Activin receptors regulate the oligodendrocyte lineage in health and disease.

The most prevalent neurological disorders of myelin include perinatal brain injury leading to cerebral palsy in infants and multiple sclerosis in adults. Although these disorders have distinct etiologies, they share a common neuropathological feature of failed progenitor differentiation into myelin-producing oligodendrocytes and lack of myelin, for which there is an unmet clinical need. Here, we reveal that a molecular pathology common to both disorders is dysregulation of activin receptors and that activin receptor signaling is required for the majority of myelin generation in development and following injury. Using a constitutive conditional knockout of all activin receptor signaling in oligodendrocyte lineage cells, we discovered this signaling to be required for myelination via regulation of oligodendrocyte differentiation and myelin compaction. These processes were found to be dependent on the activin receptor subtype Acvr2a, which is expressed during oligodendrocyte differentiation and axonal ensheathment in development and following myelin injury. During efficient myelin regeneration, Acvr2a upregulation was seen to coincide with downregulation of Acvr2b, a receptor subtype with relatively higher ligand affinity; Acvr2b was shown to be dispensable for activin receptor-driven oligodendrocyte differentiation and its overexpression was sufficient to impair the abovementioned ligand-driven responses. In actively myelinating or remyelinating areas of human perinatal brain injury and multiple sclerosis tissue, respectively, oligodendrocyte lineage cells expressing Acvr2a outnumbered those expressing Acvr2b, whereas in non-repairing lesions Acvr2b+ cells were increased. Thus, we propose that following human white matter injury, this increase in Acvr2b expression would sequester ligand and consequently impair Acvr2a-driven oligodendrocyte differentiation and myelin formation. Our results demonstrate dysregulated activin receptor signaling in common myelin disorders and reveal Acvr2a as a novel therapeutic target for myelin generation following injury across the lifespan.

Loss of Activin Receptor Type 1B Accelerates Development of Intraductal Papillary Mucinous Neoplasms in Mice With Activated KRAS.

BACKGROUND & AIMS: Activin, a member of the transforming growth factor-ß (TGFB) family, might be involved in pancreatic tumorigenesis, similar to other members of the TGFB family. Human pancreatic ductal adenocarcinomas contain somatic mutations in the activin A receptor type IB (ACVR1B) gene, indicating that ACVR1B could be a suppressor of pancreatic tumorigenesis. METHODS: We disrupted Acvr1b specifically in pancreata of mice (Acvr1b(flox/flox);Pdx1-Cre mice) and crossed them with LSL-KRAS(G12D) mice, which express an activated form of KRAS and develop spontaneous pancreatic tumors. The resulting Acvr1b(flox/flox);LSL-KRAS(G12D);Pdx1-Cre mice were monitored; pancreatic tissues were collected and analyzed by histology and immunohistochemical analyses. We also analyzed p16(flox/flox);LSL-Kras(G12D);Pdx1-Cre mice and Cre-negative littermates (controls). Genomic DNA, total RNA, and protein were isolated from mouse tissues and primary pancreatic tumor cell lines and analyzed by reverse-transcription polymerase chain reaction, sequencing, and immunoblot analyses. Human intraductal papillary mucinous neoplasm (IPMN) specimens were analyzed by immunohistochemistry. RESULTS: Loss of ACVR1B from pancreata of mice increased the proliferation of pancreatic epithelial cells, led to formation of acinar to ductal metaplasia, and induced focal inflammatory changes compared with control mice. Disruption of Acvr1b in LSL-KRAS(G12D);Pdx1-Cre mice accelerated the growth of pancreatic IPMNs compared with LSL-KRAS(G12D);Pdx1-Cre mice, but did not alter growth of pancreatic intraepithelial neoplasias. We associated perinuclear localization of the activated NOTCH4 intracellular domain to the apical cytoplasm of neoplastic cells with the expansion of IPMN lesions in Acvr1b(flox/flox);LSL-KRAS(G12D);Pdx1-Cre mice. Loss of the gene that encodes p16 (Cdkn2a) was required for progression of IPMNs to pancreatic ductal adenocarcinomas in Acvr1b(flox/flox);LSL-Kras(G12D);Pdx1-Cre mice. We also observed progressive loss of p16 in human IPMNs of increasing grades. CONCLUSIONS: Loss of ACVR1B accelerates growth of mutant KRAS-induced pancreatic IPMNs in mice; this process appears to involve NOTCH4 and loss of p16. ACVR1B suppresses early stages of pancreatic tumorigenesis; the activin signaling pathway therefore might be a therapeutic target for pancreatic cancer.

Challenges and advances in mouse modeling for human pancreatic tumorigenesis and metastasis.

Pancreatic cancer is critical for developed countries, where its rate of diagnosis has been increasing steadily annually. In the past decade, the advances of pancreatic cancer research have not contributed to the decline in mortality rates from pancreatic cancer-the overall 5-year survival rate remains about 5% low. This number only underscores an obvious urgency for us to better understand the biological features of pancreatic carcinogenesis, to develop early detection methods, and to improve novel therapeutic treatments. To achieve these goals, animal modeling that faithfully recapitulates the whole process of human pancreatic cancer is central to making the advancements. In this review, we summarize the currently available animal models for pancreatic cancer and the advances in pancreatic cancer animal modeling. We compare and contrast the advantages and disadvantages of three major categories of these models: (1) carcinogen-induced; (2) xenograft and allograft; and (3) genetically engineered mouse models. We focus more on the genetically engineered mouse models, a category which has been rapidly expanded recently for their capacities to mimic human pancreatic cancer and metastasis, and highlight the combinations of these models with various newly developed strategies and cell-lineage labeling systems.

Gene Therapy with p14/tBID Induces Selective and Synergistic Apoptosis in Mutant Ras and Mutant p53 Cancer Cells In Vitro and In Vivo.

Any gene therapy for cancer will be predicated upon its selectivity against cancer cells and non-toxicity to normal cells. Therefore, safeguards are needed to prevent its activation in normal cells. We designed a minimal p14ARF promoter with upstream Ap1 and E2F enhancer elements and a downstream MDR1 inhibitory element, TATA box, and a transcription initiation site (hereafter p14ARFmin). The modified p14ARFmin promoter was linked to bicistronic P14 and truncated BID (tBID) genes, which led to synergistic apoptosis via the intrinsic and extrinsic pathways of apoptosis when expressed. The promoter was designed to be preferentially activated by mutant Ras and completely inhibited by wild-type p53 so that only cells with both mutant Ras and mutant p53 would activate the construct. In comparison to most p53 gene therapies, this construct has selective advantages: (1) p53-based gene therapies with a constitutive CMV promoter cannot differentiate between normal cells and cancer cells, and can be toxic to normal cells; (2) our construct does not induce p21WAF/CIPI in contrast to other p53-based gene therapies, which can induce cell cycle arrest leading to increased chemotherapy resistance; (3) the modified construct (p14ARFmin-p14-tBID) demonstrates bidirectional control of its promoter, which is completely repressed by wild-type p53 and activated only in cells with both RAS and P53 mutations; and (4) a novel combination of genes (p14 and tBID) can synergistically induce potent intrinsic and extrinsic apoptosis in cancer cells.

Development, validation, and comparison of a nomogram based on radiologic findings for predicting malignancy in intraductal papillary mucinous neoplasms of the pancreas: An international multicenter study.

BACKGROUND: Although we previously proposed a nomogram to predict malignancy in intraductal papillary mucinous neoplasms (IPMN) and validated it in an external cohort, its application is challenging without data on tumor markers. Moreover, existing nomograms have not been compared. This study aimed to develop a nomogram based on radiologic findings and to compare its performance with previously proposed American and Korean/Japanese nomograms. METHODS: We recruited 3708 patients who underwent surgical resection at 31 tertiary institutions in eight countries, and patients with main pancreatic duct >10 mm were excluded. To construct the nomogram, 2606 patients were randomly allocated 1:1 into training and internal validation sets, and area under the receiver operating characteristics curve (AUC) was calculated using 10-fold cross validation by exhaustive search. This nomogram was then validated and compared to the American and Korean/Japanese nomograms using 1102 patients. RESULTS: Among the 2606 patients, 90 had main-duct type, 900 had branch-duct type, and 1616 had mixed-type IPMN. Pathologic results revealed 1628 low-grade dysplasia, 476 high-grade dysplasia, and 502 invasive carcinoma. Location, cyst size, duct dilatation, and mural nodule were selected to construct the nomogram. AUC of this nomogram was higher than the American nomogram (0.691 vs 0.664, P = .014) and comparable with the Korean/Japanese nomogram (0.659 vs 0.653, P = .255). CONCLUSIONS: A novel nomogram based on radiologic findings of IPMN is competitive for predicting risk of malignancy. This nomogram would be clinically helpful in circumstances where tumor markers are not available. The nomogram is freely available at http://statgen.snu.ac.kr/software/nomogramIPMN.

Stathmin as a surrogate marker of phosphatidylinositol-3-kinase pathway activity: Towards precision medicine in HPV-negative head & neck squamous cell carcinoma.

In order to assess Stathmin as an immunohistochemical (IHC) indicator of phosphatidylinositol 3-kinase (PI3K) pathway activity in HPV-negative head & neck squamous cell carcinoma (HNSCC), we compared Stathmin IHC to expression of other pathway components. We also evaluated the relationship between Stathmin IHC and the mutational status of four key pathway genes. Finally, we ascertained whether Stathmin IHC correlates with tumor grade or primary site. Correlation exists between high Stathmin expression and high pAKT1 expression, indicating a role for Stathmin IHC as a marker of pathway activity. Our analysis did not show correlation between Stathmin IHC and mutation of the four genes evaluated. We also observed an association between high Stathmin expression and oropharyngeal primary site. Our results suggest utility of Stathmin IHC as an indicator of PI3K pathway activity, and thereby demonstrate potential relevance of Stathmin IHC in the context of HNSCC.

Inactivation of Notch4 Attenuated Pancreatic Tumorigenesis in Mice.

Expression of the Notch family of receptors is often upregulated in pancreatic ductal adenocarcinoma (PDAC). In this study, we focused on Notch4, which had not been investigated in PDAC. We generated KC (LSL-KrasG12D;p48-Cre), N4 - / - KC (Notch4- / -;LSL-KrasG12D;p48-Cre), PKC (p16fl/fl;LSL-KrasG12D;p48-Cre), and N4 - / - PKC (Notch4-/ -; p16fl/f l;LSL-KrasG12D;p48-Cre) genetically engineered mouse models (GEMM). We performed caerulein treatment in both KC and N4 - / - KC mice, and the development of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) lesions were significantly diminished in the N4 - / - KC than in the KC GEMM (P = 0.01). This in vivo result was validated by in vitro ADM induction of the explant cultures of pancreatic acinar cells from the N4 - / - KC and KC mice (P < 0.001), confirming that Notch4 is an important contributor to early pancreatic tumorigenesis. To evaluate the role of Notch4 in the later stage of pancreatic tumorigenesis, we compared the PKC and N4 - / - PKC mice. The N4 - / - PKC mice had better overall survival (P = 0.012) and significantly reduced tumor burden (PanIN: P = 0.018 at 2 months, PDAC: P = 0.039 at 5 months) compared with the PKC GEMM. RNA-sequencing analysis of pancreatic tumor cell lines derived from the PKC and N4 - / - PKC GEMMs revealed that 408 genes were differentially expressed (FDR < 0.05) and Pcsk5 is a potential downstream effector of the Notch4 signaling pathway (P < 0.001). Low expression of Pcsk5 positively correlates with good survival in patients with PDAC (P = 0.028). We have identified a novel role for Notch4 signaling with tumor-promoting function in pancreatic tumorigenesis. Our study also uncovered a novel association between Pcsk5 and Notch4 signaling in PDAC. Significance: We demonstrated that global inactivation of Notch4 significantly improved the survival of an aggressive mouse model for PDAC and provided preclinical evidence that Notch4 and Pcsk5 are novel targets for PDAC therapies.

Dual Roles of the Activin Signaling Pathway in Pancreatic Cancer.

Activin, a member of the TGF-ß superfamily, is involved in many physiological processes, such as embryonic development and follicle development, as well as in multiple human diseases including cancer. Genetic mutations in the activin signaling pathway have been reported in many cancer types, indicating that activin signaling plays a critical role in tumorigenesis. Recent evidence reveals that activin signaling may function as a tumor-suppressor in tumor initiation, and a promoter in the later progression and metastasis of tumors. This article reviews many aspects of activin, including the signaling cascade of activin, activin-related proteins, and its role in tumorigenesis, particularly in pancreatic cancer development. The mechanisms regulating its dual roles in tumorigenesis remain to be elucidated. Further understanding of the activin signaling pathway may identify potential therapeutic targets for human cancers and other diseases.

Loss of the wild-type KRAS allele promotes pancreatic cancer progression through functional activation of YAP1.

Human pancreatic ductal adenocarcinoma (PDAC) harboring one KRAS mutant allele often displays increasing genomic loss of the remaining wild-type (WT) allele (known as LOH at KRAS) as tumors progress to metastasis, yet the molecular ramification of this WT allelic loss is unknown. In this study, we showed that the restoration of WT KRAS expression in human PDAC cell lines with LOH at KRAS significantly attenuated the malignancy of PDAC cells both in vitro and in vivo, demonstrating a tumor-suppressive role of the WT KRAS allele. Through RNA-Seq, we identified the HIPPO signaling pathway to be positively regulated by WT KRAS in PDAC cells. In accordance with this observation, PDAC cells with LOH at KRAS exhibited increased nuclear localization and activation of transcriptional co-activator YAP1. Mechanistically, we discovered that WT KRAS expression sequestered YAP1 from the nucleus, through enhanced 14-3-3zeta interaction with phosphorylated YAP1 at S127. Consistently, expression of a constitutively-active YAP1 mutant in PDAC cells bypassed the growth inhibitory effects of WT KRAS. In patient samples, we found that the YAP1-activation genes were significantly upregulated in tumors with LOH at KRAS, and YAP1 nuclear localization predicted poor survival for PDAC patients. Collectively, our results reveal that the WT allelic loss leads to functional activation of YAP1 and enhanced tumor malignancy, which explains the selection advantage of the tumor cells with LOH at KRAS during pancreatic cancer clonal evolution and progression to metastasis, and should be taken into consideration in future therapeutic strategies targeting KRAS.

PIK3CA and p53 Mutations Promote 4NQO-Initated Head and Neck Tumor Progression and Metastasis in Mice.

The PI3K signaling pathway is frequently mutated in head and neck squamous cell carcinoma (HNSCC), often via gain-of-function (GOF) mutations in the PIK3CA gene. Here, we present novel genetically engineered mouse models (GEMM) carrying a GOF allele Loxp-STOP-Loxp(LSL)-PIK3CAH1047R (E20) alone or in combination with heterozygous LSL-p53+/R172H (p53) mutation with tissue-specific expression to interrogate the role of oncogenic PIK3CA in transformation of upper aerodigestive track epithelium. We demonstrated that the GOF PIK3CA mutation promoted progression of 4-nitroquinoline 1-oxide-induced oral squamous cell carcinoma (OSCC) in both E20 single mutant and E20/p53 double mutant mice, with frequent distal metastasis detected only in E20/p53 GEMM. Similar to in human OSCC, loss of p16 was associated with progression of OSCC in these mice. RNA-seq analyses revealed that among the common genes differentially expressed in primary OSCC cell lines derived from E20, p53, and E20/p53 GEMMs compared with those from the wild-type mice, genes associated with proliferation and cell cycle were predominantly represented, which is consistent with the progressive loss of p16 detected in these GEMMs. Importantly, all of these OSCC primary cell lines exhibited enhanced sensitivity to BYL719 and cisplatin combination treatment in comparison with cisplatin alone in vitro and in vivo, regardless of p53 and/or p16 status. Given the prevalence of mutations in p53 and the PI3K pathways in HNSCC in conjunction with loss of p16 genetically or epigenetically, this universal increased sensitivity to cisplatin and BYL719 combination therapy in cancer cells with PIK3CA mutation represents an opportunity to a subset of patients with HNSCC. IMPLICATIONS: Our results suggest that combination therapy of cisplatin and PI3K inhibitor may be worthy of consideration in patients with HNSCC with PIK3CA mutation.

Risk prediction for malignant intraductal papillary mucinous neoplasm of the pancreas: logistic regression versus machine learning.

Most models for predicting malignant pancreatic intraductal papillary mucinous neoplasms were developed based on logistic regression (LR) analysis. Our study aimed to develop risk prediction models using machine learning (ML) and LR techniques and compare their performances. This was a multinational, multi-institutional, retrospective study. Clinical variables including age, sex, main duct diameter, cyst size, mural nodule, and tumour location were factors considered for model development (MD). After the division into a MD set and a test set (2:1), the best ML and LR models were developed by training with the MD set using a tenfold cross validation. The test area under the receiver operating curves (AUCs) of the two models were calculated using an independent test set. A total of 3,708 patients were included. The stacked ensemble algorithm in the ML model and variable combinations containing all variables in the LR model were the most chosen during 200 repetitions. After 200 repetitions, the mean AUCs of the ML and LR models were comparable (0.725 vs. 0.725). The performances of the ML and LR models were comparable. The LR model was more practical than ML counterpart, because of its convenience in clinical use and simple interpretability.

Blocking IL-6/GP130 Signaling Inhibits Cell Viability/Proliferation, Glycolysis, and Colony Forming Activity in Human Pancreatic Cancer Cells.

BACKGROUND: Elevated production of the pro-inflammatory cytokine interleukin-6 (IL-6) and dysfunction of IL-6 signaling promotes tumorigenesis and are associated with poor survival outcomes in multiple cancer types. Recent studies showed that the IL-6/GP130/STAT3 signaling pathway plays a pivotal role in pancreatic cancer development and maintenance. OBJECTIVE: We aim to develop effective treatments through inhibition of IL-6/GP130 signaling in pancreatic cancer. METHODS: The effects on cell viability and cell proliferation were measured by MTT and BrdU assays, respectively. The effects on glycolysis was determined by cell-based assays to measure lactate levels. Protein expression changes were evaluated by western blotting and immunoprecipitation. siRNA transfection was used to knock down estrogen receptor α gene expression. Colony forming ability was determined by colony forming cell assay. RESULTS: We demonstrated that IL-6 can induce pancreatic cancer cell viability/proliferation and glycolysis. We also showed that a repurposing FDA-approved drug bazedoxifene could inhibit the IL-6/IL-6R/GP130 complexes. Bazedoxifene also inhibited JAK1 binding to IL-6/IL-6R/GP130 complexes and STAT3 phosphorylation. In addition, bazedoxifene impeded IL-6 mediated cell viability/ proliferation and glycolysis in pancreatic cancer cells. Consistently, other IL-6/GP130 inhibitors SC144 and evista showed similar inhibition of IL-6 stimulated cell viability, cell proliferation and glycolysis. Furthermore, all three IL-6/GP130 inhibitors reduced the colony forming ability in pancreatic cancer cells. CONCLUSION: Our findings demonstrated that IL-6 stimulates pancreatic cancer cell proliferation, survival and glycolysis, and supported persistent IL-6 signaling is a viable therapeutic target for pancreatic cancer using IL-6/GP130 inhibitors.

Isolation of Extracellular Vesicles for Cancer Diagnosis and Functional Studies.

Extracellular vesicles (EVs) are a diverse category of cellular export products that are present in a variety of biofluids and cell culture media. EVs contain a wide variety of macromolecules that represent a sampling of the cytoplasmic or endosomal compartments and function in cell-to-cell paracrine and endocrine signaling; it has been demonstrated that pathological states such as oxidative stress, transformation, apoptosis, and various cell injuries induce cells to increase their EV release rate, simultaneously altering their composition to reflect the altered state of the cellular origin. Specifically, in patients with solid tumors, EVs are released from cancerous cells at a higher rate than from healthy cells and are enriched in tumor signature molecules. Because of their stability, increased concentration, and unique signatures in cancer patients, EVs have become the subject of investigation for diagnostic and prognostic purposes. Moreover, understanding EVs' biogenesis and biological role could lead to novel insights toward cellular cross talk and complex biological pathways in cancer research. To make use of EVs for diagnostic and mechanistic cancer research, standardized well-characterized methods are required. This chapter provides an overview of two EV isolation techniques and provides detailed instructions on the isolation of EVs by ultracentrifugation, the labor-intensive gold standard, and concentrated polymer precipitation, a faster, higher-yield technique that can be utilized in cancer research.

Dasatinib can enhance paclitaxel and gemcitabine inhibitory activity in human pancreatic cancer cells.

SRC and its activated form, phospho-SRC (pSRC), are aberrantly activated in pancreatic cancer and SRC represents a potential target for pancreatic cancer therapy. In this paper, we examined the inhibitory effect of dasatinib, a potent SRC inhibitor in combination with paclitaxel or gemcitabine on human and murine pancreatic cancer cell lines. The results showed that p-SRC can be highly expressed in most human and mouse pancreatic cancer cell lines compared with normal human cell lines and can be induced by paclitaxel or gemcitabine in HPAC cells. Dasatinib can enhance the efficacy of paclitaxel or gemcitabine by reducing the cell viability and inhibiting the cell proliferation. Dasatinib with paclitaxel combination exhibits statistically greater inhibition of the cell migration ability than single agent alone, paclitaxel with gemcitabine or FOLFIRINOX (combination of fluorouracil, leucovorin, irinotecan, and oxaliplatin) in HAPC, PANC-1, and BXPC-3 human pancreatic cancer cell lines as well as 8-285 APR and 8-365 APR mouse pancreatic cancer cell lines. In addition, dasatinib with gemcitabine combination also showed statistically greater inhibition of cell migration than single agent alone, paclitaxel with gemcitabine, or FOLFIRINOX in HAPC, PANC-1 and 8-285 APR cells. The combination of dasatinib with paclitaxel or gemcitabine also showed greater inhibition of the colony formation ability of pancreatic cancer cells compared with single-agent monotherapy or FOLFIRINOX. Dasatinib with paclitaxel or gemcitabine combination also inhibits p-SRC, p-STAT3, p-AKT, and/or p-ERK in these pancreatic cancer cells. Therefore, our results support that combined dasatinib and paclitaxel or gemcitabine therapy may be a viable therapeutic approach for human pancreatic cancer.

Abemaciclib Is Effective Against Pancreatic Cancer Cells and Synergizes with HuR and YAP1 Inhibition.

Mutation or promoter hypermethylation of CDKN2A is found in over 90% of pancreatic ductal adenocarcinomas (PDAC) and leads to loss of function of cell-cycle inhibitors p16 (INK4A) and p14 (ARF) resulting in unchecked proliferation. The CDK4/6 inhibitor, abemaciclib, has nanomolar IC50s in PDAC cell lines and decreases growth through inhibition of phospho-Rb (pRb), G1 cell-cycle arrest, apoptosis, and the senescent phenotype detected with ß-galactosidase staining and relevant mRNA elevations. Daily abemaciclib treatments in mouse PDAC xenograft studies were safe and demonstrated a 3.2-fold decrease in tumor volume compared with no treatment (P < 0.0001) accompanying a decrease in both pRb and Ki67. We determined that inhibitors of HuR (ELAVL1), a prosurvival mRNA stability factor that regulates cyclin D1, and an inhibitor of Yes-Associated Protein 1 (YAP1), a pro-oncogenic, transcriptional coactivator important for CDK6 and cyclin D1, were both synergistic with abemaciclib. Accordingly, siRNA oligonucleotides targeted against HuR, YAP1, and their common target cyclin D1, validated the synergy studies. In addition, we have seen increased sensitivity to abemaciclib in a PDAC cell line that harbors a loss of the ELAVL1 gene via CRISP-Cas9 technology. As an in vitro model for resistance, we investigated the effects of long-term abemaciclib exposure. PDAC cells chronically cultured with abemaciclib displayed a reduction in cellular growth rates (GR) and coresistance to gemcitabine and 5-fluorouracil (5-FU), but not to HuR or YAP1 inhibitors as compared with no treatment controls. We believe that our data provide compelling preclinical evidence for an abemaciclib combination-based clinical trial in patients with PDAC. IMPLICATIONS: Our data suggest that abemaciclib may be therapeutically relevant for the treatment in PDAC, especially as part of a combination regimen inhibiting YAP1 or HuR.

HHLA2 is a novel immune checkpoint protein in pancreatic ductal adenocarcinoma and predicts post-surgical survival.

HHLA2 is a newly identified member of the B7 immune checkpoint family, but its function and crosstalk with immune cells is not fully understood. To gain insights into the HHLA2 expression profile and to determine the clinical significance and function of HHLA2 in pancreatic cancer, we performed immunohistochemistry (IHC) analyses on tissue microarrays (TMAs) of pancreatic ductal adenocarcinoma (PDAC, n = 92) with matched peritumoral tissues as well as in cohorts of precancerous lesions: pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN). We found that HHLA2 was rarely detected in normal acinar, islet, and ductal cells but widely expressed from early pancreatic precancerous lesions to invasive PDAC. The overall HHLA2 positivity was 95% (19/20) in low grade PanIN and 70.73% (29/41) in IPMN. HHLA2 expression was detected in 77.17% (71/92) of the PDAC cases and was significantly associated with better prognosis in this cohort. Our findings suggest that HHLA2 may behave as a costimulatory ligand in pancreatic cancer, which differs from other B7 family members that are largely characterized as checkpoint inhibitors. Further investigation of the HHLA2 signaling pathway and its receptors is warranted by our data and may lead to novel therapeutic interventions.

Novel mutant-enriched sequencing identified high frequency of PIK3CA mutations in pharyngeal cancer.

We previously reported 4 PIK3CA mutations in 38 head and neck cancer samples, 3 of which were identified in 6 pharyngeal cancer samples. To determine the mutation frequency of PIK3CA in pharyngeal cancer, we studied 24 additional cases of pharyngeal squamous cell carcinoma in this study. Using both direct genomic DNA sequencing and novel mutant-enriched sequencing methods developed specifically for the 3 hot-spot mutations (H1047R, E545K and E452K) of PIK3CA, we detected 5 mutations of PIK3CA in the 24 pharyngeal cancers (20.8%). Three of the 5 mutations had been missed by the conventional sequencing method and were subsequently detected by novel mutant-enriched sequencing methods. We showed that the mutant-enriched sequencing method for the H1047R hot-spot mutation can identify the mutation in a mixed population of mutant and wild-type DNA sequences at 1:360 ratios. These novel mutant-enriched sequencing methods allow the detection of the PIK3CA hot-spot mutations in clinical specimens which often contain limited tumor tissues (i.e., biopsy specimens). The data further support that oncogenic PIK3CA may play a critical role in pharyngeal carcinogenesis, and the mutant-enriched sequencing methods for PIK3CA are sensitive and reliable ways to detect PIK3CA mutations in clinical samples. Because PIK3CA and its pathway are potential targets for chemotherapy and radiation therapy, and frequent somatic mutation of PIK3CA has been identified in many human cancer types (e.g., breast cancer, colorectal cancer), the abilities to detect PIK3CA mutations with enhanced sensitivities have great potential impacts on target therapies for many cancer types.

PIK3CA, KRAS, and BRAF mutations in intraductal papillary mucinous neoplasm/carcinoma (IPMN/C) of the pancreas.

BACKGROUND AND AIMS: Recent studies have reported high frequencies of somatic mutations in the phosphoinositide-3-kinase catalytic-alpha (PIK3CA) gene in various human tumors. Three hot-spot mutations in the exons 9 and 20 have been proven to activate the Akt signalling pathway. The Raf/MEK/ERK (mitogen-activated protein kinase) signal transduction is an important mediator of a number of cellular fates including growth, proliferation, and survival. The BRAF gene is activated by oncogenic RAS, leading to cooperative effects in cells responding to growth factor signals. Here we evaluate the mutational status of PIK3CA, KRAS, and BRAF in intraductal papillary mucinous neoplasm/carcinoma (IPMN/IPMNC) of the pancreas. MATERIALS AND METHODS: Exons 1, 4, 5, 6, 7, 9, 12, 18, and 20 of PIK3CA, exons 1 of KRAS, and exons 5, 11, and 15 of BRAF were analyzed in 36 IPMN/IPMC and two mucinous cystadenoma specimens by direct genomic DNA sequencing. RESULTS: We identified four somatic missense mutations of PIK3CA within the 36 IPMN/IPMC specimens (11%). One of the four mutations, H1047R, has been previously reported to be a hot-spot mutation. Furthermore, we found 17 (47%) KRAS mutations in exon 1 and one missense mutation (2.7%) in exon 15 of BRAF. CONCLUSION: This data is the first report of PIK3CA mutation in pancreatic cancer and it appears to be the first oncogene to be mutated in IPMN/IPMC but not in conventional ductal adenocarcinoma of the pancreas. Our data provide evidence that PIK3CA and BRAF contribute to the tumorigenesis of IPMN/IPMC, but at a lower frequency than KRAS.

Pancreatic DCLK1+ cells originate distinctly from PDX1+ progenitors and contribute to the initiation of intraductal papillary mucinous neoplasm in mice.

PanINs and IPMNs are the two most common precursor lesions that can progress to invasive pancreatic ductal adenocarcinoma (PDA). DCLK1 has been identified as a biomarker of progenitor cells in PDA progressed from PanINs. To explore the potential role of DCLK1-expressing cells in the genesis of IPMNs, we compared the incidence of DCLK1-positive cells in pancreatic tissue samples from genetically-engineered mouse models (GEMMs) for IPMNs, PanINs, and acinar to ductal metaplasia by immunohistochemistry and immunofluorescence. Mouse lineage tracing experiments in the IPMN GEMM showed that DCLK1+ cells originated from a cell lineage distinct from PDX1+ progenitors. The DCLK1+ cells shared the features of tuft cells but were devoid of IPMN tumor biomarkers. The DCLK1+ cells were detected in the earliest proliferative acinar clusters prior to the formation of metaplastic ductal cells, and were enriched in the "IPMN niches". In summary, DCLK1 labels a unique pancreatic cellular lineage in the IPMN GEMM. The clustering of DCLK1+ cells is an early event in Kras-induced pancreatic tumorigenesis and may contribute to IPMN initiation.

Disruption of transforming growth factor beta-Smad signaling pathway in head and neck squamous cell carcinoma as evidenced by mutations of SMAD2 and SMAD4.

The role of the TGF-beta-Smad signaling pathway in the carcinogenesis of head and neck cancer has not been fully evaluated genetically. In this study, we screened for mutation in the five main members of the TGF-beta -Smad signaling pathway, TGF-beta type I receptor (TGFBRI), TGF-beta type II receptor (TGFBRII), SMAD2, SMAD3 and SMAD4, in eight human head and neck squamous cell carcinoma (HNSCC) cell lines. Two mutations with presumed loss of heterozygosity (LOH) were identified. A novel missense mutation of SMAD2, located in exon 8 at codon 276 TCG (ser) -->TTG (leu), was identified in cell line SCC-15. This is the first report of a biallelic mutation of the SMAD2 gene in HNSCC. A nonsense mutation of the SMAD4 gene in exon 5 codon 245 CAG (glut) -->TAG (stop) was found in cell line CAL27. Western blotting verified that this nonsense mutation gives rise to the complete loss of the Smad4 protein in the cells. While the down-regulation and loss of expressions of the TGF-beta-Smad signaling pathway have been described frequently in HNSCC, here we offer further genetic evidence that the pathway is directly targeted for mutation during the HNSCC tumorigenesis.