Lawrence M. Knab

Treatment Trends, Risk of Lymph Node Metastasis, and Outcomes for Localized Esophageal Cancer

BACKGROUND Endoscopic resection is increasingly used to treat localized, early-stage esophageal cancer. We sought to assess its adoption, characterize the risks of nodal metastases, and define differences in procedural mortality and 5-year survival between endoscopic and surgical resection in the United States. METHODS From the National Cancer Data Base, patients with T1a and T1b lesions were identified. Treatment patterns were characterized, and hierarchical regression methods were used to define predictors and evaluate outcomes. All statistical tests were two-sided. RESULTS Five thousand three hundred ninety patients were identified and underwent endoscopic (26.5%) or surgical resection (73.5%). Endoscopic resection increased from 19.0% to 53.0% for T1a lesions (P < .001) and from 6.6% to 20.9% for T1b cancers (P < .001). The strongest predictors of endoscopic resection were depth of invasion (T1a vs T1b: odds ratio [OR] = 4.45; 95% confidence interval [CI] = 3.76 to 5.27) and patient age of 75 years or older (vs age less than 55 years: OR = 4.86; 95% CI = 3.60 to 6.57). Among patients undergoing surgery, lymph node metastasis was 5.0% for T1a and 16.6% for T1b lesions. Predictors of nodal metastases included tumor size greater than 2 cm (vs. <2 cm) and intermediate-/high-grade lesions (vs low grade). For example, 0.5% of patients with low-grade T1a lesions less than 2 cm had lymph node involvement. The risk of 30-day mortality was less after endoscopic resection (hazard ratio [HR] = 0.33; 95% CI = 0.19 to 0.58) but greater for conditional 5-year survival (HR = 1.63; 95% CI = 1.07 to 2.47). CONCLUSIONS Endoscopic resection has become the most common treatment of T1a esophageal cancer and has increased for T1b cancers. It remains important to balance the risk of nodal metastases and procedural risk when counseling patients regarding their treatment options.

GLI2-dependent c-MYC upregulation mediates resistance of pancreatic cancer cells to the BET bromodomain inhibitor JQ1

JQ1 and I-BET151 are selective inhibitors of BET bromodomain proteins that have efficacy against a number of different cancers. Since the effectiveness of targeted therapies is often limited by development of resistance, we examined whether it was possible for cancer cells to develop resistance to the BET inhibitor JQ1. Here we show that pancreatic cancer cells developing resistance to JQ1 demonstrate cross-resistance to I-BET151 and insensitivity to BRD4 downregulation. The resistant cells maintain expression of c-MYC, increase expression of JQ1-target genes FOSL1 and HMGA2, and demonstrate evidence of epithelial-mesenchymal transition (EMT). However, reverting EMT fails to sensitize the resistant cells to JQ1 treatment. Importantly, the JQ1-resistant cells remain dependent on c-MYC that now becomes co-regulated by high levels of GLI2. Furthermore, downregulating GLI2 re-sensitizes the resistant cells to JQ1. Overall, these results identify a mechanism by which cancer cells develop resistance to BET inhibitors.

BET Bromodomain Inhibitors Block Growth of Pancreatic Cancer Cells in Three-Dimensional Collagen

Pancreatic ductal adenocarcinoma (PDAC) is associated with pronounced fibrosis that contributes to chemoresistance, in part, through increased histone acetylation. Because bromodomain (BRD) and extra terminal domain (BET) proteins are “readers” of histone acetylation marks, we targeted BET proteins in PDAC cells grown in three-dimensional collagen. We show that treatment with BET inhibitors decreases growth of PDAC cells (AsPC1, CD18, and Panc1) in collagen. Transfection with siRNA against BRD4, which is increased in human PDAC tumors, also decreases growth of PDAC cells. BET inhibitors additionally decrease growth in collagen of PDAC cells that have undergone epithelial-to-mesenchymal transition or have become resistant to chemotherapy. Although BET inhibitors and BRD4 siRNA repress c-MYC only in AsPC1 and CD18 cells, downregulating c-MYC decreases growth of all three PDAC cell lines in collagen. FOSL1, which is also targeted by BET inhibitors and BRD4 siRNA in AsPC1, CD18, and Panc1 cells, additionally regulates growth of all three PDAC cell lines in collagen. BET inhibitors and BRD4 siRNA repress HMGA2, an architectural protein that modulates chromatin state and also contributes to chemoresistance, in PDAC cells grown in collagen. Importantly, we show that there is a statistically significant correlation between BRD4 and HMGA2 in human PDAC tumors. Significantly, overexpression of HMGA2 partially mitigates the effect of BET inhibitors on growth and c-MYC and/or FOSL1 expression in collagen. Overall, these results demonstrate that BET inhibitors block growth of PDAC cells in collagen and that BET proteins may be potential targets for the treatment of pancreatic cancer. Mol Cancer Ther; 13(7); 1907–17. ©2014 AACR.

Involvement of eicosanoids in the pathogenesis of pancreatic cancer: the roles of cyclooxygenase-2 and 5-lipoxygenase.

The interplay between inflammation and cancer progression is a growing area of research. A combination of clinical, epidemiological, and basic science investigations indicate that there is a relationship between inflammatory changes in the pancreas and neoplastic progression. Diets high in ω-6 polyunsaturated fatty acids provide increased substrate for arachidonic acid metabolism by cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) to form eicosanoids. These eicosanoids directly contribute to pancreatic cancer cell proliferation. Both COX-2 and 5-LOX are upregulated in multiple cancer types, including pancreatic cancer. In vitro studies using pancreatic cancer cell lines have demonstrated upregulation of COX-2 and 5-LOX at both the mRNA and protein levels. When COX-2 and 5-LOX are blocked via a variety of mechanisms, cancer cell proliferation is abrogated both in vitro and in vivo. The mechanism of COX-2 has been shown to include effects on apoptosis as well as angiogenesis. 5-LOX has been implicated in apoptosis. The use of COX-2 and 5-LOX inhibitors in clinical studies in patients with pancreatic cancer has been limited. Patient enrollment has been restricted to those with advanced disease which makes evaluation of these drugs as chemopreventive agents difficult. COX-2 and 5-LOX expression have been shown to be present during the early neoplastic changes of pancreatic cancer, well before progression to invasive disease. This indicates that the ideal role for these interventions is early in the disease process as preventive agents, perhaps in patients with chronic pancreatitis or hereditary pancreatitis.

Zileuton, 5-lipoxygenase inhibitor, acts as a chemopreventive agent in intestinal polyposis, by modulating polyp and systemic inflammation.

Purpose Leukotrienes and prostaglandins, products of arachidonic acid metabolism, sustain both systemic and lesion-localized inflammation. Tumor-associated Inflammation can also contribute to the pathogenesis of colon cancer. Patients with inflammatory bowel disease (IBD) have increased risk of developing colon cancer. The levels of 5-lipoxygenase (5-LO), the key enzyme for leukotrienes production, are increased in colon cancer specimens and colonic dysplastic lesions. Here we report that Zileuton, a specific 5-LO inhibitor, can prevent polyp formation by efficiently reducing the tumor-associated and systemic inflammation in APCΔ468 mice. Experimental Design In the current study, we inhibited 5-LO by dietary administration of Zileuton in the APCΔ468 mouse model of polyposis and analyzed the effect of in vivo 5-LO inhibition on tumor-associated and systemic inflammation. Results Zileuton-fed mice developed fewer polyps and displayed marked reduction in systemic and polyp-associated inflammation. Pro-inflammatory cytokines and pro-inflammatory innate and adaptive immunity cells were reduced both in the lesions and systemically. As part of tumor-associated inflammation Leukotriene B4 (LTB4), product of 5-LO activity, is increased focally in human dysplastic lesions. The 5-LO enzymatic activity was reduced in the serum of Zileuton treated polyposis mice. Conclusions This study demonstrates that dietary administration of 5-LO specific inhibitor in the polyposis mouse model decreases polyp burden, and suggests that Zileuton may be a potential chemo-preventive agent in patients that are high-risk of developing colon cancer.

HOIL-1L functions as the PKCζ ubiquitin ligase to promote lung tumor growth

RATIONALE Protein kinase C zeta (PKCζ) has been reported to act as a tumor suppressor. Deletion of PKCζ in experimental cancer models has been shown to increase tumor growth. However, the mechanisms of PKCζ down-regulation in cancerous cells have not been previously described. OBJECTIVES To determine the molecular mechanisms that lead to decreased PKCζ expression and thus increased survival in cancer cells and tumor growth. METHODS The levels of expression of heme-oxidized IRP2 ubiquitin ligase 1L (HOIL-1L), HOIL-1-interacting protein (HOIP), Shank-associated RH domain-interacting protein (SHARPIN), and PKCζ were analyzed by Western blot and/or quantitative real-time polymerase chain reaction in different cell lines. Coimmunoprecipitation experiments were used to demonstrate the interaction between HOIL-1L and PKCζ. Ubiquitination was measured in an in vitro ubiquitination assay and by Western blot with specific antibodies. The role of hypoxia-inducible factor (HIF) was determined by gain/loss-of-function experiments. The effect of HOIL-1L expression on cell death was investigated using RNA interference approaches in vitro and on tumor growth in mice models. Increased HOIL-1L and decreased PKCζ expression was assessed in lung adenocarcinoma and glioblastoma multiforme and documented in several other cancer types by oncogenomic analysis. MEASUREMENTS AND MAIN RESULTS Hypoxia is a hallmark of rapidly growing solid tumors. We found that during hypoxia, PKCζ is ubiquitinated and degraded via the ubiquitin ligase HOIL-1L, a component of the linear ubiquitin chain assembly complex (LUBAC). In vitro ubiquitination assays indicate that HOIL-1L ubiquitinates PKCζ at Lys-48, targeting it for proteasomal degradation. In a xenograft tumor model and lung cancer model, we found that silencing of HOIL-1L increased the abundance of PKCζ and decreased the size of tumors, suggesting that lower levels of HOIL-1L promote survival. Indeed, mRNA transcript levels of HOIL-1L were elevated in tumor of patients with lung adenocarcinoma, and in a lung adenocarcinoma tissue microarray the levels of HOIL-1L were associated with high-grade tumors. Moreover, we found that HOIL-1L expression was regulated by HIFs. Interestingly, the actions of HOIL-1L were independent of LUBAC. CONCLUSIONS These data provide first evidence of a mechanism of cancer cell adaptation to hypoxia where HIFs regulate HOIL-1L, which targets PKCζ for degradation to promote tumor survival. We provided a proof of concept that silencing of HOIL-1L impairs lung tumor growth and that HOIL-1L expression predicts survival rate in cancer patients suggesting that HOIL-1L is an attractive target for cancer therapy.

Snail Cooperates with KrasG12D In Vivo to Increase Stem Cell Factor and Enhance Mast Cell Infiltration

Pancreatic ductal adenocarcinoma (PDAC) is associated with a pronounced fibro-inflammatory stromal reaction that contributes to tumor progression. A critical step in invasion and metastasis is the epithelial-to-mesenchymal transition (EMT), which can be regulated by the Snail family of transcription factors. Overexpression of Snail (Snai1) and mutant KrasG12D in the pancreas of transgenic mice, using an elastase (EL) promoter, resulted in fibrosis. To identify how Snail modulates inflammation in the pancreas, we examined the effect of expressing Snail in EL-KrasG12D mice (KrasG12D/Snail) on mast cell infiltration, which has been linked to PDAC progression. Using this animal model system, it was demonstrated that there are increased numbers of mast cells in the pancreas of KrasG12D/Snail mice compared with control KrasG12D mice. In addition, it was revealed that human primary PDAC tumors with increased Snail expression are associated with increased mast cell infiltration, and that Snail expression in these clinical specimens positively correlated with the expression of stem cell factor (SCF/KITLG), a cytokine known to regulate mast cell migration. Concomitantly, SCF levels are increased in the KrasG12D/Snail mice than in control mice. Moreover, overexpression of Snail in PDAC cells increased SCF levels, and the media conditioned by Snail-expressing PDAC cells promoted mast cell migration. Finally, inhibition of SCF using a neutralizing antibody significantly attenuated Snail-induced migration of mast cells. Implications: Together, these results elucidate how the EMT regulator Snail contributes to inflammation associated with PDAC tumors. Mol Cancer Res; 12(10); 1440–8. ©2014 AACR.

Cancer Cell Invasion in Three-dimensional Collagen Is Regulated Differentially by Gα13 Protein and Discoidin Domain Receptor 1-Par3 Protein Signaling.

Background: The role of Gα13 in mediating invasion in three-dimensional collagen is not well known. Results: Gα13 promotes invasion in three-dimensional collagen by disrupting cell-cell adhesion, whereas DDR1-Par3 signaling counteracts the effects of Gα13 by enhancing cell-cell adhesion. Conclusion: Gα13 and DDR1-Par3 differentially regulate cell-cell junctions to mediate three-dimensional invasion. Significance: Better understanding of how cells invade in three-dimensional collagen may identify potential therapeutic targets. Cancer cells can invade in three-dimensional collagen as single cells or as a cohesive group of cells that require coordination of cell-cell junctions and the actin cytoskeleton. To examine the role of Gα13, a G12 family heterotrimeric G protein, in regulating cellular invasion in three-dimensional collagen, we established a novel method to track cell invasion by membrane type 1 matrix metalloproteinase-expressing cancer cells. We show that knockdown of Gα13 decreased membrane type 1 matrix metalloproteinase-driven proteolytic invasion in three-dimensional collagen and enhanced E-cadherin-mediated cell-cell adhesion. E-cadherin knockdown reversed Gα13 siRNA-induced cell-cell adhesion but failed to reverse the effect of Gα13 siRNA on proteolytic invasion. Instead, concurrent knockdown of E-cadherin and Gα13 led to an increased number of single cells rather than groups of cells. Significantly, knockdown of discoidin domain receptor 1 (DDR1), a collagen-binding protein that also co-localizes to cell-cell junctions, reversed the effects of Gα13 knockdown on cell-cell adhesion and proteolytic invasion in three-dimensional collagen. Knockdown of the polarity protein Par3, which can function downstream of DDR1, also reversed the effects of Gα13 knockdown on cell-cell adhesion and proteolytic invasion in three-dimensional collagen. Overall, we show that Gα13 and DDR1-Par3 differentially regulate cell-cell junctions and the actin cytoskeleton to mediate invasion in three-dimensional collagen.

Minimally Invasive Therapies for Hepatic Malignancy

The emergence of laparoscopic cholecystectomy has revolutionized the face of surgery and proved that in the appropriate circumstances the laparoscopic technique affords comparable surgical outcomes with improvements in parameters such as blood loss, postoperative pain, and hospital length of stay. The first laparoscopic liver resection (LLR) was described in 1992, and since then LLRs have expanded from partial hepatectomies and wedge resections for benign peripheral lesions to left and right hepatectomies for malignant lesions, which are now being performed at large-volume tertiary care centers. Laparoscopic liver surgery technique can be divided into 3 main categories based on The Louisville Statement: pure laparoscopy, hand-assisted laparoscopy, and the hybrid technique. Pure laparoscopy involves performing the entire liver resection using only laparoscopic ports, with a small incision used for specimen extraction. Hand-assisted laparoscopy is defined as laparoscopy with the addition of a hand-port placed to facilitate the operation, and the hybrid technique refers to a procedure which is started as purely laparoscopic or hand-assisted, and the resection is performed through a small laparotomy incision. The majority of LLR reported in a 2000 case registry study were totally laparoscopic (75.1%), followed by hand-assisted (16.5%), and then the hybrid technique (2.1%).

Comparative effectiveness in esophagogastric cancer.

Cancer of the esophagus and the gastroesophageal junction (GEJ) continues to have a dismal prognosis, with the incidence of esophageal cancer increasing in the United States. Although radical resection was initially the primary treatment for this disease process, systemic chemotherapy and radiation have been shown to play a role in prolonging survival in most patient populations. This chapter explores the evidence that guides treatment for esophageal and GEJ cancer today. Chemotherapy and radiation therapy were introduced as treatment modalities for esophageal and GEJ cancers when it became evident that surgical therapy alone provided poor long-term survival rates. A variety of treatment strategies have been explored including preoperative (neoadjuvant) and postoperative (adjuvant) chemotherapy, with and without radiation. The evidence suggests that neoadjuvant chemotherapy or chemoradiotherapy provides better outcomes compared to surgery alone for esophageal, GEJ, and gastric cancers. Studies indicate a trend towards improved survival when neoadjuvant chemoradiotherapy is compared to chemotherapy alone. When patients have undergone resection with node-positive disease without receiving neoadjuvant therapy, some form of adjuvant treatment is recommended. This chapter also explores the surgical management of esophageal, GEJ, and gastric cancers including the extent of the gastric lymph node dissection. It also includes a discussion about adherence to national guidelines in terms of gastric cancer treatment and esophageal and gastric lymph node examinations.