Impact of the cyclin-dependent kinase inhibitor p27Kip1 on resistance of tumor cells to anticancer agents.

A low proliferating fraction in solid tumors limits the effectiveness of cell cycle-dependent chemotherapeutic agents. To understand the molecular basis of such "kinetic" resistance we cultured tumor cells as multicellular spheroids and examined levels of p27Kip1, a cyclin-dependent kinase inhibitor known to be upregulated by intercellular contact in normal cells. When transferred from monolayer to three-dimensional culture, a consistent upregulation (up to 15-fold) of p27 protein was observed in a panel of mouse and human carcinoma cell lines. Antisense-oligonucleotide-mediated downregulation of p27 in EMT-6 mammary tumor cell spheroids reduced intercellular adhesion, increased cell proliferation, sensitized tumor cells to 4-hydroperoxycyclophosphamide, and restored drug- or radiation-induced cell-cycle perturbations repressed in spheroid culture. Our results implicate p27 as a regulator of drug resistance in solid tumors and suggest that tumor-targeted p27 antagonists may be useful chemosensitizers in conjunction with conventional anticancer therapy.

E-Cadherin-dependent growth suppression is mediated by the cyclin-dependent kinase inhibitor p27(KIP1).

Recent studies have demonstrated the importance of E-cadherin, a homophilic cell-cell adhesion molecule, in contact inhibition of growth of normal epithelial cells. Many tumor cells also maintain strong intercellular adhesion, and are growth-inhibited by cell- cell contact, especially when grown in three-dimensional culture. To determine if E-cadherin could mediate contact-dependent growth inhibition of nonadherent EMT/6 mouse mammary carcinoma cells that lack E-cadherin, we transfected these cells with an exogenous E-cadherin expression vector. E-cadherin expression in EMT/6 cells resulted in tighter adhesion of multicellular spheroids and a reduced proliferative fraction in three-dimensional culture. In addition to increased cell-cell adhesion, E-cadherin expression also resulted in dephosphorylation of the retinoblastoma protein, an increase in the level of the cyclin-dependent kinase inhibitor p27(kip1) and a late reduction in cyclin D1 protein. Tightly adherent spheroids also showed increased levels of p27 bound to the cyclin E-cdk2 complex, and a reduction in cyclin E-cdk2 activity. Exposure to E-cadherin-neutralizing antibodies in three-dimensional culture simultaneously prevented adhesion and stimulated proliferation of E-cadherin transfectants as well as a panel of human colon, breast, and lung carcinoma cell lines that express functional E-cadherin. To test the importance of p27 in E-cadherin-dependent growth inhibition, we engineered E-cadherin-positive cells to express inducible p27. By forcing expression of p27 levels similar to those observed in aggregated cells, the stimulatory effect of E-cadherin-neutralizing antibodies on proliferation could be inhibited. This study demonstrates that E-cadherin, classically described as an invasion suppressor, is also a major growth suppressor, and its ability to inhibit proliferation involves upregulation of the cyclin-dependent kinase inhibitor p27.

Genes expressed in human tumor endothelium.

To gain a molecular understanding of tumor angiogenesis, we compared gene expression patterns of endothelial cells derived from blood vessels of normal and malignant colorectal tissues. Of over 170 transcripts predominantly expressed in the endothelium, 79 were differentially expressed, including 46 that were specifically elevated in tumor-associated endothelium. Several of these genes encode extracellular matrix proteins, but most are of unknown function. Most of these tumor endothelial markers were expressed in a wide range of tumor types, as well as in normal vessels associated with wound healing and corpus luteum formation. These studies demonstrate that tumor and normal endothelium are distinct at the molecular level, a finding that may have significant implications for the development of anti-angiogenic therapies.

A phosphatase associated with metastasis of colorectal cancer.

To gain insights into the molecular basis for metastasis, we compared the global gene expression profile of metastatic colorectal cancer with that of primary cancers, benign colorectal tumors, and normal colorectal epithelium. Among the genes identified, the PRL-3 protein tyrosine phosphatase gene was of particular interest. It was expressed at high levels in each of 18 cancer metastases studied but at lower levels in nonmetastatic tumors and normal colorectal epithelium. In 3 of 12 metastases examined, multiple copies of the PRL-3 gene were found within a small amplicon located at chromosome 8q24.3. These data suggest that the PRL-3 gene is important for colorectal cancer metastasis and provide a new therapeutic target for these intractable lesions.

Transcriptional response to hypoxia in human tumors.

BACKGROUND: The presence of hypoxic regions within solid tumors is associated with a more malignant tumor phenotype and worse prognosis. To obtain a blood supply and protect against cellular damage and death, oxygen-deprived cells in tumors alter gene expression, resulting in resistance to therapy. To investigate the mechanisms by which cancer cells adapt to hypoxia, we looked for novel hypoxia-induced genes. METHODS: The transcriptional response to hypoxia in human glioblastoma cells was quantified with the use of serial analysis of gene expression. The time course of gene expression in response to hypoxia in a panel of various human tumor cell lines was measured by real-time polymerase chain reaction. Hypoxic regions of human carcinomas were chemically marked with pimonidazole. Immunohistochemistry and in situ hybridization were used to examine gene expression in the tumor's hypoxic regions. RESULTS: From the 24 504 unique transcripts expressed, 10 new hypoxia-regulated genes were detected-all induced, to a greater extent than vascular endothelial growth factor, a hypoxia-induced mitogen that promotes blood vessel growth. These genes also responded to hypoxia in breast and colon cancer cells and were activated by hypoxia-inducible factor 1, a key regulator of hypoxic responses. In tumors, gene expression was limited to hypoxic regions. Induced genes included hexabrachion (an extracellular matrix glycoprotein), stanniocalcin 1 (a calcium homeostasis protein), and an angiopoietin-related gene. CONCLUSIONS: We have identified the genes that are transcriptionally activated within hypoxic malignant cells, a crucial first step in understanding the complex interactions driving hypoxia response. Within our catalogue of hypoxia-responsive genes are novel candidates for hypoxia-driven angiogenesis.

Cell surface tumor endothelial markers are conserved in mice and humans.

We recently identified genes encoding tumor endothelial markers (TEMs) that displayed elevated expression during tumor angiogenesis. From both biological and clinical points of view, TEMs associated with the cell surface membrane are of particular interest. Accordingly, we have further characterized four such genes, TEM1, TEM5, TEM7, and TEM8, all of which contain putative transmembrane domains. TEM5 appears to be a seven-pass transmembrane receptor, whereas TEM1, TEM7, and TEM8 span the membrane once. We identified mouse counterparts of each of these genes, designated mTEM1, mTEM5, mTEM7, and mTEM8. Examination of these mTEMs in mouse tumors, embryos, and adult tissues demonstrated that three of them (mTEM1, mTEM5, and mTEM8) were abundantly expressed in tumor vessels as well as in the vasculature of the developing embryo. Importantly, expression of these mTEMs in normal adult mouse tissues was either undetectable or detected only in a small fraction of the vessels. These results demonstrate conservation of human and mouse tumor angiogenesis at the molecular level and support the idea that tumor angiogenesis largely reflects normal physiological neovasculaturization. The coordinate expression of TEM1, TEM5, and TEM8 on tumor endothelium in humans and mice makes these genes attractive targets for the development of antiangiogenic therapies.

Secreted and cell surface genes expressed in benign and malignant colorectal tumors.

Serial analysis of gene expression was used to identify transcripts encoding secreted or cell surface proteins that were expressed in benign and malignant tumors of the colorectum. A total of 290,394 tags were analyzed from normal, adenomatous, and cancerous colonic epithelium. Of the 21,343 different transcripts observed, 957 were found to be differentially expressed between normal tissue and adenoma or between normal tissue and cancer. Forty-nine transcripts were elevated > or =20-fold in adenomas, 40 transcripts were elevated > or =20-fold in cancers, and 9 transcripts were elevated > or =20-fold in both. Products of six of these nine transcripts (TGFBI, LYS, RDP, MIC-1, REGA, and DEHL) were predicted to be secreted or to reside on the cell surface, and these were analyzed in more detail. The abnormal expression levels predicted by serial analysis of gene expression were confirmed by quantitative PCR analyses of each of these six genes. Moreover, the cell types responsible for the elevated expression were identified by in situ hybridization and by PCR analyses of epithelial cells immunoaffinity purified from primary tumors. This study extends knowledge of the differences in gene expression that underlie various stages of neoplasia and suggests specific diagnostic approaches that may be useful for the early detection of colorectal neoplasia.

Anthrax Toxin Receptor 1 Is Essential for Arteriogenesis in a Mouse Model of Hindlimb Ischemia.

Anthrax toxin receptor 1/tumor endothelial marker 8 (Antxr1 or TEM8) is up-regulated in tumor vasculature and serves as a receptor for anthrax toxin, but its physiologic function is unclear. The objective of this study was to evaluate the role of Antxr1 in arteriogenesis. The role of Antxr1 in arteriogenesis was tested by measuring gene expression and immunohistochemistry in a mouse model of hindlimb ischemia using wild-type and ANTXR1(-/-) mice. Additional tests were performed by measuring gene expression in in vitro models of fluid shear stress and hypoxia, as well as in human muscle tissues obtained from patients having peripheral artery disease. We observed that Antxr1 expression transiently increased in ischemic tissues following femoral artery ligation and that its expression was necessary for arteriogenesis. In the absence of Antxr1, the mean arterial lumen area in ischemic tissues decreased. Antxr1 mRNA and protein expression was positively regulated by fluid shear stress, but not by hypoxia. Furthermore, Antxr1 expression was elevated in human peripheral artery disease requiring lower extremity bypass surgery. These findings demonstrate an essential physiologic role for Antxr1 in arteriogenesis and peripheral artery disease, with important implications for managing ischemia and other arteriogenesis-dependent vascular diseases.

Reversal of intrinsic and acquired forms of drug resistance by hyaluronidase treatment of solid tumors.

There are two broad categories of drug resistance encountered during cancer chemotherapy, i.e. intrinsic and acquired. They are observed in virtually every type of tumor with every known anticancer chemotherapeutic drug. As such there is an urgent need to develop innovative approaches of preventing or reversing these types of resistance. One strategy to do so is to develop completely new drugs which may be resistance free, such as direct acting angiogenesis inhibitors (T. Boehm, J. Folkman, T. Browder, M.S. O'Reilly, Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance, Nature 390 (1997) 404-407; R.S. Kerbel, Inhibition of tumor angiogenesis as a strategy to circumvent acquired resistance to anti-cancer therapeutic agents, BioEssays 13 (1991) 31-36; R.S. Kerbel, A cancer therapy resistant to resistance, Nature 390 (1997) 335-336). Another is to devise methods which will improve significantly the effectiveness of those conventional drugs already in use, such as adriamycin, cyclophosphamide and taxol. We have directed efforts towards the latter. They depend on the discovery of a new class of chemosensitizers which act as antiadhesive agents rendering solid tumors more susceptible to such conventional cytotoxic therapeutic drugs. Examples of this concept are illustrated with bovine testicular hyaluronidase and a mouse mammary tumor called EMT-6. When this enzyme preparation is used to treat intact multicellular spheroids of the EMT-6 tumor, the spheroids are substantially disaggregated. Dispersed spheroids are more susceptible to the cytotoxic effects of cyclophosphamide than intact spheroids. Moreover, this antiadhesive chemosensitizing effect can actually be reproduced in BALB/c mice when EMT-6 cells are grown intraperitoneally as an ascites tumor (consisting mostly of multicellular aggregates) and the mice are given injections of hyaluronidase and cyclophosphamide. In a similar fashion, the indifference of P-glycoprotein-positive multidrug-resistant EMT-6 spheroids to the P-glycoprotein reversal agent PSC-833 (a cyclosporin A analogue) can be reversed by disaggregation of the intact spheroids by hyaluronidase. This renders the treated cells highly sensitive to a combination of adriamycin and PSC-833 in a manner similar to the striking chemosensitization effects commonly observed in monolayer culture systems. Thus, hyaluronidase has the potential to reverse forms of both intrinsic and acquired drug resistance in solid tumors, such as EMT-6, which are sensitive to its antiadhesive effects.

Induction and reversal of cell adhesion-dependent multicellular drug resistance in solid breast tumors.

Although there are a number of chemotherapeutic drugs available for the treatment of breast cancer, eg. adriamycin, cyclophosphamide and taxol, their effectiveness is severely limited by expression of intrinsic resistance in some patients and by acquired resistance in others. There is thus an urgent need to develop innovative methods to try and make these drugs more effective than is currently the case. One such method is to combine them with novel "chemosensitizers", i.e., drugs which themselves lack anti-tumor cytotoxic properties but which will increase the efficacy of those which do. In this regard we hae been studying the hypothesis that the resistance of solid tumors, including breast cancer, can be expressed at the prototissue/multicellular level, and that this "multicellular resistance" can be minimized or reversed by the appropriate use of so-called "anti-adhesive" agents. RESULTS/BACKGROUND: It is well known that monolayer cultures of tumor cells-including murine breast cancer-are generally much more intrinsically chemosensitive than the same cells grown as solid tumors in vivo. However, the relative resistance of solid tumors can often be recapitulated in tissue culture simply by growth of the tumor cells as three dimensional multicellular spheroids. There are cases where this is also true with respect to acquired drug resistance. This "multicellular resistance" could be due to such factors as insufficient drug penetration, a reduced growth fraction, or a decreased sensitivity to drug induced apoptosis mediated by cell-cell interaction survival signals. Can such multicellular resistance mechanisms in solid tumors be reversed? With respect to this question, we have recently found that the relative intrinsic resistance of intact murine EMT-6 mouse mammary carcinoma spheroids can be significantly reversed by the anti-adhesive (disaggregating) effects of hyaluronidase. Moreover, this novel method of chemosensitization appears to depend on increased recruitment of disaggregated cells into the cycling pool, thus rendering them more sensitive to a cell cycle dependent drug such as cyclophosphamide. The reduced growth fraction observed in spheroids appears to be due to a marked cell contact-dependent upregulation of the cyclin dependent kinase inhibitor, p27Kipl. FUTURE OBJECTIVE: The overall goal of our current and future research is to determine whether solid tumors, including human breast cancer, express intrinsic or acquired resistance at the multicellular level to such drugs as taxol or cyclophosphamide, and if so, determine whether it can be reversed by the chemosensitizing effect of anti-adhesive agents. This will require a search for effective anti-adhesive agents for human cancers as hyaluronidase has not been found to possess anti-adhesive function against such tumors to date. In addition, the counter-intuitive and innovative idea of downregulating p27kipl in human breast cancers as a means of cytotoxic drug chemosensitization is also being evaluated.

Cell adhesion and drug resistance in cancer.

The impact of cell adhesion in the pathobiology of tumors has been studied almost exclusively in the context of invasion, angiogenesis, and metastasis. Here we review data supporting a major role for cell-cell adhesion in the regulation of intrinsic or acquired resistance of solid tumors to various anticancer therapeutics. Cell-cell interactions are known to protect cells from apoptosis, and may help to explain chemoresistance of solid tumors. Recent data implicates p27KIP1, a cyclin-dependent kinase inhibitor, as a possible mediator of adhesion-dependent drug resistance. A model is described whereby cell-cell interactions signal the upregulation of p27, which in turn causes growth arrest in the G1 phase of the cell cycle and resistance to apoptosis induced by anticancer agents that target rapidly dividing cells. We focus on E-cadherin, a homophilic cell-cell adhesion molecule capable of upregulating p27, as one potential mediator of intrinsic resistance of carcinomas. A clearer understanding of how cell-cell adhesion suppresses cell growth and apoptosis should aid in the development of novel, more effective anticancer strategies.

Consequences of angiogenesis for tumor progression, metastasis and cancer therapy.

The growth of solid tumors to a clinically relevant size is dependent upon an adequate blood supply. This is achieved by the process of tumor stroma generation where the formation of new capillaries is a central event. Progressive recruitment of blood vessels to the tumor site and reciprocal support of tumor expansion by the resulting neovasculature are thought to result in a self-perpetuating loop helping to drive the growth of solid tumors. The development of new vasculature also allows an 'evacuation route' for metastatically-competent tumor cells, enabling them to depart from the primary site and colonize initially unaffected organs. Several molecular and cellular mechanisms have been identified by which tumor parenchyma may exert its angiogenic effect on host endothelial cells. As a result of this paracrine influence, tumor-associated endothelial cells acquire an 'immature' phenotype manifested by rapid proliferation, migration, release of proteases and expression of cytokines, endothelial-specific tyrosine kinases (e.g. flk-1, tek and others) as well as numerous other molecular alterations. Consequently a network of structurally and functionally aberrant blood vessels is formed within the tumor mass. There is also evidence that endothelial cells themselves, and likewise other stromal cells, may act reciprocally to alter the behavior of adjacent tumor cells in a paracrine or cell contact mediated fashion. For example, production of interleukin 6(IL-6) by endothelial cells may have a differential effect on human melanoma cells expressing different degrees of aggressiveness. In this manner endothelial derived cytokines could conceivably contribute to tumor progression by suppressing the growth of the less aggressive tumor cells and promoting dominance of their malignant counterparts in 'strategic' perivascular zones. Distinct biological features expressed by tumor-associated vasculature may serve as potential prognostic markers of disease progression as well as novel targets for therapeutic intervention.