Bonnie F. Sloane

Enhanced levels of cathepsin B mRNA in murine tumors

Relative amounts of mRNA for cathepsin B were measured in normal murine liver and three murine tumors, an invasive liver tumor (hepatoma, Hepa cl 9) and two melanoma variants (B16‐F1 and B16 amelanotic melanoma, B16a). Using a human cDNA to the cathepsin B coding region as a hybridization probe, we detected two species of cathepsin B specific RNA transcripts (2.2 and 4.1 kb) in total RNA preparation of all four tissues. The concentrations of the 2.2 and 4.1 kb species were 3.6 and 2.7‐fold greater in the highly metastatic B16a melanoma than in normal liver. The concentration of the 2.2 kb species in the invasive hepatoma wa 1.7‐fold greater than in normal liver. The increased levels of the 2.2 kb message were reflected in increases in activity of cathepsin B in both Hepa cl 9 and B16a.

Prostacyclin/Thromboxanes and Tumor Cell Metastasis

The ability to metastasize is a characteristic which distinguishes benign from malignant neoplasms. Metastasis can be simply defined as a loss of contiguity between a tumor cell or a clump of tumor cells and the primary tumor with successful transfer to, and growth at, a spatially separate site. The overall process can be regarded as a series of sequential events representing complex interactions between the tumor cell and the host (Figure 1).

Calcium Channel Blockers: Inhibitors of Tumor Cell-Platelet-Endothelial Cell Interactions

The movement of calcium ions across cellular membranes (e. g., plasma membrane, endoplasmic or sarcoplasmic reticulum) can serve as a molecular messenger that modulates biochemical processes appropriate to the specific cell type. Calcium antagonists by preventing release of Ca2+ from intracellular stores, by peventing influx of external Ca2+ or by inhibiting the effects of Ca2+ through interactions with Ca2+-binding proteins such as calmodulin can interfere with these biochemical functions. The primary action of one subgroup of calcium antagonists is to interact with the channels in the cell membrane through which calcium enters the cells. This class of compounds originally described by Fleckenstein has been called by a variety of names including Ca2+-channel blockers, Ca2+-entry blockers, Ca2+-channel inhibitors and slow-channel blockers (for review see 1–3). We will use the terminology calcium channel blockers (CCB) in this chapter.

Proteolytic Enzymes and Arachidonic Acid Metabolites

Borgeat and Sirois (1) have summarized the relationship between arachidonic acid (20:4) metabolites and lysosomal enzyme release thusly: agents which increase cellular levels of cAMP inhibit lysosomal enzyme release, whereas agents which increase cGMP induce release. In 3T3L1 fibroblasts the most potent stimulator of increased cAMP is PGI2 followed by PGE1 (2). However, the relative potency of the prostaglandins for cAMP accumulation appears to be a function of the cell type. Thromboxane A2 prevents an increase in cellular cAMP due to an external stimulus (3). In contrast to the cyclooxygenase products (PGI2, PGE1, etc.), products of the lipoxygenase pathway have been shown to increase cellular cGMP (4).

Cathepsin B-Like Cysteine Proteinases and Metastasis

During the process of metastasis tumor cells traverse several extracellular matrix barriers in order to gain entry to the vascular space at the site of the primary tumor and to the perivascular space at the sites of metastatic tumors. A number of in vitro model systems have been designed to study tumor cell invasion through extracellular matrices. Several of these model systems including systems to study the concomitant digestion of these matrices by tumor cells are discussed elsewhere in this volume (see Chapters 18–21). A focus of much recent research is the basement membrane which underlies endothelial cells. The physical location of the basement membrane suggests that it must be traversed for tumor cells to form hematogenous metastases. Since the basement membrane in contrast to other extracellular matrix barriers contains type IV collagen a number of investigators have hypothesized that tumor cells must be able to degrade type IV collagen in order to invade through the basement membrane (see also Chapter 19). Metastatic variants of the B16 melanoma have been shown to secrete a type IV collagenase (1). However, type IV collagenase is secreted by tumor cells in a latent form which requires activation (2) suggesting that type IV collagenase is not by itself sufficient for basement membrane invasion by tumor cells and that additional proteinases or a proteolytic cascade may participate in basement membrane invasion.

Tumor Cysteine Proteinases, Platelet Aggregation and Metastasis

Cysteine proteinases are a subclass of endopeptidases which require activation by thiol reagents (1). This group of enzymes includes the plant proteinase papain, the lysosomal cysteine proteinases (cathepsins B, H and L) and the cytosolic calcium-activated neutral proteinases (CANP). Sequence homologies among the cysteine proteinases suggest that they may have a common evolutionary origin. Takio et al. (2) compared the amino acid sequences of papain and of rat liver cathepsins B and H and found substantial sequence homologies among the three. Surrounding the active site cysteine, cathepsin B has 10 of 11 and cathepsin H 9 of 11 amino acids found in papain. Overall, however, cathepsin H is more closely homologous to papain than to cathepsin B. A peptide containing the active site cysteine residue of chicken skeletal muscle CANP was isolated and found to consist of 7 residues of which 3 are common to the active sites of papain and cathepsin B (3) and cathepsin H (2). Bajkowski and Frankfater (4, 5) have provided evidence that there are functional homologies as well as structural homologies between the active sites of cathepsin B and papain.

Abstract B28: Induced expression of Sprouty4 in breast invasive ductal carcinoma cells inhibits ERK MAP kinase and reduces malignant phenotype

Our previous work has identified increased expression of Sprouty4 in 3D models of breast ductal carcinoma in situ (DCIS). Sprouty4 in other systems has been shown to function as a negative regulator of the mitogen activated protein kinase (MAPK/ERK) pathway. We hypothesize that Sprouty4 is an endogenous inhibitor of ERK/MAPK signaling in DCIS, and that its loss or reduced expression is one mechanism by which triple-negative lesions progress toward invasive ductal carcinoma (IDC). Using immunohistochemistry our labs have found that Sprouty4 was highly expressed in some human premalignant breast tissue samples, and that this expression was reduced in malignant triple-negative samples. These results correspond with immunoblot data from our 3D culture model of breast cancer progression in which Sprouty4 expression was higher during DCIS than in the IDC stage. Efficient over-expression of Sprouty4 reduced both MAPK/ERK activity as well as the aggressive phenotype of MCF10.CA1d IDC cells. Immunofluorescence experiments done in IDC cells overexpressing Sprouty4 revealed relocation of E-cadherin back to the cell surface and the restoration of adherens junctions. To determine if these effects were due to changes in MAPK/ERK signaling, IDC cells were treated with MEK162, an allosteric MEK inhibitor. Nanomolar concentrations of drug restored an epithelial-like phenotype similar to that produced by Sprouty4 over-expression. From these data we conclude that Sprouty4 may act to control MAPK/ERK signaling in a subset of DCIS, thus limiting the progression of these premalignant breast cancers. Further, if in vivo data correspond with our in vitro work, this may argue for the investigation of MEK inhibitors as an adjunct treatment in triple-negative disease, where options are limited. Citation Format: Ryan M. Jackson, Ethan J. Brock, Seema Shah, Mansoureh Sameni, Bonnie F. Sloane, Quanwen Li, Raymond R. Mattingly. Induced expression of Sprouty4 in breast invasive ductal carcinoma cells inhibits ERK MAP kinase and reduces malignant phenotype. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B28.