Galen Hostetter

High frequency of BRAF mutations in nevi.

To evaluate the timing of mutations in BRAF (v-raf murine sarcoma viral oncogene homolog B1) during melanocytic neoplasia, we carried out mutation analysis on microdissected melanoma and nevi samples. We observed mutations resulting in the V599E amino-acid substitution in 41 of 60 (68%) melanoma metastases, 4 of 5 (80%) primary melanomas and, unexpectedly, in 63 of 77 (82%) nevi. These data suggest that mutational activation of the RAS/RAF/MAPK pathway in nevi is a critical step in the initiation of melanocytic neoplasia but alone is insufficient for melanoma tumorigenesis.

A Transforming Mutation in the Pleckstrin Homology Domain of Akt1 in Cancer.

Although AKT1 (v-akt murine thymoma viral oncogene homologue 1) kinase is a central member of possibly the most frequently activated proliferation and survival pathway in cancer, mutation of AKT1 has not been widely reported. Here we report the identification of a somatic mutation in human breast, colorectal and ovarian cancers that results in a glutamic acid to lysine substitution at amino acid 17 (E17K) in the lipid-binding pocket of AKT1. Lys 17 alters the electrostatic interactions of the pocket and forms new hydrogen bonds with a phosphoinositide ligand. This mutation activates AKT1 by means of pathological localization to the plasma membrane, stimulates downstream signalling, transforms cells and induces leukaemia in mice. This mechanism indicates a direct role of AKT1 in human cancer, and adds to the known genetic alterations that promote oncogenesis through the phosphatidylinositol-3-OH kinase/AKT pathway. Furthermore, the E17K substitution decreases the sensitivity to an allosteric kinase inhibitor, so this mutation may have important clinical utility for AKT drug development.

Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma.

Gene expression profiling identified human melanoma cells demonstrating increased cell motility and invasiveness. The gene WNT5A best determined in vitro invasive behavior. Melanoma cells were transfected with vectors constitutively overexpressing Wnt5a. Consistent changes included actin reorganization and increased cell adhesion. No increase in beta-catenin expression or nuclear translocation was observed. There was, however, a dramatic increase in activated PKC. In direct correlation with Wnt5a expression and PKC activation, there was an increase in melanoma cell invasion. Blocking this pathway using antibodies to Frizzled-5, the receptor for Wnt5a, inhibited PKC activity and cellular invasion. Furthermore, Wnt5a expression in human melanoma biopsies directly correlated to increasing tumor grade. These observations support a role for Wnt5a in human melanoma progression.

Germline mutations in the ribonuclease L gene in families showing linkage with HPC1.

Although prostate cancer is the most common non-cutaneous malignancy diagnosed in men in the United States, little is known about inherited factors that influence its genetic predisposition. Here we report that germline mutations in the gene encoding 2′-5′-oligoadenylate(2-5A)–dependent RNase L (RNASEL) segregate in prostate cancer families that show linkage to the HPC1 (hereditary prostate cancer 1) region at 1q24–25 (ref. 9). We identified RNASEL by a positional cloning/candidate gene method, and show that a nonsense mutation and a mutation in an initiation codon of RNASEL segregate independently in two HPC1-linked families. Inactive RNASEL alleles are present at a low frequency in the general population. RNASEL regulates cell proliferation and apoptosis through the interferon-regulated 2-5A pathway and has been suggested to be a candidate tumor suppressor gene. We found that microdissected tumors with a germline mutation showed loss of heterozygosity and loss of RNase L protein, and that RNASEL activity was reduced in lymphoblasts from heterozyogous individuals compared with family members who were homozygous with respect to the wildtype allele. Thus, germline mutations in RNASEL may be of diagnostic value, and the 2-5A pathway might provide opportunities for developing therapies for those with prostate cancer.

Tissue factor expression, angiogenesis, and thrombosis in pancreatic cancer.

Purpose: Hemostatic activation is common in pancreatic cancer and may be linked to angiogenesis and venous thromboembolism. We investigated expression of tissue factor (TF), the prime initiator of coagulation, in noninvasive and invasive pancreatic neoplasia. We correlated TF expression with vascular endothelial growth factor (VEGF) expression, microvessel density, and venous thromboembolism in resected pancreatic cancer. Experimental Design: Tissue cores from a tri-institutional retrospective series of patients were used to build tissue microarrays. TF expression was graded semiquantitatively using immunohistochemistry in normal pancreas (n = 10), intraductal papillary mucinous neoplasms (n = 70), pancreatic intraepithelial neoplasia (n = 40), and resected or metastatic pancreatic adenocarcinomas (n = 130). Results: TF expression was observed in a majority of noninvasive and invasive pancreatic neoplasia, including 77% of pancreatic intraepithelial neoplasias, 91% of intraductal papillary mucinous neoplasms, and 89% of pancreatic cancers, but not in normal pancreas. Sixty-six of 122 resected pancreatic cancers (54%) were found to have high TF expression (defined as grade ≥2, the median score). Carcinomas with high TF expression were more likely to also express VEGF (80% versus 27% with low TF expression, P < 0.0001) and had a higher median MVD (8 versus 5 per tissue core with low TF expression, P = 0.01). Pancreatic cancer patients with high TF expression had a venous thromboembolism rate of 26.3% compared with 4.5% in patients with low TF expression (P = 0.04). Conclusions: TF expression occurs early in pancreatic neoplastic transformation and is associated with VEGF expression, increased microvessel density, and possibly clinical venous thromboembolism in pancreatic cancer. Prospective studies evaluating the role of TF in pancreatic cancer outcomes are warranted.

Perineural invasion and associated pain in pancreatic cancer

Perineural invasion (PNI) is a prominent characteristic of pancreatic cancer. PNI is a process whereby cancer cells invade the surrounding nerves, thus providing an alternative route for metastatic spread and pain generation. PNI is thought to be an indicator of aggressive tumour behaviour and has been shown to correlate with poor prognosis of patients with pancreatic cancer. Recent studies demonstrated that some signalling molecules and pathways that are involved in PNI are also involved in pain generation. Targeting these signalling pathways has shown some promise in alleviating pain and reducing PNI, which could potentially improve treatment outcomes for patients with pancreatic cancer.

Increased Fibroblast Growth Factor-Inducible 14 Expression Levels Promote Glioma Cell Invasion via Rac1 and Nuclear Factor-κB and Correlate with Poor Patient Outcome

Glial tumors progress to malignant grades by heightened proliferation and relentless dispersion throughout the central nervous system. Understanding genetic and biochemical processes that foster these behaviors is likely to reveal specific and effective targets for therapeutic intervention. Our current report shows that the fibroblast growth factor-inducible 14 (Fn14), a member of the tumor necrosis factor (TNF) receptor superfamily, is expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Forced Fn14 overexpression stimulates glioma cell migration and invasion, and depletion of Rac1 by small interfering RNA inhibits this cellular response. Activation of Fn14 signaling by the ligand TNF-like weak inducer of apoptosis (TWEAK) stimulates migration and up-regulates expression of Fn14; this TWEAK effect requires Rac1 and nuclear factor-kappaB (NF-kappaB) activity. The Fn14 promoter region contains NF-kappaB binding sites, which mediate positive feedback causing sustained overexpression of Fn14 and enduring glioma cell invasion. Furthermore, Fn14 gene expression levels increase with glioma grade and inversely correlate with patient survival. These results show that the Fn14 cascade operates as a positive feedback mechanism for elevated and sustained Fn14 expression. Such a feedback loop argues for aggressive targeting of the Fn14 axis as a unique and specific driver of glioma malignant behavior.

Targeting the Tumor Microenvironment in Cancer: Why Hyaluronidase Deserves a Second Look

Increased extracellular matrix (ECM) deposition is a characteristic observed in many solid tumors. Increased levels of one ECM component-namely, hyaluronan (HA)-leads to reduced elasticity of tumor tissue and increased interstitial fluid pressure. Multiple initial reports showed that the addition of hyaluronidase (HYAL) to chemotherapeutic regimens could greatly improve efficacy. Unfortunately, the bovine HYAL used in those studies was limited therapeutically by immunologic responses to treatment. Newly developed recombinant human HYAL has recently been introduced into clinical trials. In this article, we describe the role of HA in cancer, methods of targeting HA, and clinical studies performed to date, and we propose that targeting HA could now be an effective treatment option for patients with many different types of solid tumors.

Hypoxia Triggers Hedgehog-Mediated Tumor–Stromal Interactions in Pancreatic Cancer

Pancreatic cancer is characterized by a desmoplastic reaction that creates a dense fibroinflammatory microenvironment, promoting hypoxia and limiting cancer drug delivery due to decreased blood perfusion. Here, we describe a novel tumor-stroma interaction that may help explain the prevalence of desmoplasia in this cancer. Specifically, we found that activation of hypoxia-inducible factor-1α (HIF-1α) by tumor hypoxia strongly activates secretion of the sonic hedgehog (SHH) ligand by cancer cells, which in turn causes stromal fibroblasts to increase fibrous tissue deposition. In support of this finding, elevated levels of HIF-1α and SHH in pancreatic tumors were determined to be markers of decreased patient survival. Repeated cycles of hypoxia and desmoplasia amplified each other in a feed forward loop that made tumors more aggressive and resistant to therapy. This loop could be blocked by HIF-1α inhibition, which was sufficient to block SHH production and hedgehog signaling. Taken together, our findings suggest that increased HIF-1α produced by hypoxic tumors triggers the desmoplasic reaction in pancreatic cancer, which is then amplified by a feed forward loop involving cycles of decreased blood flow and increased hypoxia. Our findings strengthen the rationale for testing HIF inhibitors and may therefore represent a novel therapeutic option for pancreatic cancer.

Improved procedure for fluorescence in situ hybridization on tissue microarrays

BACKGROUND The recently developed tissue microarray (TMA) technology allows the arrangement of up to a thousand tissue specimens on a single microscope slide. This technology enables researchers to perform gene copy number studies on very large series of archival formalin-fixed tissues using fluorescence in situ hybridization (FISH). However, the hybridization properties of individual archival specimens can vary considerably. Therefore a highly optimized protocol is needed to fulfill the task of producing evaluable hybridization signals simultaneously in hundreds of specimens in a TMA. METHODS The performance of two different FISH protocols, the standard protocol for paraffin embedded tissues and our new optimized protocol, was tested on TMAs using probes for the HER-2 and ZNF217 genes as well as the chromosome 17 centromere. RESULTS The new protocol resulted in greatly increased signal intensity and an almost 30% increase in the number of tissue samples with evaluable hybridization signals. CONCLUSIONS Our improved protocol for FISH on TMAs provides standardized hybridization conditions leading to high-quality hybridization signals in the majority of specimens. The increases in the signal intensity and the number of evaluable samples are extremely important for the successful analyses of TMAs by FISH and will allow the utilization of the TMA technology in its full potential.