S/TP motifs. Although the major mitotic kinases in Xenopus egg extracts, Cdk1, MAPK, Plx1, and RSK2, are able to phosphorylate the five mitotic regulators, they are neither sufficient nor required to produce the M-phase supershift. Accordingly, inhibition of the four major mitotic kinase activities in Xenopus oocytes did not inhibit the M-phase supershift in okadaic acid-induced oocyte maturation. These findings indicate that the M-phase supershift is produced by a previously unrecognized category of mitotic phosphorylation that likely plays important roles in M-phase induction.

Vestigial-like 1 is a shared targetable cancer-placenta antigen expressed by pancreatic and basal-like breast cancers.

Cytotoxic T lymphocyte (CTL)-based cancer immunotherapies have shown great promise for inducing clinical regressions by targeting tumor-associated antigens (TAA). To expand the TAA landscape of pancreatic ductal adenocarcinoma (PDAC), we performed tandem mass spectrometry analysis of HLA class I-bound peptides from 35 PDAC patient tumors. This identified a shared HLA-A*0101 restricted peptide derived from co-transcriptional activator Vestigial-like 1 (VGLL1) as a putative TAA demonstrating overexpression in multiple tumor types and low or absent expression in essential normal tissues. Here we show that VGLL1-specific CTLs expanded from the blood of a PDAC patient could recognize and kill in an antigen-specific manner a majority of HLA-A*0101 allogeneic tumor cell lines derived not only from PDAC, but also bladder, ovarian, gastric, lung, and basal-like breast cancers. Gene expression profiling reveals VGLL1 as a member of a unique group of cancer-placenta antigens (CPA) that may constitute immunotherapeutic targets for patients with multiple cancer types.

Bone secreted factors induce cellular quiescence in prostate cancer cells.

Disseminated tumor cells (DTCs) undergo a dormant state in the distant metastatic site(s) before becoming overt metastatic diseases. In prostate cancer (PCa), bone metastasis can occur years after prostatectomy, suggesting that bone may provide dormancy-inducing factors. To search for these factors, we prepared conditioned media (CM) from calvariae. Using live-cell imaging, we found that Calvarial-CM treatment increased cellular quiescence in C4-2B4 PCa cells. Mass spectrometry analysis of Calvarial-CM identified 132 secreted factors. Western blot and ELISA analyses confirmed the presence of several factors, including DKK3, BMP1, neogenin and vasorin in the Calvarial-CM. qRT-PCR analysis of total calvariae versus isolated osteoblasts showed that DKK3, BMP1, vasorin and neogenin are mainly expressed by osteoblasts, while MIA, LECT1, NGAL and PEDF are expressed by other calvarial cells. Recombinant human DKK3, BMP1, vasorin, neogenin, MIA and NGAL treatment increased cellular quiescence in both C4-2b and C4-2B4 PCa cells. Mechanistically, DKK3, vasorin and neogenin, but not BMP1, increased dormancy through activating the p38MAPK signaling pathway. Consistently, DKK3, vasorin and neogenin failed to induce dormancy in cells expressing dominant-negative p38αMAPK while BMP1 remained active, suggesting that BMP1 uses an alternative dormancy signaling pathway. Thus, bone secretes multiple dormancy-inducing factors that employ distinct signaling pathways to induce DTC dormancy in bone.

Hypervirulent group A Streptococcus emergence in an acaspular background is associated with marked remodeling of the bacterial cell surface.

Inactivating mutations in the control of virulence two-component regulatory system (covRS) often account for the hypervirulent phenotype in severe, invasive group A streptococcal (GAS) infections. As CovR represses production of the anti-phagocytic hyaluronic acid capsule, high level capsule production is generally considered critical to the hypervirulent phenotype induced by CovRS inactivation. There have recently been large outbreaks of GAS strains lacking capsule, but there are currently no data on the virulence of covRS-mutated, acapsular strains in vivo. We investigated the impact of CovRS inactivation in acapsular serotype M4 strains using a wild-type (M4-SC-1) and a naturally-occurring CovS-inactivated strain (M4-LC-1) that contains an 11bp covS insertion. M4-LC-1 was significantly more virulent in a mouse bacteremia model but caused smaller lesions in a subcutaneous mouse model. Over 10% of the genome showed significantly different transcript levels in M4-LC-1 vs. M4-SC-1 strain. Notably, the Mga regulon and multiple cell surface protein-encoding genes were strongly upregulated-a finding not observed for CovS-inactivated, encapsulated M1 or M3 GAS strains. Consistent with the transcriptomic data, transmission electron microscopy revealed markedly altered cell surface morphology of M4-LC-1 compared to M4-SC-1. Insertional inactivation of covS in M4-SC-1 recapitulated the transcriptome and cell surface morphology. Analysis of the cell surface following CovS-inactivation revealed that the upregulated proteins were part of the Mga regulon. Inactivation of mga in M4-LC-1 reduced transcript levels of multiple cell surface proteins and reversed the cell surface alterations consistent with the effect of CovS inactivation on cell surface composition being mediated by Mga. CovRS-inactivating mutations were detected in 20% of current invasive serotype M4 strains in the United States. Thus, we discovered that hypervirulent M4 GAS strains with covRS mutations can arise in an acapsular background and that such hypervirulence is associated with profound alteration of the cell surface.

Genome-wide identification and differential analysis of translational initiation.

Translation is principally regulated at the initiation stage. The development of the translation initiation (TI) sequencing (TI-seq) technique has enabled the global mapping of TIs and revealed unanticipated complex translational landscapes in metazoans. Despite the wide adoption of TI-seq, there is no computational tool currently available for analyzing TI-seq data. To fill this gap, we develop a comprehensive toolkit named Ribo-TISH, which allows for detecting and quantitatively comparing TIs across conditions from TI-seq data. Ribo-TISH can also predict novel open reading frames (ORFs) from regular ribosome profiling (rRibo-seq) data and outperform several established methods in both computational efficiency and prediction accuracy. Applied to published TI-seq/rRibo-seq data sets, Ribo-TISH uncovers a novel signature of elevated mitochondrial translation during amino-acid deprivation and predicts novel ORFs in 5'UTRs, long noncoding RNAs, and introns. These successful applications demonstrate the power of Ribo-TISH in extracting biological insights from TI-seq/rRibo-seq data.

Proteomics Profiling of Exosomes from Primary Mouse Osteoblasts under Proliferation versus Mineralization Conditions and Characterization of Their Uptake into Prostate Cancer Cells.

Osteoblasts communicate both with normal cells in the bone marrow and with tumor cells that metastasized to bone. Here we show that osteoblasts release exosomes, we termed osteosomes, which may be a novel mechanism by which osteoblasts communicate with cells in their environment. We have isolated exosomes from undifferentiated/proliferating (D0 osteosomes) and differentiated/mineralizing (D24 osteosomes) primary mouse calvarial osteoblasts. The D0 and D24 osteosomes were found to be vesicles of 130-140 nm by dynamic light scattering analysis. Proteomics profiling using tandem mass spectrometry (LC-MS/MS) identified 206 proteins in D0 osteosomes and 336 in D24 osteosomes. The proteins in osteosomes are mainly derived from the cytoplasm (∼47%) and plasma membrane (∼31%). About 69% of proteins in osteosomes are also found in Vesiclepedia, and these canonical exosomal proteins include tetraspanins and Rab family proteins. We found that there are differences in both protein content and levels in exosomes isolated from undifferentiated and differentiated osteoblasts. Among the proteins that are unique to osteosomes, 169 proteins are present in both D0 and D24 osteosomes, 37 are unique to D0, and 167 are unique to D24. Among those 169 proteins present in both D0 and D24 osteosomes, 10 proteins are likely present at higher levels in D24 than D0 osteosomes based on emPAI ratios of >5. These results suggest that osteosomes released from different cellular state of osteoblasts may mediate distinct functions. Using live-cell imaging, we measured the uptake of PKH26-labeled osteosomes into C4-2B4 and PC3-mm2 prostate cancer cells. In addition, we showed that cadherin-11, a cell adhesion molecule, plays a role in the uptake of osteosomes into PC3-mm2 cells as osteosome uptake was delayed by neutralizing antibody against cadherin-11. Together, our studies suggest that osteosomes could have a unique role in the bone microenvironment under both physiological and pathological conditions.

SLC45A2: A Melanoma Antigen with High Tumor Selectivity and Reduced Potential for Autoimmune Toxicity.

Cytotoxic T lymphocyte (CTL)-based immunotherapies have had remarkable success at generating objective clinical responses in patients with advanced metastatic melanoma. Although the melanocyte differentiation antigens (MDA) MART-1, PMEL, and tyrosinase were among the first melanoma tumor-associated antigens identified and targeted with immunotherapy, expression within normal melanocytes of the eye and inner ear can elicit serious autoimmune side effects, thus limiting their clinical potential as CTL targets. Using a tandem mass spectrometry (MS) approach to analyze the immunopeptidomes of 55 melanoma patient-derived cell lines, we identified a number of shared HLA class I-bound peptides derived from the melanocyte-specific transporter protein SLC45A2. Antigen-specific CTLs generated against HLA-A*0201- and HLA-A*2402-restricted SLC45A2 peptides effectively killed a majority of HLA-matched cutaneous, uveal, and mucosal melanoma cell lines tested (18/25). CTLs specific for SLC45A2 showed significantly reduced recognition of HLA-matched primary melanocytes that were, conversely, robustly killed by MART1- and PMEL-specific T cells. Transcriptome analysis revealed that SLC45A2 mRNA expression in normal melanocytes was less than 2% that of other MDAs, therefore providing a more favorable melanoma-to-melanocyte expression ratio. Expression of SLC45A2 and CTL sensitivity could be further upregulated in BRAF(V600E)-mutant melanoma cells upon treatment with BRAF or MEK inhibitors, similarly to other MDAs. Taken together, our study demonstrates the feasibility of using tandem MS as a means of discovering shared immunogenic tumor-associated epitopes and identifies SLC45A2 as a promising immunotherapeutic target for melanoma with high tumor selectivity and reduced potential for autoimmune toxicity. Cancer Immunol Res; 5(8); 618-29. ©2017 AACR.

Dihydrodiol dehydrogenase in drug resistance and sensitivity of human carcinomas.

We previously reported (UroOncology 1:165, 2001) cross-resistance and collateral-sensitivity to 2-chlorodeoxyadenosine (CldAdo) and fludarabine (FaraA), respectively, in a human renal cell carcinoma selected for resistance to 2'-deoxytubercidin (Caki-dTub). Insofar that these drugs generally demonstrate cross resistance rather than collateral sensitivity, we further examined the bases for this phenomenon. Both CldAdo and FaraA induce apoptosis, as the triphosphates, via binding to Apaf-1. In the presence of cytochrome c, this binding leads to activation of procaspase 9 to active caspase 9 that induces apoptosis through its activation of caspase 3. CldAdo and FaraA induced caspase 3 activities in wild type and Caki-dTub cell lines in a dose-dependent manner that paralleled the cross-resistance (CldAdo, 200-fold) or collateral sensitivity (FaraA, 20-fold) with regard to cell viability. The activation of caspase 3 was inhibited by the caspase 9 inhibitor, Z-LEHD-FMK, suggesting that both drugs act via the same pathway. By differential display and direct enzyme analysis, dihydrodiol dehydrogenase (DDH) was observed to be profoundly underexpressed in the Caki-dTub compared to wild-type Caki-1 cells. Stable transfection of the Caki-dTub cells with a vector encoding the enzyme led to partial reversal of the resistance to CldAdo. Resistance to cisplatin has recently been ascribed to overexpression of DDH in a human ovarian carcinoma cell line (Deng et al. in J Biol Chem 227:15035, 2002). It is tempting to speculate a mutation in the Apaf-1 nucleotide binding site that reduces (CldAdo) or increases (FaraA) toxicity in the Caki-dTub cells; however, the recent finding by others in a human ovarian carcinoma cell line suggests that DDH expression mediates the cross-resistance and perhaps, collateral-sensitivity.

Suicide gene therapy using E. coli beta-galactosidase.

PURPOSE: Suicide gene therapy offers the potential to increase the selective toxicity of antitumor agents by intratumoral expression of exogenous enzymes that convert nontoxic prodrugs to toxic products. The use of herpes simplex virus thymidine kinase with ganciclovir, and E. coli cytosine deaminase with 5-fluorocytosine are well-known examples of this approach. The purpose of this study was to investigate a novel suicide gene therapy using E. coli beta-galactosidase (beta-gal) as the prodrug-activating enzyme. Advantages of this approach include: (1) the ability to use prodrugs that are cleaved by beta-gal to agents that are known to possess activity against human solid tumors, and (2) the extensive experience gained with targeting beta-gal to specific tumors in experimental animals and in humans. METHODS: Two different structural types of anthracycline prodrugs, N-[4"-(beta- D-galactopyranosyl)-3"-nitrobenzyloxycarbonyl]daunomycin (Daun02) and N-[(4" R,S)-4"-ethoxy-4"-(1"'- O-beta- D-galactopyranosyl)butyl]daunorubicin (gal-DNC4) were investigated. The prodrugs were evaluated as substrates for beta-gal. Cytotoxicity studies of Daun02 were conducted against a murine tumor (Panc02), two human breast tumors (MCF-7 and T47D), and three human prostate tumors (PC3, DU145 and LNCAP) that had been transduced to express beta-gal. Antitumor studies of Daun02 were conducted against mouse tumor Panc02 xenografts implanted subcutaneously. RESULTS: Daun02 was a good substrate for beta-gal. By comparison, gal-DCN4 was a poor substrate. Except for PC3, the beta-gal-transduced tumors showed 3- to 60-fold increased sensitivity to Daun02 compared with mock-transduced control cells. Daunomycin was formed from Daun02 in tissue culture medium containing beta-gal-transduced cell lines but was not observed in the medium from mock-transduced controls. In vivo therapeutic studies of Daun02 against the Panc02 tumor in athymic mice showed no significant inhibition of tumor growth. Pharmacokinetic studies showed limited distribution of the prodrug beyond the vascular space. CONCLUSIONS: E. coli beta-gal may be useful as a prodrug-activating enzyme in suicide gene therapy and has the potential to increase the selective toxicity of conventional antitumor agents. Although this approach worked well against tumor cells in vitro, it was not effective against a xenograft model in vivo, apparently because of poor drug-tissue distribution.