David S. Zuzga

VEGF links hippocampal activity with neurogenesis, learning and memory

An enriched environment is associated with hippocampal plasticity, including improved cognitive performance and increased neurogenesis. Here, we show that hippocampal expression of vascular endothelial growth factor (VEGF) is increased by both an enriched environment and performance in a spatial maze. Hippocampal gene transfer of VEGF in adult rats resulted in ∼2 times more neurogenesis associated with improved cognition. In contrast, overexpression of placental growth factor, which signals through Flt1 but not kinase insert domain protein receptors (KDRs), had negative effects on neurogenesis and inhibited learning, although it similarly increased endothelial cell proliferation. Expression of a dominant-negative mutant KDR inhibited basal neurogenesis and impaired learning. Coexpression of mutant KDR antagonized VEGF-enhanced neurogenesis and learning without inhibiting endothelial cell proliferation. Furthermore, inhibition of VEGF expression by RNA interference completely blocked the environmental induction of neurogenesis. These data support a model in which VEGF, acting through KDR, mediates the effect of the environment on neurogenesis and cognition.

Tumor Epithelial Cell Matrix Metalloproteinase 9 Is a Target for Antimetastatic Therapy in Colorectal Cancer

Background: The current paradigm suggests that matrix metalloproteinase 9 (MMP-9) expressed by stromal cells is a therapeutic target in human colorectal tumors which presumably regulates metastatic disease progression. Conversely, whereas cancer cells within those tumors may induce stromal cells to produce MMP-9 and may be targets for MMP-9 activity, they are not the source of MMP-9 underlying metastasis. Methods: MMP-9 expression in matched colorectal tumors and normal adjacent mucosa from patients and human colon cancer cell lines was examined by real-time reverse transcription-PCR, laser capture microdissection, immunoelectron microscopy, and immunoblot analysis. The role of colon cancer cell MMP-9 in processes underlying metastasis was explored in vitro by examining degradation of extracellular matrix components by gelatin zymography and formation of locomotory organelles by cell spreading analysis and in vivo by quantifying hematogenous tumor cell seeding of mouse lungs. Results: Primary colorectal tumors overexpress MMP-9 compared with matched normal adjacent mucosa. In contrast to the current paradigm, MMP-9 is expressed equally by cancer and stromal cells within human colon tumors. Cancer cell MMP-9 regulates metastatic behavior in vitro, including degradation of extracellular matrix components and formation of locomotory organelles. Moreover, this MMP-9 critically regulates hematogenous seeding of mouse lungs by human colon cancer cells in vivo. Conclusions: These observations reveal that MMP-9 produced by human colon cancer, rather than stromal, cells is central to processes underlying metastasis. They underscore the previously unrecognized potential of specifically targeting tumor cell MMP-9 in interventional strategies to reduce mortality from metastatic colorectal cancer.

The Paracrine Hormone Hypothesis of Colorectal Cancer

Colorectal carcinogenesis originates in the context of dysregulated epithelial cell homeostasis, wherein hyperproliferation, hypodifferentiation, metabolic reprogramming, and mesenchymal remodeling reflect recursive mutually reinforcing mechanisms contributing to progressive genomic instability. Although genotypic and phenotypic elements characterizing the terminal integration of these pathophysiological processes defining cancer are well enumerated, events initiating, coordinating, and sustaining this hierarchical maladaptive systems evolution remain elusive for most tumors. In the intestine, guanylyl cyclase C (GCC) and its paracrine ligands organize and regulate the homeostatic integrity of the crypt–villus axis, forming a hormonal tumor suppressor signaling sequence, whose dysfunction defines the initiation of neoplastic transformation and creates a permissive niche for tumor progression.

Guanylyl cyclase C prevents colon cancer metastasis by regulating tumor epithelial cell matrix metalloproteinase-9.

Matrix metalloproteinase-9 (MMP-9) produced by colorectal cancer cells is a critical determinant of metastatic disease progression and an attractive target for antimetastatic strategies to reduce colon cancer mortality. Cellular signaling by cyclic GMP (cGMP) regulates MMP-9 dynamics in various cell systems, and the bacterial enterotoxin receptor guanylyl cyclase C (GCC), the principle source of cGMP in colonocytes, which is overexpressed in colorectal cancers, inhibits tumor initiation and progression in the intestine. Here, we show that ligand-dependent GCC signaling through cGMP induces functional remodeling of cancer cell MMP-9 reflected by a compartmental redistribution of this gelatinase, in which intracellular retention resulted in reciprocal extracellular depletion. Functional remodeling of MMP-9 by GCC signaling reduced the ability of colon cancer cells to degrade matrix components, organize the actin cytoskeleton to form locomotory organelles and spread, and hematogenously seed distant organs. Of significance, GCC effects on cancer cell MMP-9 prevented establishment of metastatic colonies by colorectal cancer cells in the mouse peritoneum in vivo. Because endogenous hormones for GCC are uniformly deficient in intestinal tumors, reactivation of dormant GCC signaling with exogenous administration of GCC agonists may represent a specific intervention to target MMP-9 functions in colon cancer cells. The notion that GCC-mediated regulation of cancer cell MMP-9 disrupts metastasis, in turn, underscores the unexplored utility of GCC hormone replacement therapy in the chemoprevention of colorectal cancer progression.

Phosphorylation of Vasodilator-Stimulated Phosphoprotein Ser239 Suppresses Filopodia and Invadopodia in Colon Cancer

In colorectal cancer, the antitumorigenic guanylyl cyclase C (GCC) signalome is defective reflecting ligand deprivation from downregulation of endogenous hormone expression. Although the proximal intracellular mediators of that signal transduction system, including cyclic guanosine monophosphate (cGMP) and cGMP‐dependent protein kinase (PKG), are well characterized, the functional significance of its distal effectors remain vague. Dysregulation of ligand‐dependent GCC signaling through vasodilator‐stimulated phosphoprotein (VASP), an actin‐binding protein implicated in membrane protrusion dynamics, drastically reduced cGMP‐dependent VASP phosphorylation levels in colorectal tumors from patients. Restoration of cGMP‐dependent VASP phosphorylation by GCC agonists suppressed the number and length of locomotory (filopodia) and invasive (invadopodia) actin‐based organelles in human colon cancer cells. Membrane organelle disassembly reflected specific phosphorylation of VASP Ser239, the cGMP/PKG preferred site, and rapid VASP removal from tumor cell protrusions. Importantly, VASP Ser239 phosphorylation inhibited the proteolytic function of invadopodia, reflected by suppression of the cancer cell ability to digest DQ‐collagen IV embedded in Matrigel. These results demonstrate a previously unrecognized role for VASP Ser239 phosphorylation, a single intracellular biochemical reaction, as an effective mechanism which opposes tumor cell shape promoting colon cancer invasion and metastasis. Reconstitution of physiological cGMP circuitry through VASP, in turn, represents an attractive targeted approach for patients with colorectal cancer.

Colorectal Cancer Is a Paracrine Deficiency Syndrome Amenable to Oral Hormone Replacement Therapy

The most commonly lost gene products in colorectal carcinogenesis include the paracrine hormones guanylin and uroguanylin, the endogenous ligands for guanylyl cyclase C (GCC), the intestinal receptor for diarrheagenic bacterial enterotoxins. Recently, GCC‐cyclic guanosine monophosphate (GMP) signaling has emerged as a principal regulator of proliferation, genetic integrity, and metabolic programming in normal human enterocytes and colon cancer cells. Elimination of GCC in mice produced hyperplasia of the proliferating compartment associated with increases in the rapidly cycling progenitor cells and reprogrammed enterocyte metabolism, with a shift from oxidative phosphorylation to glycolysis. In addition, in the colons of mice carrying mutations in adenomatous polyposis coli gene Apc (ApcMin/+) or exposed to the carcinogen azoxymethane, elimination of GCC increased tumor initiation and promotion by disrupting genomic integrity and releasing cell cycle restriction. These previously unrecognized roles for GCC as a fundamental regulator of intestinal homeostasis and as an intestinal tumor suppressor suggest that receptor dysregulation reflecting paracrine hormone insuffciency is a key event during the initial stages of colorectal tumorigenesis. Together with the uniform overexpression of GCC in human tumors, these novel roles for GCC underscore the potential of oral replacement with GCC ligands for a targeted prevention and therapy of colorectal cancer.

Enterotoxin preconditioning restores calcium-sensing receptor-mediated cytostasis in colon cancer cells

Guanylyl cyclase C (GCC), the receptor for diarrheagenic bacterial heat-stable enterotoxins (STs), inhibits colorectal cancer cell proliferation by co-opting Ca(2+) as the intracellular messenger. Similarly, extracellular Ca(2+) (Ca(2+)(o)) opposes proliferation and induces terminal differentiation in intestinal epithelial cells. In that context, human colon cancer cells develop a phenotype characterized by insensitivity to cytostasis imposed by Ca(2+)(o). Here, preconditioning with ST, mediated by GCC signaling through cyclic nucleotide-gated channels, restored Ca(2+)(o)-dependent cytostasis, reflecting posttranscriptional regulation of calcium-sensing receptors (CaRs). ST-induced GCC signaling deployed CaRs to the surface of human colon cancer cells, whereas elimination of GCC signaling in mice nearly abolished CaR expression in enterocytes. Moreover, ST-induced Ca(2+)(o)-dependent cytostasis was abrogated by CaR-specific antisense oligonucleotides. Importantly, following ST preconditioning, newly expressed CaRs at the cell surface represented tumor cell receptor targets for antiproliferative signaling by CaR agonists. Since expression of the endogenous paracrine hormones for GCC is uniformly lost early in carcinogenesis, these observations offer a mechanistic explanation for the Ca(2+)(o)-resistant phenotype of colon cancer cells. Restoration of antitumorigenic CaR signaling by GCC ligand replacement therapy represents a previously unrecognized paradigm for the prevention and treatment of human colorectal cancer employing dietary Ca(2+) supplementation.

Overexpression of matrix metalloproteinase 9 in tumor epithelial cells correlates with colorectal cancer metastasis.

Colorectal cancer mortality largely reflects metastasis, the spread of the disease to distant organs. Matrix metalloproteinase 9 (MMP‐9) is a key regulator of metastasis and a target for anticancer strategies in colon cancer. Here, the overexpression of MMP‐9 in pure tumor epithelial, but nor stromal, cell populations was associated with metastatic progression of colorectal cancer, as defined by reverse transcriptase‐polymerase chain reaction (qRT‐PCR) and confirmed by immunostaining. Thus, cancer cell MMP‐9 represents a novel, selective prognostic and predictive factor that may be exploited for more effective disease stage stratification and therapeutic regimen selection in patients with colorectal cancer.

Abstract 5805: Myosin 1e colocalization with β1-intergin and association with tumor progression in colorectal cancer

For colorectal cancer (CRC), the risk of relapse among stage II and stage III a/b patients is 20-30% and the benefit of treating these patients with chemotherapy is uncertain. As a result, many patients are undertreated, putting them at increased risk for disease relapse, or overtreated, exposing them to unnecessary and harmful chemotherapy with little potential benefit. Therefore, defining prognostic biomarkers to more accurately determine each person’s risk of relapse and need for chemotherapy is a priority in CRC research. Promising targets for biomarker discovery are actin-binding proteins. As a broad class, these proteins regulate the actin cytoskeleton and serve as direct, proximal regulators of invasive and metastatic phenotypes. Further, the prognostic utility of several of these proteins has recently been reported in the literature. Myosin 1e (Myo1e) a long-tailed, class I myosin, is one such protein with proposed clinical utility as a prognostic biomarker. In kidney podocyte cells, Myo1e regulates endocytosis, adhesion, migration, and invadosome dynamics. Despite its expression in numerous cancers, the functional role of Myo1e specifically in cancer cells and its association with tumor progression remains elusive. To define a role in cancer cells, the localization of Myo1e was examined in vitro by immunofluorescence in T84 human CRC cells. Myo1e co-localized with actin, cortactin and β1 integrin at membrane ruffles, which regulate integrin endocytosis and trafficking. Interestingly, both Myo1e and β1 integrin also co-localized with caveolin-1, a regulator of integrin endocytosis, suggesting Myo1e expression may regulate caveolae-dependent integrin endocytosis and trafficking. To evaluate potential prognostic utility, Myo1e expression in colorectal tumors and matched normal adjacent tissue (NAT) was examined by immunohistochemistry in a tissue microarray constructed from duplicate tissue cores from 119 CRC patients. The patient cohort was well balanced across TNM stage: stage 0 (10.1%), stage 1 (20.2%), stage 2 (26.9%), stage 3 (31.1%), and stage 4 (12.6%), and the majority (68.9%) of CRC tissues examined were of moderate tumor grade. The ratio of Myo1e expression in tumors compared to NAT was significantly correlated with clinicopathologic indicators of disease progression, including clinical stage, depth of invasion at the primary tumor (T-score), and lymph node metastasis. Together, these data suggest Myo1e expression correlates with tumor progression and may regulate invasive phenotypes through integrin trafficking pathways. Examining the prognostic utility and biologic function of Myo1e may a define a novel biomarker with translational utility for improved clinical management of colorectal cancer patients. Citation Format: Jeffrey Pfannenstein, Filippo Bori, Alessandro Bombonati, David Zuzga. Myosin 1e colocalization with β1-intergin and association with tumor progression in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5805. doi:10.1158/1538-7445.AM2017-5805

Abstract A8: Vasodilator-stimulated phosphoprotein Ser239 regulates matrix metalloproteinase-9 in colon cancer

Cancer invasion and spreading is promoted by membrane protrusion formation driven by actin polymerization and filament (F)-actin elongation, a process controlled by the actin-binding protein vasodilator-stimulated phosphoprotein (VASP). Tumor epithelial cell matrix metalloproteinase-9 (MMP-9) is a key promoter of carcinogenesis and invasion in colon cancer. Interestingly, both VASP and MMP-9 activities are regulated by intracellular cGMP signaling in colon cancer cells. However, direct functional relationships between VASP and MMP-9 are unrecognized. Here, human colon carcinoma cells were examined by immunoblotting, sucrose-gradient fractionation, confocal microscopy and imaging analysis. MMP-9 localizes at membrane protrusions and lipid rafts, cell surface microdomains specialized in signal transduction, in a cGMP-dependent manner. Activation of cGMP signaling disrupted membrane protrusion dynamics and induced intracellular MMP-9 redistribution. Strikingly, cancer cells expressing the serine-to-alanine VASP phosphomutant at Ser239, resistant to cGMP-dependent VASP phosphorylation, were insensitive to cGMP effects on membrane protrusions and MMP-9. Importantly, cGMP signaling through VASP Ser239, but not Ser157, critically regulates the ability of cancer cells to release MMP-9 in the extracellular environment. Together, these observations indicate tumor epithelial cell MMP-9, a key promoter of cancer invasion, is regulated by VASP Ser239 phosphorylation, a previously unappreciated paradigm in colon cancer biology. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A8.