James Warneke

Notch1 is an effector of Akt and hypoxia in melanoma development

Melanomas are highly aggressive neoplasms resistant to most conventional therapies. These tumors result from the interaction of altered intracellular tumor suppressors and oncogenes with the microenvironment in which these changes occur. We previously demonstrated that physiologic skin hypoxia contributes to melanomagenesis in conjunction with Akt activation. Here we show that Notch1 signaling is elevated in human melanoma samples and cell lines and is required for Akt and hypoxia to transform melanocytes in vitro. Notch1 facilitated melanoma development in a xenograft model by maintaining cell proliferation and by protecting cells from stress-induced cell death. Hyperactivated PI3K/Akt signaling led to upregulation of Notch1 through NF-kappaB activity, while the low oxygen content normally found in skin increased mRNA and protein levels of Notch1 via stabilization of HIF-1alpha. Taken together, these findings demonstrate that Notch1 is a key effector of both Akt and hypoxia in melanoma development and identify the Notch signaling pathway as a potential therapeutic target in melanoma treatment.

The stress-response proteins poly(ADP-ribose) polymerase and NF-κB protect against bile salt-induced apoptosis

Bile salts induce apoptosis and are implicated as promoters of colon cancer. The mechanisms by which bile salts produce these effects are poorly understood. We report that the cytotoxic bile salt, sodium deoxycholate (NaDOC), activates the key stress response proteins, NF-κB and poly(ADP-ribose) polymerase (PARP). The activation of NF-κB and PARP, respectively, indicates that bile salts induce oxidative stress and DNA damage. The pre-treatment of cells with specific inhibitors of these proteins [pyrrolidine dithiocarbamate (NF-κB inhibitor) and 3-aminobenzamide (PARP inhibitor)] sensitizes cells to the induction of apoptosis by NaDOC, indicating that these stress response pathways are protective in nature. Colon cancer risk has been reported to be associated with resistance to apoptosis. We found an increase in activated NF-κB at the base of human colon crypts that exhibit apoptosis resistance. This provides a link between an increased stress response and colon cancer risk. The implications of these findings with respect to apoptosis and to colon carcinogenesis are discussed.

A phase 2 trial of complete resection for stage IV melanoma: Results of Southwest Oncology Group Clinical Trial S9430

BACKGROUND On the basis of retrospective experience at individual centers, it appears that patients with stage IV melanoma who undergo complete resection have a favorable outcome compared with patients with disseminated stage IV disease. The Southwest Oncology Group (SWOG) performed a prospective trial in patients with metastatic melanoma who were enrolled before complete resection of their metastatic disease and provided prospective outcomes in the cooperative group setting. METHODS Based on their physical examination and radiologic imaging studies, patients with a stage IV melanoma judged amenable to complete resection underwent surgery within 28 days of enrollment. All eligible patients were followed with scans (computed tomography or positron emission tomography) every 6 months until relapse and death. RESULTS Seventy-seven patients were enrolled from 18 different centers. Of those, 5 patients were ineligible; 2 had stage III disease alone; and 3 had no melanoma in their surgical specimen. In addition, 8 eligible patients had incompletely resected tumor. Therefore, the primary analysis included 64 completely resected patients. Twenty patients (31%) had visceral disease. With a median follow-up of 5 years, the median relapse-free survival was 5 months (95% CI, 3-7 months) whereas median overall survival was 21 months (95% CI, 16-34 months). Overall survivals at 3 and 4 years were 36% and 31%, respectively. CONCLUSIONS In a prospective multicenter setting, appropriately selected patients with stage IV melanoma achieved prolonged overall survival after complete surgical resection. Although median relapse-free survival was only 5 months, patients could still frequently undergo subsequent surgery for isolated recurrences. This patient population is appropriate for aggressive surgical therapy and for trials evaluating adjuvant therapy.

Decreased expression of eukaryotic initiation factor 3f deregulates translation and apoptosis in tumor cells

The eukaryotic initiation factor 3f (eIF3f) is the p47 subunit of the multi-subunit eIF3 complex. eIF3 plays an important role in translation initiation. In the present study, we investigate the biological function of eIF3f in translation and apoptosis in tumor cells. We demonstrated for the first time that eIF3f is downregulated in most human tumors using a cancer profiling array and confirmed by real-time reverse transcription PCR in melanoma and pancreatic cancer. Overexpression of eIF3f inhibits cell proliferation and induces apoptosis in melanoma and pancreatic cancer cells. Silencing of eIF3f protects melanoma cells from apoptosis. We further investigated the biological function of eIF3f. In vitro translation studies indicate that eIF3f is a negative regulator of translation and that the region between amino acids 170 and 248 of eIF3f is required for its translation regulatory function. Ectopic expression of eIF3f inhibits translation and overall cellular protein synthesis. Ribosome profile and ribosomal RNA (rRNA) fragmentation assays revealed that eIF3f reduces ribosomes, which may be associated with rRNA degradation. We propose that eIF3f may play a role in ribosome degradation during apoptosis. These data provide critical insights into the cellular function of eIF3f and in linking translation initiation and apoptosis.

Abdominal wall hernias in patients with abdominal aortic aneurysmal versus aortoiliac occlusive disease

BACKGROUND This study was undertaken to determine the incidence of ventral incisional hernias (VIHs) and inguinal hernias (IHs) in patients with abdominal aortic aneurysmal (AAA) versus those with aortoiliac occlusive disease (AIOD). PATIENTS AND METHODS The medical records of 193 patients (128 with AAA and 65 with AIOD) who had undergone elective aortic reconstruction were reviewed to determine the number and location of abdominal wall hernias (AWHs). RESULTS Forty-one AWHs (28 IHs and 13 VIHs) were detected in patients with AAA compared to 13 (11 IHs and 2 VIHs) in patients with AIOD. There was a significantly greater incidence of VIHs in patients with AAA versus patients with AIOD (10% versus 3%, P < 0.05) and recurrent AWHs (28% versus 19%, P < 0.01), but not of IHs (22% versus 17%). CONCLUSION Patients with AAA have a higher incidence of VIHs and recurrent AWHs--without a corresponding increase in patient-related risk factors--than patients without aneurysm, suggesting that as yet unidentified etiologic factors may contribute to the development of AWHs in these patients.

Assessment of Apoptosis by Immunohistochemical Markers Compared to Cellular Morphology in Ex Vivo-stressed Colonic Mucosa

Apoptosis competence is central to the prevention of cancer. Frequency of apoptotic cells, after a sample of colonic tissue is stressed, can be used to gauge apoptosis competence and, thus, possible susceptibility to colon cancer. The gold standard for assessment of apoptosis is morphological evaluation, but this requires an experienced microscopist. Easier-to-use immunohistochemical markers of apoptosis, applicable in archived paraffin-embedded tissue, have been commercially developed. Potentially useful apoptosis markers include cleaved cytokeratin-18 (c-CK18), cleaved caspase-3 (c-cas-3), cleaved lamin A (c-lam-A), phosphorylated histone H2AX (γH2AX), cleaved poly(ADP ribose) polymerase (c-PARP), and translocation of apoptosis-inducing factor (AIF). When tissue samples from freshly resected colon segments were challenged ex vivo with the bile acid deoxycholate, ∼50% of goblet cells became apoptotic by morphologic criteria. This high level of morphologic apoptosis allowed quantitative comparison with the usefulness and specificity of immunohistochemical markers of apoptosis. The antibody to c-CK18 was almost as useful and about as specific as morphology for identifying apoptotic colonic epithelial cells. Antibodies to c-cas-3, c-lam-A, and γH2AX, though specific for apoptotic cells, were less useful. The antibody to c-PARP, though specific for apoptotic cells, had low usefulness, and the antibody to AIF was relatively nonspecific, under our conditions.

Investigating Sun-damaged Skin and Actinic Keratosis with Optical Coherence Tomography: A Pilot Study

Actinic Keratosis (AK) arises from sun-damaged skin and is the first clinical manifestation in the multistep process of skin carcinogenesis to invasive squamous cell carcinoma. Thus, it is an ideal target for chemopreventive efforts. Noninvasive measures of AK severity are needed to assess the efficacy of chemoprevention agents. We performed a pilot study on 20 participants to investigate the OCT appearance of sun-protected skin of the upper inner arm as well as sun-damaged skin and early AKs of the dorsal forearms, and to determine if features or quantitative measures in Optical Coherence Tomography (OCT) images could be used to reliably differentiate between these categories. OCT images of upper inner arm (normal appearing skin) showed skin layers and features (stratum corneum, epidermis, dermis, blood vessels) seen in previous studies; additionally in this participant group the subcutaneous fat layer was usually identified. Sun-damaged skin was characterized by increased signal in the epidermis and rapid attenuation of light. AKs were diverse in appearance but frequently characterized by high surface reflection, the presence of a low-signal band in the stratum corneum, and heterogeneous appearance in the epidermis/dermis. Significant differences were found between skin categories using measures of stratum corneum and epidermal/dermal depths and intensities. The presence of a dark band in the stratum corneum was 79% sensitive and 100% specific for AK. This study indicates that OCT holds promise as a useful technique for identifying and characterizing AKs and monitoring their response to chemoprevention agents.

Sequence motifs associated with hepatotoxicity of locked nucleic acid—modified antisense oligonucleotides

Fully phosphorothioate antisense oligonucleotides (ASOs) with locked nucleic acids (LNAs) improve target affinity, RNase H activation and stability. LNA modified ASOs can cause hepatotoxicity, and this risk is currently not fully understood. In vitro cytotoxicity screens have not been reliable predictors of hepatic toxicity in non-clinical testing; however, mice are considered to be a sensitive test species. To better understand the relationship between nucleotide sequence and hepatotoxicity, a structure–toxicity analysis was performed using results from 2 week repeated-dose-tolerability studies in mice administered LNA-modified ASOs. ASOs targeting human Apolipoprotien C3 (Apoc3), CREB (cAMP Response Element Binding Protein) Regulated Transcription Coactivator 2 (Crtc2) or Glucocorticoid Receptor (GR, NR3C1) were classified based upon the presence or absence of hepatotoxicity in mice. From these data, a random-decision forest-classification model generated from nucleotide sequence descriptors identified two trinucleotide motifs (TCC and TGC) that were present only in hepatotoxic sequences. We found that motif containing sequences were more likely to bind to hepatocellular proteins in vitro and increased P53 and NRF2 stress pathway activity in vivo. These results suggest in silico approaches can be utilized to establish structure–toxicity relationships of LNA-modified ASOs and decrease the likelihood of hepatotoxicity in preclinical testing.

Functional protein pathway activation mapping of the progression of normal skin to squamous cell carcinoma.

Reverse phase protein microarray analysis was used to identify cell signaling derangements in squamous cell carcinoma (SCC) compared with actinic keratosis (AK) and upper inner arm (UIA). We analyzed two independent tissue sets with isolation and enrichment of epithelial cells by laser capture microdissection. Set 1 served as a pilot and a means to identify protein pathway activation alterations that could be further validated in a second independent set. Set 1 was comprised of 4 AK, 13 SCC, and 20 UIA. Set 2 included 15 AK, 9 SCCs, and 20 UIAs. Activation of 51 signaling proteins, known to be involved in tumorigenesis, were assessed for set 1 and showed that the MEK–ERK [mitogen-activated protein (MAP)/extracellular signal-regulated (ERK; MEK)] pathway was activated in SCC compared with AK and UIA, and that epidermal growth factor receptor (EGFR) and mTOR pathways were aberrantly activated in SCC. Unsupervised two-way hierarchical clustering revealed that AK and UIA shared a common signaling network activation architecture while SCC was dramatically different. Statistical analysis found that prosurvival signaling through phosphorylation of ASK and 4EBP1 as well as increased Bax and Bak expression was higher in AK compared with UIA. We expanded pathway network activation mapping in set 2 to 101 key signaling proteins, which corroborated activation of MEK–ERK, EGFR, and mTOR pathways through discovery of a number of upstream and downstream signaling molecules within these pathways to conclude that SCC is indeed a pathway activation–driven disease. Pathway activation mapping of SCC compared with AK revealed several interconnected networks that could be targeted with drug therapy for potential chemoprevention and therapeutic applications. Cancer Prev Res; 5(3); 403–13. ©2012 AACR.

Aspiration pneumonia following surgically placed feeding tubes

BACKGROUND The enteral route is preferred in surgical patients requiring nutritional support; however, controversy surrounds the choice of location of feeding tube placement. Although jejunostomy has been commonly accepted as superior to gastrostomy for long-term nutritional support because of an assumed lower risk of aspiration pneumonia, recent studies suggest that reevaluation of common practices of surgical tube placement is warranted. PATIENTS AND METHODS We conducted a retrospective chart review of gastrostomy and jejunostomy procedures from 1986 to 1993. Demographic information and complications related to the procedure were reviewed. Aspiration pneumonia was defined as respiratory symptoms, leukocytosis, and infiltrate on chest radiograph. RESULTS Sixty-nine gastrostomies and 86 jejunostomies were performed during the study period. Six patients were diagnosed with aspiration pneumonia; 2 cases of which occurred with jejunostomy and 4 cases occurred with gastrostomy (P = not significant). CONCLUSIONS There was no difference in rates of pulmonary aspiration or other complications between gastrostomy and jejunostomy. We suggest that when a surgically placed feeding tube is required, the determination of appropriate procedure be based on clinical factors such as the technical difficulty of the operation or long-term feeding goals.