Faculty Bibliography

The bulk of the pancreas primarily comprises long-lived acinar cells that are not considered a bona fide source for stem cells. However, certain acinar subpopulations have a repopulating capacity during regeneration, raising the hypothesis as to the presence of regenerative progenitor-like populations in the adult pancreas. Here, we describe recent discoveries based on fate-mapping techniques that support the existence of progenitor-like acinar subpopulations, including active progenitor-like cells that maintain tissue homeostasis and facultative progenitor-like cells that drive tissue regeneration. A possible link between progenitor-like acinar cells and cancer initiators is proposed. Further analysis of these cellular components is needed, because it would help uncover possible cellular sources for regeneration and cancer, as well as potential targets for therapy.

Neuro-glial activation is a recently identified hallmark of growing cancers. Targeting tumor hyperinnervation in preclinical and small clinical trials has yielded promising antitumor effects, highlighting the need of systematic analysis of neural influences in cancer (NIC). Here, we outline the strategies translating these findings from bench to the clinic.

Mounting evidence indicates that the nervous system plays a central role in cancer pathogenesis. In turn, cancers and cancer therapies can alter nervous system form and function. This Commentary seeks to describe the burgeoning field of "cancer neuroscience" and encourage multidisciplinary collaboration for the study of cancer-nervous system interactions.

In many solid tumors, parasympathetic input is provided by the vagus nerve, which has been shown to modulate tumor growth. However, whether cholinergic signaling directly regulates progression of pancreatic cancer (PDAC) has not been defined. Here, we found that subdiaphragmatic vagotomy in LSL-Kras+/G12D;Pdx1-Cre (KC) mice accelerated PDAC development, whereas treatment with the systemic muscarinic agonist bethanechol restored the normal KC phenotype, thereby suppressing the accelerated tumorigenesis caused by vagotomy. In LSL-Kras+/G12D;LSL-Trp53+/R172H;Pdx1-Cre (KPC) mice with established PDAC, bethanechol significantly extended survival. These effects were mediated in part through the CHRM1, which inhibited downstream MAPK/EGFR and PI3K/AKT pathways in PDAC cells. Enhanced cholinergic signaling led to a suppression of the CSC compartment, CD11b+ myeloid cells, TNF-α levels, and metastatic growth in the liver. Therefore, these data suggest that cholinergic signaling directly and indirectly suppresses growth of PDAC cells, and therapies that stimulate muscarinic receptors may be useful in the treatment of PDAC.

BACKGROUND:Our next generation sequencing (NGS)-based human papillomavirus (HPV) genotyping assay showed a high degree of concordance with the Roche Linear Array (LA) with as little as 1.25 ng formalin-fixed paraffin-embedded-derived genomic DNA in head and neck and cervical cancer samples. This sensitive genotyping assay uses barcoded HPV PCR broad-spectrum general primers 5+/6+ (BSGP)5+/6+ applicable to population studies, but it's diagnostic performance has not been tested in cases with multiple concurrent HPV infections. METHODS:We conducted a cross-sectional study to compare the positive and negative predictive value (PPV and NPV), sensitivity and specificity of the NGS assay to detect HPV genotype infections as compared to the LA. DNA was previously extracted from ten anal swab samples from men who have sex with men in Nigeria enrolled on the TRUST/RV368 cohort study. Two-sample tests of proportions were used to examine differences in the diagnostic performance of the NGS assay to detect high vs. low-risk HPV type-specific infections. RESULTS:In total there were 94 type-specific infections detected in 10 samples with a median of 9.5, range (9 to 10) per sample. Using the LA as the gold standard, 84.4% (95% CI: 75.2-91.2) of the same anal type-specific infections detected on the NGS assay had been detected by LA. The PPV and sensitivity differed significantly for high risk (PPV: 90%, 95% CI: 79.5-96.2; sensitivity: 93.1%, 95% CI: 83.3-98.1) as compared to low risk HPV (PPV: 73%, 95% CI: 54.1-87.7; sensitivity: 61.1, 95% CI: 43.5-76.9) (all p < 0.05). The NPV for all types was 92.5% (95% CI: 88.4-95.4). The NPV and specificity were similar for high and low risk HPVs (all p > 0.05). The NGS assay detected 10 HPV genotypes that were not among the 37 genotypes found on LA (30, 32, 43, 44, 74, 86, 87, 90, 91, 114). CONCLUSIONS:The NGS assay accurately detects multiple HPV infections in individual clinical specimens with limited sample volume and has extended coverage compared to LA.

Available clinical human papilloma virus (HPV) diagnostics for head and neck cancer have limited sensitivity and/or fail to define the HPV genotype. Common HPV genotyping assays are costly and labor intensive. We sought to develop a next-generation sequencing (NGS)-based HPV genotyping assay that was sensitive enough to work on formalin-fixed paraffin-embedded (FFPE) samples. We developed an ion torrent NGS HPV genotyping assay using barcoded HPV PCR broad-spectrum general primers 5(+)/6(+) (BSGP)5(+)/6(+). To validate genotype specificity and use in archived clinical FFPE tumor samples, we compared NGS HPV genotyping at 2 sequencing centers with typing by Roche Linear Array assay in 42 oropharyngeal and cervical cancer specimens representing 10 HPV genotypes, as well as HPV-negative cases. To demonstrate the detection of a broad range of HPV genotypes, we genotyped a cohort of 266 cervical cancers. A comparison of NGS genotyping of FFPE cancer specimens with genotyping by Linear Array showed concordant results in 34/37 samples (92%) at sequencing site 1 and 39/42 samples (93%) at sequencing site 2. Concordance between sites was 92%. Designed for use with 10 ng genomic DNA, the assay detected HPV using as little as 1.25 ng FFPE-derived genomic DNA. In 266 cervical cancer specimens, the NGS assay identified 20 different HPV genotypes, including all 13 carcinogenic genotypes. This novel NGS assay provides a sensitive and specific high-throughput method to detect and genotype HPV in a range of clinical specimens derived from FFPE with low per-sample cost.

Squamous cell carcinomas (SCCs) originate in stratified epithelia, with a small subset becoming metastatic. Epithelial stem cells are targets for driver mutations that give rise to SCCs, but it is unknown whether they contribute to oncogenic multipotency and metastasis. We developed a mouse model of SCC by targeting two frequent genetic mutations in human SCCs, oncogene Kras(G12D) activation and Smad4 deletion, to mouse keratin 15-expressing (K15+) stem cells. We show that transgenic mice developed multilineage tumors, including metastatic SCCs. Among cancer stem cell-enriched (CSC-enriched) populations, those with increased side population (SP) cells correlated with epithelial-mesenchymal transition (EMT) and lung metastasis. We show that microRNA-9 (miR-9) contributed to SP expansion and metastasis, and miR-9 inhibition reduced the number of SP cells and metastasis. Increased miR-9 was detected in metastatic human primary SCCs and SCC metastases, and miR-9-transduced human SCC cells exhibited increased invasion. We identified α-catenin as a predominant miR-9 target. Increased miR-9 in human SCC metastases correlated with α-catenin loss but not E-cadherin loss. Our results demonstrate that stem cells with Kras(G12D) activation and Smad4 depletion can produce tumors that are multipotent and susceptible to EMT and metastasis. Additionally, tumor initiation and metastatic properties of CSCs can be uncoupled, with miR-9 regulating the expansion of metastatic CSCs.

CREB-binding protein (CREBBP) is important for the cell-autonomous regulation of hematopoiesis, including the stem cell compartment. In the present study, we show that CREBBP plays an equally pivotal role in microenvironment-mediated regulation of hematopoiesis. We found that the BM microenvironment of Crebbp(+/-) mice was unable to properly maintain the immature stem cell and progenitor cell pools. Instead, it stimulates myeloid differentiation, which progresses into a myeloproliferation phenotype. Alterations in the BM microenvironment resulting from haploinsufficiency of Crebbp included a marked decrease in trabecular bone that was predominantly caused by increased osteoclastogenesis. Although CFU-fibroblast (CFU-F) and total osteoblast numbers were decreased, the bone formation rate was similar to that found in wild-type mice. At the molecular level, we found that the known hematopoietic modulators matrix metallopeptidase-9 (MMP9) and kit ligand (KITL) were decreased with heterozygous levels of Crebbp. Lastly, potentially important regulatory proteins, endothelial cell adhesion molecule 1 (ESAM1) and cadherin 5 (CDH5), were increased on Crebbp(+/-) endothelial cells. Our findings reveal that a full dose of Crebbp is essential in the BM microenvironment to maintain proper hematopoiesis and to prevent excessive myeloproliferation.

Global expression analysis of fetal liver hematopoietic stem cells (FL HSCs) revealed the presence of unspliced pre-mRNA for a number of genes in normal FL HSCs. In a subset of these genes, Crebbp+/- FL HSCs had less unprocessed pre-mRNA without a corresponding reduction in total mRNA levels. Among the genes thus identified were the key regulators of HSC function Itga4, Msi2 and Tcf4. A similar but much weaker effect was apparent in Ep300+/- FL HSCs, indicating that, in this context as in others, the two paralogs are not interchangeable. As a group, the down-regulated intronic probe sets could discriminate adult HSCs from more mature cell types, suggesting that the underlying mechanism is regulated with differentiation stage and is active in both fetal and adult hematopoiesis. Consistent with increased myelopoiesis in Crebbp hemizygous mice, targeted reduction of CREBBP abundance by shRNA in the multipotent EML cell line triggered spontaneous myeloid differentiation in the absence of the normally required inductive signals. In addition, differences in protein levels between phenotypically distinct EML subpopulations were better predicted by taking into account not only the total mRNA signal but also the amount of unspliced message present. CREBBP thus appears to selectively influence the timing and degree of pre-mRNA processing of genes essential for HSC regulation and thereby has the potential to alter subsequent cell fate decisions in HSCs.

Transforming growth factor beta (TGFβ) is a key regulator of epithelial cell proliferation, immune function and angiogenesis. Because TGFβ signaling maintains epithelial homeostasis, dysregulated TGFβ signaling is common in many malignancies, including head and neck squamous cell carcinoma (HNSCC). Defective TGFβ signaling in epithelial cells causes hyperproliferation, reduced apoptosis and increased genomic instability, and the compensatory increase in TGFβ production by tumor epithelial cells with TGFβ signaling defects further promotes tumor growth and metastases by increasing angiogenesis and inflammation in tumor stromal cells. Here, we review the mouse models that we used to study TGFβ signaling in HNSCC.