Rama Khokha

Steps in tumor metastasis: new concepts from intravital videomicroscopy

SummaryMetastases are responsible for the majority of failures in cancer treatment. Clarifying steps in metastasis and their molecular mechanisms will be important for the development of anti-metastasis therapeutic strategies. Considerable progress has been made in identifying molecules involved in metastasis. However, because of the nature of assays that have been available, conclusions about steps in metastasis and their molecular bases have been drawn primarily from inference. In order to complete the picture of how metastases form, a technique is needed to directly watch the processin vivo as it occurs over time. We have developed an intravital videomicroscopy (IVVM) procedure to make such observations possible. Results from IVVM are providing us with new conceptual understanding of the metastatic process, as well as the nature and timing of the contributions of molecules implicated in metastasis (e.g. adhesion molecules and proteinases). Our findings suggest that early steps in metastasis, including hemodynamic destruction and extravasation, may contribute less to metastatic inefficiency than previously believed. Instead, our results suggest that the control of post-extravasation growth of individual cancer cells is a significant contributor to metastatic inefficiency. Thus, this stage may be an appropriate target for design of novel strategies to prevent metastases.

TIMP-3 deficiency leads to dilated cardiomyopathy.

Background—Despite the mounting clinical burden of heart failure, the biomolecules that control myocardial tissue remodeling are poorly understood. TIMP-3 is an endogenous inhibitor of matrix metalloproteinases (MMPs) that has been found to be deficient in failing human myocardium. We hypothesized that TIMP-3 expression prevents maladaptive tissue remodeling in the heart, and accordingly, its deficiency in mice would alone be sufficient to trigger progressive cardiac remodeling and dysfunction similar to human heart failure. Methods and Results—Mice with a targeted timp-3 deficiency were evaluated with aging and compared with age-matched wild-type littermates. Loss of timp-3 function triggered spontaneous LV dilatation, cardiomyocyte hypertrophy, and contractile dysfunction at 21 months of age consistent with human dilated cardiomyopathy. Its absence also resulted in interstitial matrix disruption with elevated MMP-9 activity, and activation of the proinflammatory tumor necrosis factor-&agr; cytokine system, molecular hallmarks of human myocardial remodeling. Conclusions—TIMP-3 deficiency disrupts matrix homeostasis and the balance of inflammatory mediators, eliciting the transition to cardiac dilation and dysfunction. Therapeutic restoration of myocardial TIMP-3 may provide a novel approach to limit cardiac remodeling and the progression to failure in patients with dilated cardiomyopathy.

Up-regulation of TIMP-1 expression in B16-F10 melanoma cells suppresses their metastatic ability in chick embryo

Clonal B16-F10 cell lines with increased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) have been generated by transfection with a TIMP-1-containing expression vector. The parental B16-F10 and control 1–2 cells, and two TIMP-1 up-regulated clones (2–10, 6–5), were studied for their growth characteristics in tissue culture and their experimental metastatic ability in the chick embryo. Both of the TIMP-1 up-regulated clones showed slower in vitro growth and had lower saturation densities. Both clones were also less metastatic following intravenous injection into chick embryos, and formed significantly fewer metastatic tumors in the chorioallantoic membrane and in the liver than did parental B16-F10 and control cells. Furthermore, the size of tumors formed by TIMP-1 up-regulated cells was significantly reduced in comparison to the tumors produced by B16-F10 or control cells. Our results show that malignant cell lines genetically modified to express increased levels of TIMP-1 exhibit a suppressed experimental metastatic ability in vivo. We propose that TIMP-1 suppresses metastatic ability by decreasing both invasive and growth abilities.

Utilization of transgenic mice in the study of matrix degrading proteinases and their inhibitors

The extracellular matrix (ECM) acts as both a structural scaffold and an informational medium. Its dynamic status is determined by cells that secrete its constituent molecules and, in most cases, also secrete enzymes that catalyze degradation of these molecules. A stasis between ECM degrading enzymes and their inhibitors maintains the integrity of the matrix. While controlled ECM remodelling is fundamental to several normal processes, uncontrolled disruption underlies diverse pathological conditions. Transgenic mice with specific modulations or a total lack of expression of certain metalloproteinases, serine proteinases or their inhibitors have been generated to elucidate endogenous expression patterns, identify regulatory elements of these genes, and study the physiological consequences of their deregulated expression. With these models we enhance our understanding of the role of proteinases and their inhibitors in diverse normal processes and pathologies including mammary gland development, hemostasis, emphysema and cancer.

Increased proteinase expression during tumor progression of cell lines down-modulated for TIMP levels: a new transformation paradigm? [corrected].

We have reported that down-modulation of tissue inhibitor of metalloproteinases (TIMP) by means of antisense RNA converts non-tumorigenic Swiss 3T3 cells into malignant cells capable of forming metastasizing tumors in nude mice [Science 243:947 (1989)]. We now describe changes in the expression of specific genes associated with tumor progression of two lines down-modulated with TIMP, LA1 and LA7. Six independent variant cell lines, generated from different primary tumors produced by LA1 and LA7, lacked (like LA1 and LA7) many characteristics of typical transformed cells. However, their tumorigenicity in nude mice was enhanced; tumors appeared with a shorter lag (1-3 weeks versus 8-10 weeks for the parental clones, LA1 and LA7) and grew very rapidly. Increases, substantial in some cases, in the expression of a cysteine proteinase, cathepsin L, and metalloproteinases homologous to rat transin (stromelysin) and transin-2 were characteristic of these variant clones. The mRNA levels encoding the transformation-associated secreted phosphoprotein (osteopontin) and the calcium-binding protein calcyclin were also augmented. No evidence for gene amplification was found, and we did not detect any change in the mRNA levels of the proto-oncogenes that were examined. These novel cell lines represent a new paradigm for the transformed cell. Our data suggest that a reduction in TIMP secretion enhances the cells oncogenic capacity by altering the extracellular environment in a way conducive to further changes in gene expression necessary for tumor progression.

Hypotriglyceridemic effects of levonorgestrel in rats

The progestin, levonorgestrel administered orally to fed female rats significantly lowers both plasma total and very low density lipoprotein triglycerides. In contrast, plasma total cholesterol and low density lipoprotein cholesterol rose significantly. Suspensions of isolated hepatocytes were used to study the effects of levonorgestrel on triglyceride synthesis by examining the incorporation of labelled precursors [( 9,10- 3H]palmitate and [U-14C]glycerol) into triglycerides. Levonorgestrel (10(-4) M) significantly inhibited the incorporation of both precursors into hepatocyte triglycerides and also reduced their incorporation into the triglycerides (nearly all in d less than 1.006) released into the medium. These results suggest that inhibition of hepatic triglyceride synthesis and release can account at least for part of the lowering of plasma VLDL which occurs during administration of levonorgestrel.

Effects of levonorgestrel on enzymes responsible for synthesis of triacylglycerols in rat liver

The effects of levonorgestrel treatment (4 micrograms/day per kg body weight 0.75 for 18 days) on rate-limiting enzymes of hepatic triacylglycerol synthesis, namely glycerol-3-phosphate acyltransferase and phosphatidic acid phosphatase were investigated in microsomal, mitochondrial and cytosolic fractions of rat liver. Levonorgestrel treatment resulted in a significant reduction (26%) of hepatic microsomal glycerol-3-phosphate acyltransferase specific activity. Hepatic mitochondrial glycerol-3-phosphate acyltransferase specific activity was unchanged. Levonorgestrel treatment also significantly reduced (by 20%) the specific activity of hepatic microsomal magnesium-independent phosphatidic acid phosphatase. However, magnesium-dependent phosphatic acid phosphatase specific activities in microsomal and cytosolic fractions were unaffected. Cytosolic magnesium-independent phosphatidic acid phosphatase activity was also unchanged. These studies are consistent with the view that levonorgestrel lowers serum triacylglycerol levels, at least in part, by inhibition of the glycerol-3-phosphate acyltransferase (EC 2.3.1.15) step in hepatic triacylglycerol synthesis.