Vitamin D as a Primer for Oncolytic Viral Therapy in Colon Cancer Models.

Oncolytic viroimmunotherapy is an exciting modality that can offer lasting anti-tumor immunity for aggressive malignancies like colon cancer. The impact of oncolytic viruses may be extended by combining them with agents to prime a tumor for viral susceptibility. This study investigates vitamin D analogue as an adjunct to oncolytic viral therapy for colon cancer. While vitamin D (VD) has historically been viewed as anti-viral, our in vitro investigations using human colon cancer cell lines showed that VD does not directly inhibit replication of recombinant chimeric poxvirus CF33. VD did restrict growth in HT29 but not HCT116 human colon cancer cells. In vivo investigations using HCT116 and HT29 xenograft models of colon cancer demonstrated that a VD analogue, calcipotriol, was additive with CF33-based viral therapy in VD-responsive HT29 but not in HCT116 tumors. Analyses of RNA-sequencing and gene expression data demonstrated a downregulation in the Jak-STAT signaling pathway with the addition of VD to viral therapy in HT29 models suggesting that the anti-inflammatory properties of VD may enhance the effects of viral therapy in some models. In conclusion, VD may prime oncolytic viral therapy in certain colon cancers.

Proteomic analysis of oropharyngeal carcinomas reveals novel HPV-associated biological pathways.

Oropharyngeal carcinoma (OPC) can be classified into two equally prevalent subtypes depending on the presence of human papillomavirus (HPV). Patients with HPV-positive (HPV+) OPC represent a unique cohort with a distinct tumor biology and clinical behavior compared to HPV-negative (HPV-) OPC. Genetic studies have demonstrated chromosomal and gene expression changes associated with distinct subclasses of OPC; however, the proteomic consequences of HPV infection are not known. We analyzed sets of ten HPV+ and ten HPV- OPCs and ten normal adult oral epithelia using a standardized global proteomic analysis platform. This analysis yielded a total of 2,653 confidently identified proteins from which we chose 31 proteins on the basis of expression differences between HPV+, HPV- and normal epithelium for targeted protein quantitation. Analysis of differentially expressed proteins by HPV status revealed enrichment of proteins involved in epithelial cell development, keratinization and extracellular matrix organization in HPV- OPC, whereas enrichment of proteins in DNA initiation and replication and cell cycle control was found for HPV+ OPC. Enrichment analysis for transcription factor targets identified transcription factors E2F1 and E2F4 to be highly expressed in HPV+ OPC. We also found high expression of argininosuccinate synthase 1 in HPV+ OPC, suggesting that HPV+ OPC is more dependent on conditionally essential amino acid, arginine, and this was confirmed on a OPC-specific tissue microarray. These identified proteomic changes reveal novel driving molecular pathways for HPV+ and HPV- OPCs that may be pertinent in therapeutic strategies and outcomes of OPC.

Advanced glycation end product (AGE) targeting antibody SIWA318H is efficacious in preclinical models for pancreatic cancer.

SIWA318H is a novel monoclonal antibody that selectively targets an advanced glycation end product biomarker found in damaged/dysfunctional cells exhibiting (a) aerobic glycolysis, and (b) oxidative stress. Cells with this biomarker are dysfunctional and are associated with stresses and/or damages relating to aging, cancer and other disease processes. In this study, we evaluated the biological effects and antitumor activity of SIWA318H in preclinical models for pancreatic cancer. SIWA318H binds to pancreatic cancer cells and cancer-associated fibroblasts, as well as tumor xenografts derived from pancreatic cancer patients. Furthermore, SIWA318H induced significant antibody-dependent cell-mediated cytotoxicity (ADCC) against pancreatic cancer cells. In a humanized CD34+ NSG mouse xenograft model for pancreatic cancer, tumors in mice treated with SIWA318H grew significantly slower compared to those in control mice (p < 0.001). After 3 weeks of treatment with SIWA318H, the tumor growth was suppressed by 68.8% and 61.5% for the high and low dose regimens, respectively, when compared to the isotype antibody control (ANOVA p < 0.002). Moreover, a significant increase in complete remission (CR) rate was observed in mice receiving the high dose (60%, p < 0.04) or low dose (77.8%, p < 0.02) of SIWA318H treatment compared with control mice (6.7%). Immunohistochemical analyses of the tumor tissues showed a significant decrease in senescent cells in the tumor microenvironment of SIWA318H treated mice compared to that of control treated mice (p < 0.05). These results provide compelling evidence that SIWA318H is a promising novel therapeutic against pancreatic cancer.

Tumor suppressor Lzap regulates cell cycle progression, doming, and zebrafish epiboly.

Initial stages of embryonic development rely on rapid, synchronized cell divisions of the fertilized egg followed by a set of morphogenetic movements collectively called epiboly and gastrulation. Lzap is a putative tumor suppressor whose expression is lost in 30% of head and neck squamous cell carcinomas. Lzap activities include regulation of cell cycle progression and response to therapeutic agents. Here, we explore developmental roles of the lzap gene during zebrafish morphogenesis. Lzap is highly conserved among vertebrates and is maternally deposited. Expression is initially ubiquitous during gastrulation, and later becomes more prominent in the pharyngeal arches, digestive tract, and brain. Antisense morpholino-mediated depletion of Lzap resulted in delayed cell divisions and apoptosis during blastomere formation, resulting in fewer, larger cells. Cell cycle analysis suggested that Lzap loss in early embryonic cells resulted in a G2/M arrest. Furthermore, the Lzap-deficient embryos failed to initiate epiboly--the earliest morphogenetic movement in animal development--which has been shown to be dependent on cell adhesion and migration of epithelial sheets. Our results strongly implicate Lzap in regulation of cell cycle progression, adhesion and migratory activity of epithelial cell sheets during early development. These functions provide further insight into Lzap activity that may contribute not only to development, but also to tumor formation.

Priming stroma with a vitamin D analog to optimize viroimmunotherapy for pancreatic cancer.

Pancreatic cancer resistance to immunotherapies is partly due to deficits in tumor-infiltrating immune cells and stromal density. Combination therapies that modify stroma and recruit immune cells are needed. Vitamin D analogs such as calcipotriol (Cal) decrease fibrosis in pancreas stroma, thus allowing increased chemotherapy delivery. OVs infect, replicate in, and kill cancer cells and recruit immune cells to immunodeficient microenvironments. We investigated whether stromal modification with Cal would enhance oncolytic viroimmunotherapy using recombinant orthopoxvirus, CF33. We assessed effect of Cal on CF33 replication using pancreas ductal adenocarcinoma (PDAC) cell lines and in vivo flank orthotopic models. Proliferation assays showed that Cal did not alter viral replication. Less replication was seen in cell lines whose division was slowed by Cal, but this appeared proportional to cell proliferation. Three-dimensional in vitro models demonstrated decreased myofibroblast integrity after Cal treatment. Cal increased vascular lumen size and immune cell infiltration in subcutaneous models of PDAC and increased viral delivery and replication. Cal plus serial OV dosing in the syngeneic Pan02 model caused more significant tumor abrogation than other treatments. Cal-treated tumors had less dense fibrosis, enhanced immune cell infiltration, and decreased T cell exhaustion. Calcipotriol is a possible adjunct for CF33-based oncolytic viroimmunotherapy against PDAC.

DNMT1 and p38γ are inversely expressed in reactive non-metastatic lymph nodes burdened with colorectal adenocarcinoma.

Lymph nodes are important front-line defense immune tissues, which also act against inflammatory diseases and cancer. Lymph nodes undergo extensive upheavals within newly formed germinal centers (GCs) when exposed to antigens, the molecular mechanisms of which remain elusive. Recently, p38γ was identified as an important target for multiple cancers, including cutaneous T-cell lymphoma (CTCL). We previously observed that p38γ is overexpressed in CTCL versus normal cells, but it is not clear if p38γ is expressed in B or T lymphocytes of GCs of patients in response to a stress such as cancer. Therefore, in this study, we obtained non-metastatic reactive lymph nodes adjacent to cancer lesions (colorectal adenocarcinoma), then performed multicolor immunohistochemical staining for p38γ and other relevant markers. We observed for the first time that p38γ was expressed in the light zone of activated B cells and T helper cells in GCs, whereas DNA-methyltransferase 1 (DNMT1), a marker for GC B cells, was highly expressed in centrocytes and in the dark zone of GCs. This inverse relationship suggests a novel function for p38γ in T cells that cross-talk to B cells in response to stress.

Heart-specific ablation of Prkar1a causes failure of heart development and myxomagenesis.

BACKGROUND: Protein kinase A signaling has long been known to play an important role in cardiac function. Dysregulation of the protein kinase A system, caused by mutation of the protein kinase A regulatory subunit gene PRKAR1A, causes the inherited tumor syndrome Carney complex, which includes cardiac myxomas as one of its cardinal features. Mouse models of this genetic defect have been unsatisfactory because homozygote null animals die early in development and heterozygotes do not exhibit a cardiac phenotype. METHODS AND RESULTS: To study the cardiac-specific effects resulting from complete loss of Prkar1a, we used cre-lox technology to generate mice lacking this protein specifically in cardiomyocytes. Conditional knockout mice died at day 11.5 to 12.5 of embryogenesis with thin-walled, dilated hearts. These hearts showed elevated protein kinase A activity and decreased cardiomyocyte proliferation before demise. Analysis of the expression of transcription factors required for cardiogenesis revealed downregulation of key cardiac transcription factors such as the serum response factor, Gata4, and Nkx2-5. Although heart wall thickness was reduced overall, specific areas exhibited morphological changes consistent with myxomatous degeneration in the walls of knockout hearts. CONCLUSIONS: Loss of Prkar1a from the heart causes a failure of proper myocardial development with subsequent cardiac failure and embryonic demise. These changes appear to be due to suppression of cardiac-specific transcription by increased protein kinase A activity. These biochemical changes lead to myxoma-like changes, indicating that these mice may be a good model with which to study the formation of these tumors.

Pituitary-specific knockout of the Carney complex gene Prkar1a leads to pituitary tumorigenesis.

Carney complex (CNC) is an inherited neoplasia syndrome characterized by spotty skin pigmentation, myxomas, endocrine tumors, and schwannomas. Among the endocrine tumors that comprise the syndrome, GH-producing pituitary tumors are seen in approximately 10% of patients, although biochemical abnormalities of the GH axis are much more common. To explore the role of loss of the CNC gene PRKAR1A on pituitary tumorigenesis, we produced a tissue-specific knockout (KO) of this gene in the mouse. For these studies, we generated a mouse line expressing the cre recombinase in pituitary cells using the rat GHRH receptor promoter. These mice were then crossed with Prkar1a conditional null animals to produce tissue-specific KOs. Although prolactinomas were observed in KO and control mice, the KO mice exhibited a significantly increased frequency of pituitary tumors compared with wild-type or conventional Prkar1a(+/-) mice. Characterization of the tumors demonstrated they were composed of cells of the Pit1 lineage that stained for GH, prolactin, and TSH. At the biochemical level, levels of GH in the serum of KO animals were markedly elevated compared with controls, regardless of the presence of a frank tumor. These data indicate that complete loss of Prkar1a is sufficient to allow the formation of pituitary tumors and abnormalities of the GH axis, in close analogy to human patients with CNC.

Development of a pituitary-specific cre line targeted to the Pit-1 lineage.

Tissue-specific expression of the Cre recombinase is a well-established genetic tool to analyze gene function in specific tissues and cell types. In this report, we describe the generation of a new transgenic line that expresses Cre under the control of the rat growth hormone releasing hormone receptor (rGhrhr) promoter. This promoter, chosen to target the anterior pituitary, drives cre-mediated recombination in cells of the Pit1 lineage, including somatotrophs, lactotrophs, and thyrotrophs. Cre activity is first detected at embryonic day 13.5, and gradually increases to reach high level expression by postnatal day 2. In addition to the pituitary, rGhrhr-cre expression was detected in vibrissae and in hair follicles of the proximal limb, but not in other tissues. The rGhrhr-cre line will be a valuable tool for the study of the development of the pituitary Pit1 lineage and for the study of tumorigenesis involving these cells.

Genotoxic effects of particles of surgical cobalt chrome alloy on human cells of different age in vitro.

Humans are exposed to metals from industry, the environment and from wear debris from worn orthopaedic joint replacements. Patients exposed to worn cobalt chrome hip replacements show an increase of chromosome aberrations in the bone marrow adjacent to the implant and an increase of chromosome translocations and aneuploidy in the peripheral blood. This study has tested whether particles of surgical cobalt chrome alloy are able to induce similar DNA damage and chromosome aberrations in human cells in vitro. Because increasingly young patients are receiving hip replacements it has also tested whether the response is altered at different cellular age in vitro. Primary human fibroblasts, were tested at different pre senescent population doublings (PD10 (young) and PD35 (older)) to particles of cobalt chrome alloy for up to 15 days. As in patients there was an increase of aneuploidy, chromosome translocations and DNA damage after exposure to the cobalt chrome particles in vitro. The overall level of DNA damage and numerical and structural aberrations was approximately the same in young and older cells. However, the cellular reaction to the DNA damage was different. Older cells showed a greater loss of viability and induction of senescence and a lesser rate of mitosis and cell growth than young cells. They showed less change in transcription, particularly of p38 and caspase 10 mRNA levels, than young cells. They showed more complex aneuploidy in association with unseparated or prematurely separated chromatids. This study suggests that at least part of the chromosome changes in patients with worn implants may be due to direct effects of the metal wear particles from the implant. It would be of interest to test whether the altered reaction of the human cells at different in vitro age might correspond with a different incidence of chromosome aberrations in patients at different ages.

Antagonizing Bcl-2 family members sensitizes neuroblastoma and Ewing's sarcoma to an inhibitor of glutamine metabolism.

Neuroblastomas (NBL) and Ewing's sarcomas (EWS) together cause 18% of all pediatric cancer deaths. Though there is growing interest in targeting the dysregulated metabolism of cancer as a therapeutic strategy, this approach has not been fully examined in NBL and EWS. In this study, we first tested a panel of metabolic inhibitors and identified the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) as the most potent chemotherapeutic across all NBL and EWS cell lines tested. Myc, a master regulator of metabolism, is commonly overexpressed in both of these pediatric malignancies and recent studies have established that Myc causes cancer cells to become "addicted" to glutamine. We found DON strongly inhibited tumor growth of multiple tumor lines in mouse xenograft models. In vitro, inhibition of caspases partially reversed the effects of DON in high Myc expressing cell lines, but not in low Myc expressing lines. We further showed that induction of apoptosis by DON in Myc-overexpressing cancers is via the pro-apoptotic factor Bax. To relieve inhibition of Bax, we tested DON in combination with the Bcl-2 family antagonist navitoclax (ABT-263). In vitro, this combination caused an increase in DON activity across the entire panel of cell lines tested, with synergistic effects in two of the N-Myc amplified neuroblastoma cell lines. Our study supports targeting glutamine metabolism to treat Myc overexpressing cancers, such as NBL and EWS, particularly in combination with Bcl-2 family antagonists.

The genomic landscape of childhood and adolescent melanoma.

Despite remarkable advances in the genomic characterization of adult melanoma, the molecular pathogenesis of pediatric melanoma remains largely unknown. We analyzed 15 conventional melanomas (CMs), 3 melanomas arising in congenital nevi (CNMs), and 5 spitzoid melanomas (SMs), using various platforms, including whole genome or exome sequencing, the molecular inversion probe assay, and/or targeted sequencing. CMs demonstrated a high burden of somatic single-nucleotide variations (SNVs), with each case containing a TERT promoter (TERT-p) mutation, 13/15 containing an activating BRAF V600 mutation, and >80% of the identified SNVs consistent with UV damage. In contrast, the three CNMs contained an activating NRAS Q61 mutation and no TERT-p mutations. SMs were characterized by chromosomal rearrangements resulting in activated kinase signaling in 40%, and an absence of TERT-p mutations, except for the one SM that succumbed to hematogenous metastasis. We conclude that pediatric CM has a very similar UV-induced mutational spectrum to that found in the adult counterpart, emphasizing the need to promote sun protection practices in early life and to improve access to therapeutic agents being explored in adults in young patients. In contrast, the pathogenesis of CNM appears to be distinct. TERT-p mutations may identify the rare subset of spitzoid melanocytic lesions prone to disseminate.

Thyroid-specific ablation of the Carney complex gene, PRKAR1A, results in hyperthyroidism and follicular thyroid cancer.

Thyroid cancer is the most common endocrine malignancy in the population, and the incidence of this cancer is increasing at a rapid rate. Although genetic analysis of papillary thyroid cancer (PTC) has identified mutations in a large percentage of patients, the genetic basis of follicular thyroid cancer (FTC) is less certain. Thyroid cancer, including both PTC and FTC, has been observed in patients with the inherited tumor predisposition Carney complex, caused by mutations in PRKAR1A. In order to investigate the role of loss of PRKAR1A in thyroid cancer, we generated a tissue-specific knockout of Prkar1a in the thyroid. We report that the resulting mice are hyperthyroid and developed follicular thyroid neoplasms by 1 year of age, including FTC in over 40% of animals. These thyroid tumors showed a signature of pathway activation different from that observed in other models of thyroid cancer. In vitro cultures of the tumor cells indicated that Prkar1a-null thyrocytes exhibited growth factor independence and suggested possible new therapeutic targets. Overall, this work represents the first report of a genetic mutation known to cause human FTC that exhibits a similar phenotype when modeled in the mouse. In addition to our knowledge of the mechanisms of human follicular thyroid tumorigenesis, this model is highly reproducible and may provide a viable mechanism for the further clinical development of therapies aimed at FTC.

Differential role of PKA catalytic subunits in mediating phenotypes caused by knockout of the Carney complex gene Prkar1a.

The Carney complex is an inherited tumor predisposition caused by activation of the cAMP-dependent protein kinase [protein kinase A (PKA)] resulting from mutation of the PKA-regulatory subunit gene PRKAR1A. Myxomas and tumors in cAMP-responsive tissues are cardinal features of this syndrome, which is unsurprising given the important role played by PKA in modulating cell growth and function. Previous studies demonstrated that cardiac-specific knockout of Prkar1a causes embryonic heart failure and myxomatous degeneration in the heart, whereas limited Schwann cell-specific knockout of the gene causes schwannoma formation. In this study, we sought to determine the role of PKA activation in this phenotype by using genetic means to reduce PKA enzymatic activity. To accomplish this goal, we introduced null alleles of the PKA catalytic subunits Prkaca (Ca) or Prkacb (Cb) into the Prkar1a-cardiac knockout (R1a-CKO) or limited Schwann cell knockout (R1a-TEC3KO) line. Heterozygosity for Prkaca rescued the embryonic lethality of the R1a-CKO, although mice had a shorter than normal lifespan and died from cardiac failure with atrial thrombosis. In contrast, heterozygosity for Prkacb only enabled the mice to survive 1 extra day during embryogenesis. Biochemical analysis indicated that reduction of Ca markedly reduced PKA activity in embryonic hearts, whereas reduction of Cb had minimal effects. In R1a-TEC3KO mice, tumorigenesis was completely suppressed by a heterozygosity for Prkaca, and by more than 80% by heterozygosity for Prkacb. These data suggest that both developmental and tumor phenotypes caused by Prkar1a mutation result from excess PKA activity due to PKA-Ca.

Neural crest-specific loss of Prkar1a causes perinatal lethality resulting from defects in intramembranous ossification.

The cranial neural crest (CNC) undergoes complex molecular and morphological changes during embryogenesis in order to form the vertebrate skull, and nearly three quarters of all birth defects result from defects in craniofacial development. The molecular events leading to CNC differentiation have been extensively studied; however, the role of the cAMP-dependent protein kinase [protein kinase A (PKA)] during craniofacial development has only been described in palate formation. Here, we provide evidence that strict PKA regulation in postmigratory CNC cells is essential during craniofacial bone development. Selective inactivation of Prkar1a, a regulatory subunit of the PKA holoenzyme, in the CNC results in perinatal lethality caused by dysmorphic craniofacial development and subsequent asphyxiation. Additionally, aberrant differentiation of CNC mesenchymal cells results in anomalous intramembranous ossification characterized by formation of cartilaginous islands in some areas and osteolysis of bony trabeculae with fibrous connective tissue stabilization in others. Genetic interaction studies revealed that genetic reduction of the PKA catalytic subunit C(alpha) was able to rescue the phenotype, whereas reduction in Cbeta had no effect. Overall, these observations provide evidence of the essential role of proper regulation of PKA during the ossification of the bones of the skull. This knowledge may have implications for the understanding and treatment of craniofacial birth defects.

The Carney complex gene PRKAR1A plays an essential role in cardiac development and myxomagenesis.

Cardiac myxomas are the most common primary tumors of the heart, although little is known about their etiology. Mutations of the protein kinase A regulatory subunit gene PRKAR1A cause inherited myxomas in the setting of the Carney complex tumor syndrome, providing a possible window for understanding their pathogenesis. We recently reported that cardiac-specific knockout of this gene causes myxomatous changes in the heart, although the mice die during gestation from cardiac failure. In this review, we discuss these findings and place them in the larger understanding of how protein kinase A dysregulation might affect cardiac function and cause myxomagenesis.

Mouse models of altered protein kinase A signaling.

Protein kinase A (PKA) is an evolutionarily conserved protein which has been studied in model organisms from yeast to man. Although the cAMP-PKA signaling system was the first mammalian second messenger system to be characterized, many aspects of this pathway are still not well understood. Owing to findings over the past decade implicating PKA signaling in endocrine (and other) tumorigenesis, there has been renewed interest in understanding the role of this pathway in physiology, particularly as it pertains to the endocrine system. Because of the availability of genetic tools, mouse modeling has become the pre-eminent system for studying the physiological role of specific genes and gene families as a means to understanding their relationship to human diseases. In this review, we will summarize the current data regarding mouse models that have targeted the PKA signaling system. These data have led to a better understanding of both the complexity and the subtlety of PKA signaling, and point the way for future studies, which may help to modulate this pathway for therapeutic effect.