Ashleigh Pulkoski-Gross

Targeting matrix metalloproteinases in cancer: Bringing new life to old ideas

Since the identification of matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, as being a driving factor for cancer progression and patient prognosis, MMPs have been studied extensively. Although early programs targeting MMPs were largely unsuccessful in clinical trials, they remain a viable and highly desirable therapeutic target based on preclinical studies and their role in disease progression. As information regarding the structure and function of these proteinases is compiled and biotechnology evolves, tools to develop better inhibitors are within our grasp. Improved methods for high throughput screening and in silico drug design programs have identified compounds which are highly potent, have high binding affinities, and exhibit favorable pharmacokinetic profiles. More recently, advances in drug delivery methods or compounds which bind outside the active site have brought new light to the field. In this review, we highlight the role of MMPs in cancer, clinical trials for MMP inhibitors, and novel approaches to targeting MMPs in cancer.

Inhibition of matrix metalloproteinase-14 (MMP-14)-mediated cancer cell migration

Matrix metalloproteinases (MMPs) have been shown to be key players in both extracellular matrix remodeling and cell migration during cancer metastasis. MMP-14, a membrane-anchored MMP, in particular, is closely associated with these processes. The hemopexin (PEX) domain of MMP-14 has been proposed as the modulating region involved in the molecular cross-talk that initiates cell migration through homodimerization of MMP-14 as well as heterodimerization with the cell surface adhesion molecule CD44. In this study, minimal regions required for function within the PEX domain were investigated through a series of substitution mutations. Blades I and IV were found to be involved in cell migration. We found that blade IV is necessary for MMP-14 homodimerization and that blade I is required for CD44 MMP-14 heterodimerization. Cross-talk between MMP-14 and CD44 results in phosphorylation of EGF receptor and downstream activation of the MAPK and PI3K signaling pathways involved in cell migration. Based on these mutagenesis analyses, peptides mimicking the essential outermost strand motifs within the PEX domain of MMP-14 were designed. These synthetic peptides inhibit MMP-14-enhanced cell migration in a dose-dependent manner but have no effect on the function of other MMPs. Furthermore, these peptides interfere with cancer metastasis without affecting primary tumor growth. Thus, targeting the MMP-14 hemopexin domain represents a novel approach to inhibit MMP-14-mediated cancer dissemination.

Unraveling the Role of KIAA1199, a Novel Endoplasmic Reticulum Protein, in Cancer Cell Migration

BACKGROUND Cell migration is a critical determinant of cancer metastasis, and a better understanding of the genes involved will lead to the identification of novel targets aimed at preventing cancer dissemination. KIAA1199 has been shown to be upregulated in human cancers, yet its role in cancer progression was hitherto unknown. METHODS Clinical relevance was assessed by examining KIAA1199 expression in human cancer specimens. In vitro and in vivo studies were employed to determine the function of KIAA1199 in cancer progression. Cellular localization of KIAA1199 was microscopically determined. SNAP-tag pull-down assays were used to identify binding partner(s) of KIAA1199. Calcium levels were evaluated using spectrofluorometric and fluorescence resonance energy transfer analyses. Signaling pathways were dissected by Western blotting. Student t test was used to assess differences. All statistical tests were two-sided. RESULTS KIAA1199 was upregulated in invasive breast cancer specimens and inversely associated with patient survival rate. Silencing of KIAA1199 in MDA-MB-435 cancer cells resulted in a mesenchymal-to-epithelial transition that reduced cell migratory ability in vitro (75% reduction; P < .001) and decreased metastasis in vivo (80% reduction; P < .001). Gain-of-function assays further demonstrated the role of KIAA1199 in cell migration. KIAA1199-enhanced cell migration required endoplasmic reticulum (ER) localization, where it forms a stable complex with the chaperone binding immunoglobulin protein (BiP). A novel ER-retention motif within KIAA1199 that is required for its ER localization, BiP interaction, and enhanced cell migration was identified. Mechanistically, KIAA1199 was found to mediate ER calcium leakage, and the resultant increase in cytosolic calcium ultimately led to protein kinase C alpha activation and cell migration. CONCLUSIONS KIAA1199 serves as a novel cell migration-promoting gene and plays a critical role in maintaining cancer mesenchymal status.

Repurposing the antipsychotic trifluoperazine as an antimetastasis agent.

Because cancer cell invasion is a critical determinant of metastasis, targeting invasion is a viable approach to prevent metastasis. Utilizing a novel three-dimensional high-throughput invasion assay, we screened a National Cancer Institute compound library and discovered compounds demonstrating inhibitory effects on cancer cell invasion. One hit, trifluoperazine, suppresses invasion of human cancer cell lines while displaying a limited cytotoxicity profile. This inhibition is due to the interference with cancer cell migratory ability but not proteolytic activity. Treatment of cancer cells with trifluoperazine significantly reduces angiogenesis and prevents cancer cell invasion through a chorioallantoic basement membrane. Mechanistically, treatment results in decreased phosphorylated AKT (Ser473 and Thr308) and β-catenin (Ser552). Lack of phosphorylation of Ser552 of β-catenin prevents β-catenin nuclear relocation, resulting in decreased expression of vascular endothelial growth factor, likely mediated through dopamine receptor D2. Taken together, we demonstrated that trifluoperazine is responsible for reducing the angiogenic and invasive potential of aggressive cancer cells through dopamine receptor D2 to modulate the β-catenin pathway and propose that trifluoperazine may be used as an antimetastasis chemotherapeutic.

Conversion of Stationary to Invasive Tumor Initiating Cells (TICs): Role of Hypoxia in Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) Trafficking

Emerging evidence has implicated the role of tumor initiating cells (TICs) in the process of cancer metastasis. The mechanism underlying the conversion of TICs from stationary to invasive remains to be characterized. In this report, we employed less invasive breast cancer TICs, SK-3rd, that displays CD44high/CD24low with high mammosphere-forming and tumorigenic capacities, to investigate the mechanism by which stationary TICs are converted to invasive TICs. Invasive ability of SK-3rd TICs was markedly enhanced when the cells were cultured under hypoxic conditions. Given the role of membrane type 1-matrix metalloproteinase (MT1-MMP) in cancer invasion/metastasis, we explored a possible involvement of MT1-MMP in hypoxia-induced TIC invasion. Silencing of MT1-MMP by a shRNA approach resulted in diminution of hypoxia-induced cell invasion in vitro and metastasis in vivo. Under hypoxic conditions, MT1-MMP redistributed from cytoplasmic storage pools to the cell surface of TICs, which coincides with the increased cell invasion. In addition, CD44, a cancer stem-like cell marker, inversely correlated with increased cell surface MT1-MMP. Interestingly, cell surface MT1-MMP gradually disappeared when the hypoxia-treated cells were switched to normoxia, suggesting the plasticity of TICs in response to oxygen content. Furthermore, we dissected the pathways leading to upregulated MT1-MMP in cytoplasmic storage pools under normoxic conditions, by demonstrating a cascade involving Twist1-miR10b-HoxD10 leading to enhanced MT1-MMP expression in SK-3rd TICs. These observations suggest that MT1-MMP is a key molecule capable of executing conversion of stationary TICs to invasive TICs under hypoxic conditions and thereby controlling metastasis.

Epithelial to Mesenchymal Transition (EMT) and Intestinal Tumorigenesis

Colorectal cancer is the world’s third most common cancer and has one of the highest rates of morbidity. From studying familial occurrences of colon cancer, significant advances were made in understanding the molecular basis of the disease, even those of a sporadic nature. While there have been great strides in understanding the generation of colorectal cancer, the largest determinant of morbidity, metastasis, is far less understood. Metastasis is primarily the cause of death of patients with colorectal cancer and more attention is being paid to the molecular determinants of cell invasion and migration, as these are critical steps in the metastatic cascade. The conversion of epithelial cells to mesenchymal-like ones that are capable of leaving the primary tumor site requires the coordination of many signal transduction pathways and changes in transcriptional activity. These reversible changes are referred to as epithelial-to-mesenchymal transition (EMT). As EMT is a crucial program that determines metastatic capacity, recent efforts have been made to identify inhibitors of EMT in order to augment current chemotherapies and improve patient outcome.

Regulation of Cancer Cell Metabolism by Hypoxia

The growth of a tumor usually results in the development of hypoxia, which is primarily a consequence of the tumor outgrowing existing vasculature and the disorganized nature of vascular growth induced by the tumor itself. The low oxygen tension at the site of neoplastic growth has a significant effect on the metabolic status of the cells involved. In order for the cells to survive the harsh conditions of low oxygen and nutrition, the metabolism of the cell switches from an aerobic type of metabolism to an anaerobic one, relying primarily on glycolysis for the production of energy and metabolic intermediates that feed various biosynthetic pathways. Because this phenotype is associated with increased cell survival, drug resistance, and ultimately poor patient prognosis, the metabolic components and the mediators of the hypoxic response are viable targets in the war on cancer. Currently, a variety of drugs are being explored that influence the mediators of the hypoxic response, such as hypoxia-inducible factor-1 (HIF-1), and those that target metabolic enzymes directly. These agents show promise in improving the current standard of care by acting in a synergistic manner with current cancer therapies.

Abstract 4313: A novel endoplasmic reticulum protein promotes cancer invasion/metastasis

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Although considerable progress has been made in our understanding of cancer cell invasion, much of this complex cellular process remains a mystery, ultimately hindering advancements in treatment strategies targeting metastasis. Here we report the vital role of a novel endoplasmic reticulum (ER) resident protein identified by a PCR-subtraction hybridization technique, designated Cancer ER Invasion Gene (CERIG), in cancer cell migration and invasion. Clinical relevance of CERIG is highlighted by upregulation of CERIG in human invasive breast cancer specimens examined by immunohistochemistry and real-time RT-PCR, as well as increased expression in various forms of human cancer as determined by data mining. This up-regulation of CERIG correlates with poor prognosis of patients with breast cancer assessed by a DNA microarray data-mining approach from three publicly available cohorts containing a total of 696 breast cancer patients. Mechanistically, increased CERIG in solid tumors is linked to hypoxia-induced activation of CERIG promoter via the hypoxia-inducible factor-2α. Silencing of CERIG in an aggressive breast cancer cell line disrupted mesenchymal morphogenesis in vitro, resulting in a mesenchymal-to-epithelial transition, and interfered with cancer metastasis in vivo. Employing both gain- and loss-of-function approaches, CERIG was found to induce cancer cell migration and invasion. Enhanced cell migration required CERIG retention within the ER, where this 160 kDa protein forms a complex with an ER chaperone protein, GRP78/BiP. The minimal protein domain required for ER retention was identified using a mutagenesis approach. Furthermore, fusion of the ER-retention domain to a soluble matrix metalloproteinase (MMP) prevents the secretion of the MMP and retained it in the ER. Overexpression of CERIG in cells results in diminished ER and increased cytoplasmic calcium levels, suggesting a role for CERIG in ER calcium leakage. Furthermore, chelating intracellular calcium abrogates CERIG induced cell migration. The induction of cell migration via CERIG was found to involve a CERIG-BiP-ER calcium leakage-PKC-MAPK pathway. Our data indicate that CERIG, as a novel ER resident protein, plays an important role in cancer progression, serving as a novel cell migration-promoting gene. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4313. doi:1538-7445.AM2012-4313