Invasive Potential Of Cancer Cells Research Articles

Signaling pathways responsible for cancer cell invasion as targets for cancer therapy.

Migration of cancer cells is one of the key factors responsible for cancer metastasis. The elucidation of mechanisms responsible for the highly invasive potential of cancer cells can help to identify specific targets for the treatment of cancer patients. Highly invasive cancers are usually characterized by aberrant activity of specific intra- or extracellular molecules such as protein kinases, phosphatases, transcriptional factors, proteolytic enzymes, and others. Protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) are responsible for the constitutive activity of transcriptional factors NF-kappaB and AP-1 in some of the highly invasive cancers. Furthermore, NF-kappaB and AP-1 control the expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR), and expression of both uPA and uPAR correlates with invasive cancer cell phenotype and poor prognosis. The inhibition of PKC and PI3K signaling (through NF-kappaB and AP-1) suppressed the secretion of uPA, resulting in the inhibition of motility of highly invasive breast cancer cells. Therefore, inhibition of specific target molecules in common signaling pathway(s) responsible for metastatic spread can have potential clinical relevance. This review will summarize different approaches to targeting distinct signaling molecules involved in cancer invasion and metastasis.

IN VIVO AND IN VITRO ANTITUMOR EFFECT OF NUTRIENT SYNERGY ON HUMAN OSTEOSARCOMA CELL LINE MNNG-HOS

Current treatment of osteosarcoma is associated with poor prognosis, especially due to the increased risk of developing other cancers with chemotherapy. Therefore, new safe effective treatment strategies are needed. This prompted us to investigate the synergistic effect of a nutrient mixture (NS) of lysine, proline, arginine, ascorbic acid, and epigallocatechin gallate on the growth of human osteosarcoma xenografts in athymic nude mice. We also investigated the effect of NS on human osteosarcoma cell line MNNG-HOS in vitro by measuring: cytotoxicity, modulation of MMP-2 and -9, cancer cell invasive potential, and angiogenesis. After one-week of isolation, 5-6 week old athymic male nude mice (n=12) were inoculated with 3x106 osteosarcoma cells MNNG-HOS. After injection, the mice were randomly divided into two subgroups; group A was fed a regular diet and group B was fed a regular diet supplemented with 0.5% of the nutrient mixture. Four weeks later, the mice were sacrificed, and their tumors were excised, weighed, and processed for histology. Cell proliferation was evaluated by MTT assay, MMP expression by gelatinase zymography, and invasion through Matrigel. Cells were also treated with phorbol 12-myristate 13-acetate (PMA) to study enhanced MMP expression. Results showed that the nutrient mixture (NS) inhibited the growth and reduced the size of tumors in nude mice. Furthermore, the mitotic index was decreased in the supplemented group (4-5) in contrast to the control group (12-15). The invasion of osteosarcoma MNNG-HOS cells through Matrigel was significantly reduced in a dose-dependent fashion, with 100% inhibition of invasion of MNNG cells at 50 μg/ml concentration of the nutrient mixture. No significant anti-proliferative effect by NS was seen with MNNG cells. Zymography showed dose-dependent inhibition of MMP secretion by MNNG in the presence of NS. Nutrient synergy strongly suppressed the growth of tumors without any adverse effects in nude mice, suggesting the nutrient combination has potential as an anticancer agent. In vitro studies demonstrated inhibition of cancer cell invasion and secretion of MMPs -critical parameters for cancer control and prevention.

Quantum dot-based cell motility assay

Motility and migration are measurable characteristics of cells that are classically associated with the invasive potential of cancer cells, but in vitro assays of invasiveness have been less than perfect. We previously developed an assay to monitor cell motility and migration using water-soluble CdSe/ZnS nanocrystals; cells engulf the fluorescent nanocrystals as they crawl across them and leave behind a fluorescent-free trail. We show here that semiconductor nanocrystals can also be used as a sensitive two-dimensional in vitro invasion assay. We used this assay to compare the behavior of seven different adherent human cell lines, including breast epithelial MCF 10A, breast tumor MDA-MB-231, MDA-MB-435S, MCF 7, colon tumor SW480, lung tumor NCI H1299, and bone tumor Saos-2, and observed two distinct behaviors of cancer cells that can be used to further categorize these cells. Some cancer cell lines demonstrate fibroblastic behaviors and leave long fluorescent-free trails as they migrate across the dish, whereas other cancer cells leave clear zones of varying sizes around their periphery. This assay uses fluorescence detection, requires no processing, and can be used in live cell studies. These features contribute to the increased sensitivity of this assay and make it a powerful new tool for discriminating between non-invasive and invasive cancer cell lines.

Grb7-based molecular therapeutics in cancer.

Traditional anti-cancer drugs preferentially kill rapidly growing tumour cells rather than normal cells. However, most of these drugs have no preferential selection towards cancer cells and are taken up by the whole body, resulting in significant adverse side effects. Therapeutic molecules that could specifically inhibit undesirable phenotypes are an attractive way of eliminating cancer cells. There is a widespread effort to develop inhibitors against signal transduction molecules that play a key role in the proliferative, migratory and invasive properties of a cancer cell. Grb7 is an adaptor-type signalling protein that is recruited via its Src-homology 2 (SH2) domain to a variety of tyrosine kinases. Grb7 is overexpressed in breast, oesophageal and gastric cancers, and may contribute to the invasive potential of cancer cells. Molecular interactions involving Grb7 therefore provide attractive targets for therapeutic intervention.

Increased Furin Activity Enhances the Malignant Phenotype of Human Head and Neck Cancer Cells

Many proteins are synthesized as inactive proforms requiring a proteolytic processing to render them active. A variety of proteases catalyze these cleavage reactions. Proprotein convertases are a family of serine proteases capable of activating substrates that will subsequently intervene in extracellular matrix (ECM) degradation, cell growth, differentiation and viral pathogenesis. Furin, the prototype of this family, has been implicated in many physiological and pathological processes. Some of its substrates such as TGF-beta, MT-MMP's, and IGFR-1 have been identified. Overexpression of furin has been observed in several human tumors. In this report we demonstrate that overexpression of furin causes a significant increase in the invasive potential of human tumor cells of low and moderate aggressive potential in vitro and in vivo. SCC12 and SCC15 were transfected with furin cDNA, resulting in efficient processing of furin substrates. An in vivo invasion assay showed enhancement of invasive ability. Inhibition of furin activity with the synthetic inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl-ketone, CMK, showed a significant decrease in both processing and in vitro invasiveness. A moderate enhancement in proliferation rate was observed when cells were transfected with furin. CMK treatment resulted in a marked reduction of this effect. Tumors obtained after subcutaneous (s.c.) inoculation of furin-overexpressing cells were larger and developed earlier than the controls. Furin overexpression caused an imbalance in the activation of invasion and proliferation-related substrates leading to the acquisition of an advanced malignant phenotype. In addition, inhibition of furin activity decreases substrate activation, proliferation rate, and invasive potential of cancer cells, suggesting that furin is a potentially useful target for therapeutics.

IL-8 expression and its possible relationship with estrogen-receptor-negative status of breast cancer cells.

Estrogen-receptor (ER) status is an important parameter in breast cancer management as ER-positive breast cancers have a better prognosis than ER-negative tumors. This difference comes essentially from the lower aggressiveness and invasiveness of ER-positive tumors. Here, we demonstrate, that interleukin-8 (IL-8) was clearly overexpressed in most ER-negative breast, ovary cell lines and breast tumor samples tested, whereas no significant IL-8 level could be detected in ER-positive breast or ovarian cell lines. We have also cloned human IL-8 from ER-negative MDA-MB-231 cells, and we show that IL-8 produced by breast cancer cells is identical to monocyte-derived IL-8. Interestingly, the invasion potential of ER-negative breast cancer cells is associated at least in part with expression of IL-8, but not with IL-8 receptor levels. Moreover, IL-8 increases the invasiveness of ER-positive breast cancer cells by two fold, thus confirming the invasion-promoting role of IL-8. On the other hand, exogenous expression of estrogen receptors in ER-negative cells led to a decrease of IL-8 levels. In summary, our data show that IL-8 expression is negatively linked to ER status of breast and ovarian cancer cells. We also support the idea that IL-8 expression is associated with a higher invasiveness potential of cancer cells in vitro, which suggests that IL-8 could be a novel marker of tumor aggressiveness.

Matrix-directed regulation of pericellular proteolysis and tumor progression

The microenvironment of cancer cells, composed of extracellular matrix (ECM) macromolecules, plays a pivotal function in tumor progression. ECM preexisting modules or cryptic sites revealed by partial enzymatic hydrolysis positively or negatively regulate matrix metalloproteinase (MMP) expression and activation, further influencing matrix invasion by cancer cells. Pericellular activation of gelatinase A (MMP-2) proceeds via the formation of a complex involving its inhibitor, TIMP-2, its activator(s), MT-MMPs and αv β3 integrin forming a docking system. This proteinase has been invariably linked to cancer cell invasive potential and is often predictive of a poor survival. MMP-2 degrades most ECM macromolecules and appears to act as a main ‘decryptase’. ECM modulation of MMP-2 activation pathway thus influences angiogenesis and tumor growth. For instance the noncollagenous domain of α3 chain of type IV collagen, through αv β3 integrin binding, inhibits both MT1-MMP and αv β3 integrin expression from melanoma cells and empedes cell migration and proliferation. At the opposite, a particular module in elastin (VGVAPG) with type VIII β turn conformation stimulates MT1-MMP and proMMP-2 activation through binding to S-gal elastin receptor, and increases the matrix invasive capacity of several cancer cell lines and endothelial cells. Endocytosis emerges as a main mechanism controlling MMP-2, and also other MMPs; it proceeds via the formation of a MMP-thrombospondin(s) complex further recognized by the LRP scavenger receptor. ECM undergoes conspicuous variations with aging linked to alterations of tissue organization and post-translational modifications of matrix constituents that modify cell–matrix interactions and MMP-2 activation pathway.

Identification of Novel Non-phosphorylated Ligands, Which Bind Selectively to the SH2 Domain of Grb7

Grb7 is an adapter-type signaling protein, which is recruited via its SH2 domain to a variety of receptor tyrosine kinases (RTKs), including ErbB2 and ErbB3. It is overexpressed in breast, esophageal, and gastric cancers, and may contribute to the invasive potential of cancer cells. Molecular interactions involving Grb7 therefore provide attractive targets for therapeutic intervention. We have utilized phage display random peptide libraries as a source of small peptide ligands to the SH2 domain of Grb7. Screening these libraries against purified Grb7 SH2 resulted in the identification of Grb7-binding peptide phage clones that contained a non-phosphorylated Tyr-X-Asn (YXN) motif. The tyrosine-phosphorylated form of this motif is characteristic of Grb7 SH2 domain binding sites identified in RTKs and other signaling proteins such as Shc. Peptides that are non-phosphorylated have greater potential in the development of therapeutics because of the instability of a phosphate group in vivo. Using a biased library approach with this conserved YXN motif, we identified seven different peptide phage clones, which bind specifically to the SH2 domain of Grb7. These peptides did not bind to the SH2 domain of Grb2 (which also selects for Asn at pY(+2)) or Grb14, a closely related family member. The cyclic structure of the peptides was required to bind to the Grb7 SH2 domain. Importantly, the synthetic Grb7-binding peptide G7-18 in cell lysates was able to specifically inhibit the association of Grb7 with the ErbB family of RTKs, in particular ErbB3, in a dose-dependent manner. These peptides will be useful in the development of targeted molecular therapeutics for cancers overexpressing Grb7 and in the development of Grb7-specific inhibitors to gain a complete understanding of the physiological role of Grb7.

Production of bioengineered cancer tissue constructs in vitro: epithelium-mesenchyme heterotypic interactions.

A few models have been established to study cancer cells in vitro. However, the cellular interactions have rarely been studied specifically using bioengineered cancer constructs combining human carcinoma cells and tumor-associated fibroblasts. We developed an in vitro model of tridimensional bioengineered cancer tissue constructs (bCTC) by seeding mammary epithelial cancer cells or normal keratinocytes over a mesenchymal layer containing tumor-derived fibroblastic cells or normal skin fibroblasts. After the introduction of epithelial cells, each construct was cultured for another 10 d. Histologic analyses showed that carcinoma cell lines could invade the subjacent mesenchymal layer and that the capacity to migrate was related to the invasive potential of cancer cells and the type of fibroblasts used, while noninvasive populations did not. Of the tested epithelial cells, MDA-MB-231 and, to a lesser degree, HDQ-P1 cell lines were invasive, and the invasion was deeper into the mesenchymal component containing tumor-derived fibroblasts. However, with normal skin fibroblasts, the mesenchymal layer was degraded twice faster than with tumor-derived fibroblastic cells. MDA-MB-231 cells and normal keratinocytes induced the highest level of gelatinase B, and the level was lowest with the MCF-7 cell line. The activated form of gelatinase B was, however, induced to the highest levels in the keratinocyte-seeded bCTC containing tumor-derived but not normal fibroblasts. MDA-MB-231 was the only epithelial cancer cell line whose activity of gelatinase A was reduced when cocultured with tumor-derived fibroblasts but not under normal fibroblast stimulation. Finally, a 50/48-kDa gelatinase band has been observed in bCTCs with noninvasive epithelial cells only. Our study demonstrates the selective secretion of gelatinases according to the phenotype of the cells seeded in the various bCTCs.

Clinicopathologic significance of laminin-5 gamma2 chain expression in squamous cell carcinoma of the tongue - immunohistochemical analysis of 67 lesions

The laminin-5 γ2 chain plays an important role in cell migration during tumour inva-sion and tissue remodelling. Laminin-5 γ2 chain expression in squamous cell carcino-mas of the tongue in 67 patients with Stage II, III or IVA,B (excluding the cases with distant metastasis) was examined immuno-histochemically to determine its associa-tions with the clinicopathological features of each tumour. The predominant staining patterns were categorised as follows: A, few or no tumour cells were positive; B, part of the tumour nest periphery was positive; C, the tumour nest periphery was circumferentially positive; or D, almost all the tumour cells were positive. Laminin-5 γ2 chain expression was observed clearly in tumour cell cytoplasm. Of the 67 tumours examined, 6(9%), 31 (46%), 19 (28%) and 11 (17%) showed staining patterns A, B, C and D, respectively. With progression from staining pattern A to D, the number of immunopositive tumour cells increased significantly (P<0.0001), and the tumour histology showed significantly more infiltrative growth (P<0.0001) and poorer differentiation (P=0.0021). Furthermore, both univariate (P=0.0019) and multivariate (P=0.0003; hazard ratio=3.132) analyses of the patients’ survival revealed that the prognosis became significantly poorer with progression from staining pattern A to D. Increased laminin-5 γ2 chain immunore-activity, which may reflect a high invasive potential of cancer cells, is a factor indica-tive of a poor prognosis for patients with squamous cell carcinoma of the tongue. Henk Tideman

One thousand faces of Langerhans islets.

Studies with different strains of Syrian hamsters and Syrian golden hamsters have revealed the remarkable potential of islet cells to undergo orthotopic and heterotopic metaplasia. The most common spontaneous change included the development of hepatocytes in aged and malnourished hamsters. Of the many other alterations that occurred during carcinogenesis, most of the metaplastic changes originated within the islet periphery and progressed inside and outside the islets. The development of ductular structures within islets and their progression either to structures identical to human serous cystadenoma or to highly invasive adenocarcinomas were the most common alterations. The remarkably greater invasive potential of cancer cells arising within the islets contrasted sharply with the slow growth of the tumors developing within ducts (intraductal tumors). Studies in human tissue also showed development of malignant cells within islets, and, in some cases, transition of islet cells to malignant cells was suggested. The overall results, along with recent findings in other studies in cultured human and hamster islets, indicate the enormous potential of islet cells to differentiate and undergo malignant transformation. Whether the metaplastic and malignant cells derive from stem cells embedded within islets or from transdifferentiated islet cells remains to be seen.

Clinicopathologic significance of laminin-5 ?2 chain expression in squamous cell carcinoma of the tongue

The laminin-5 gamma2 chain plays an important role in cell migration during tumor invasion and tissue remodeling. Laminin-5 gamma2 chain expression in squamous cell carcinomas of the tongue in 67 patients with Stage II, III, or IVA,B (excluding the cases with distant metastasis) was examined immunohistochemically to determine its associations with the clinicopathologic features of each tumor. The predominant staining patterns were categorized as follows: A, few or no tumor cells were positive; B, part of the tumor nest periphery was positive; C, the tumor nest periphery was circumferentially positive; or D, almost all the tumor cells were positive. Laminin-5 gamma2 chain expression was observed clearly in tumor cell cytoplasm. Of the 67 tumors examined, 6 (9%), 31 (46%), 19 (28%), and 11 (17%) showed staining patterns A, B, C, and D, respectively. With progression from staining pattern A to D, the number of immunopositive tumor cells increased significantly (P<0.0001), and the tumor histology showed significantly more infiltrative growth (P<0.0001) and poorer differentiation (P = 0.0021). Furthermore, both univariate (P = 0.0019) and multivariate (P = 0.0003; hazard ratio = 3.132) analysis of the patients' survival revealed that the prognosis became significantly poorer with progression from staining pattern A to D. Increased laminin-5 gamma2 chain immunoreactivity, which may reflect a high invasive potential of cancer cells, is a factor indicative of a poor prognosis for patients with squamous cell carcinoma of the tongue.

Effects of urinary trypsin inhibitor on the invasion of reconstituted basement membranes by ovarian cancer cells

Using the human ovarian cancer cell line HOC-1, we investigated the effects of urinary trypsin inhibitor (UTI) purified from human urine and its related synthetic peptides on the invasive potential of cancer cells in an in vitro assay. Invasiveness of tumor cells was determined using a modified Boyden chamber and a reconstituted basement membrane Matrigel. Three peptides (peptide 1, peptide 2, and peptide 3), representing sequences within UTI, were synthesized. HOC-1 cells showed detectable and reproducible levels of expression of surface urokinase-type plasminogen activator (uPA) and plasminogen/plasmin by cell ELISAs and enzyme assays. UTI was found to strongly inhibit plasmin and human leukocyte elastase (HLE). Peptide 2 and peptide 3 specifically inhibit HLE and plasmin activity respectively. Peptide 1 has essentially no inhibitory activity. Treatment with UTI and peptide 3 reduces the incidence of invasion, whereas peptide 1 and peptide 2 do not affect invasion. The inhibitory effect on cell invasion is dose-dependent. The proteolytic enzyme plasmin may be involved in human ovarian cancer invasion in extracellular matrix degradation, and the use of UTI and peptide 3 that inhibits plasmin specifically reduces invasion by tumor cells.