Tumor kinase activity in locally advanced rectal cancer: angiogenic signaling and early systemic dissemination.

Tumor hypoxia is a common determinant of resistance to cytotoxic therapies and metastatic behavior. In rectal cancer patients receiving preoperative chemoradiotherapy, tyrosine kinase activities in tumors with poor and good treatment responses were found to differ. Given that tyrosine kinase signaling mediates hypoxic tissue adaptation, the present study examined whether tumor kinase activity might also correlate with systemic dissemination of rectal cancer. Immunomagnetic selection of disseminated tumor cells (DTC) from bone marrow aspirates was undertaken in 55 patients with locally advanced rectal cancer. Using peptide arrays with 144 tyrosine kinase substrates, phosphopeptide signatures were generated from patients' baseline tumor biopsies, to study association between DTC and tumor tyrosine kinase activity regulated ex vivo by sunitinib. Disseminated tumor cells were detected in 60% of cases, and these patients had significantly poorer metastasis-free survival than patients without DTC. Phosphorylation of 31 array tyrosine kinase substrates by tumor samples was significantly more strongly inhibited by sunitinib in the DTC-negative patients, with a number of phosphosubstrates representing angiogenic factors. In this cohort of rectal cancer patients, tumor phenotypes defined by a subset of tyrosine kinase activities correlating with weak ex vivo inhibition by sunitinib, was associated with early systemic dissemination.

Coexpression and nuclear colocalization of metastasis-promoting protein S100A4 and p53 without mutual regulation in colorectal carcinoma.

Nuclear localization of the metastasis-associated protein S100A4 has been shown to correlate with advanced disease stage in primary colorectal carcinomas (CRC), but nuclear function and its relevance for the metastatic capacity of tumor cells is still unclear. Among several nuclear interacting protein partners suggested for S100A4, the tumor suppressor protein p53 has attracted particular interest, and previous studies suggest direct and indirect modes of interaction between the two proteins. The present study was undertaken to assess coexpression and potential interaction in CRC. TP53 mutational status and S100A4 expression were investigated in a selected series of primary CRC specimens (n = 40) and cell lines (n = 17) using DNA sequencing, western blot, and double immunostaining. Additionally, S100A4 and p53 were experimentally up- and down-regulated in vitro to assess reciprocal effects. For the first time, S100A4 and p53 coexpression was demonstrated in individual CRC cells, with nuclear colocalization as a particularly interesting feature. In contrast to previous studies, no correlation was observed between TP53 mutational status and S100A4 expression, and no evidence was obtained to support reciprocal regulation between the two molecules in the HCT116 isogenic cell line model. In conclusion, S100A4 and p53 were shown to be colocalized in individual nuclei of CRC cells, and it might be speculated whether the proteins interact in this subcellular compartment.

Inhibitory effects of oxaliplatin in experimental radiation treatment of colorectal carcinoma: does oxaliplatin improve 5-fluorouracil-dependent radiosensitivity?

BACKGROUND AND PURPOSE: New chemoradiotherapy regimens for rectal cancer with integration of oxaliplatin with 5-fluorouracil-based therapy are being actively investigated. However, only limited preclinical data are available on oxaliplatin as radiosensitizer in colorectal carcinoma. MATERIALS AND METHODS: A human colorectal carcinoma cell line (HT29) was exposed to ionizing radiation with or without oxaliplatin and/or 5-fluorouracil, upon which clonogenicity and cell cycle profiles were analyzed. HT29 xenografts were treated for two weeks with daily radiation and/or the oral 5-fluorouracil analog capecitabine with or without oxaliplatin once weekly, and tumor volumes were followed for up to 60 days. RESULTS: Pretreatment of HT29 cells with oxaliplatin for 2h, followed by radiation and a 48-h exposure to 5-fluorouracil, resulted in increased radiocytotoxicity, and combination index analysis indicated synergistic effects. Ionizing radiation and oxaliplatin induced cell cycle G(2)/M phase arrest in HT29 cells at distinctly different time points. Growth of HT29 xenografts was clearly inhibited by radiation. Capecitabine and oxaliplatin together significantly improved the inhibitory effect, but oxaliplatin did not add to the growth inhibitory response induced by radiation plus capecitabine. CONCLUSIONS: The combination of oxaliplatin and 5-fluorouracil sensitized colorectal carcinoma cells to ionizing radiation in vitro. In vivo, however, oxaliplatin did not convincingly improve the increased radiocytotoxicity conferred by capecitabine treatment. In the absence of conclusive clinical evidence, the integration of OXA into combined-modality treatment of rectal cancer must remain controversial.

Specific isolation of disseminated cancer cells: a new method permitting sensitive detection of target molecules of diagnostic and therapeutic value.

Molecular studies of rare cells, such as circulating cancer cells, require efficient pre-enrichment steps to obtain a pure population of target cells for further characterization. We have developed a two-step approach, starting with immunomagnetic enrichment, followed by specific isolation of individual, easily identifiable bead-rosetted target cells using a new semi-automated CellPick system. With this procedure, 1-50 live target cells can now be isolated. As a model system, we spiked a small number of tumor cells into millions of normal mononuclear cells (MNCs). Efficient isolation of pure target cells was obtained by use of the CellPick system, and the nature of isolated, bead-rosetted cells was verified by use of FISH. Single breast cancer cells were picked directly into an RNA preserving lysis buffer, reverse transcribed, and PCR amplified with two cDNA specific primer sets. With the isolated cells we consistently obtained both ubiquitously expressed and tumor cell specific PCR products. We also performed a successful mutation analysis of single cells using PCR and cycling temperature capillary electrophoresis (CTCE). This may have significant clinical implications in cancer and in other diseases, e.g. in characterizing micrometastatic cancer cells in blood and lymph nodes to help identifying patients who most likely will respond to therapies like tyrosine kinase inhibitors and compounds targeting specific mutations. By use of the CellPick system it is possible to specifically isolate bead-rosetted or otherwise labelled target cells from a heterogeneous cell population for further molecular characterization.

Radiosensitization of colorectal carcinoma cell lines by histone deacetylase inhibition.

BACKGROUND: The tumor response to preoperative radiotherapy of locally advanced rectal cancer varies greatly, warranting the use of experimental models to assay the efficacy of molecular targeting agents in rectal cancer radiosensitization. Histone deacetylase (HDAC) inhibitors, agents that cause hyperacetylation of histone proteins and thereby remodeling of chromatin structure, may override cell cycle checkpoint responses to DNA damage and amplify radiation-induced tumor cell death. METHODS: Human colorectal carcinoma cell lines were exposed to ionizing radiation and HDAC inhibitors, and cell cycle profiles and regulatory factors, as well as clonogenicity, were analyzed. RESULTS: In addition to G(2)/M phase arrest following irradiation, the cell lines displayed cell cycle responses typical for either intact or defective p53 function (the presence or absence, respectively, of radiation-induced expression of the cell cycle inhibitor p21 and subsequent accumulation of G(1) phase cells). In contrast, histone acetylation was associated with complete depletion of the G1 population of cells with functional p53 but accumulation of both G(1) and G(2)/M populations of cells with defective p53. The cellular phenotypes upon HDAC inhibition were consistent with the observed repression of Polo-like kinase-1, a regulatory G(2)/M phase kinase. Following pre-treatment with HDAC inhibitors currently undergoing clinical investigation, the inhibitory effect of ionizing radiation on clonogenicity was significantly amplified. CONCLUSION: In these experimental models, HDAC inhibition sensitized the tumor cells to ionizing radiation, which is in accordance with the concept of increased probability of tumor cell death when chromatin structure is modified.

Activin A suppresses neuroblastoma xenograft tumor growth via antimitotic and antiangiogenic mechanisms.

The tumor suppressor function of activin A, together with our findings that activin A is an inhibitor of angiogenesis, which is down-regulated by the N-MYC oncogene, prompted us to investigate in more detail its role in the malignant transformation process of neuroblastomas. Indeed, neuroblastoma cells with restored activin A expression exhibited a diminished proliferation rate and formed smaller xenograft tumors with reduced vascularity, whereas lung metastasis rate remained unchanged. In agreement with the decreased vascularity of the xenograft tumors, activin A inhibited several crucial angiogenic responses of cultured endothelial cells, such as proteolytic activity, migration, and proliferation. Endothelial cell proliferation, activin A, or its constitutively active activin receptor-like kinase 4 receptor (ALK4T206D), increased the expression of CDKN1A (p21), CDKN2B (p15), and CDKN1B (p27) CDK inhibitors and down-regulated the expression of vascular endothelial growth factor receptor-2, the receptor of a key angiogenic factor in cancer. The constitutively active forms of SMAD2 and SMAD3 were both capable of inhibiting endothelial cell proliferation, whereas the dominant-negative forms of SMAD3 and SMAD4 released the inhibitory effect of activin A on endothelial cell proliferation by only 20%. Thus, the effects of activin A on endothelial cell proliferation seem to be conveyed via the ALK4/SMAD2-SMAD3 pathways, however, non-SMAD cascades may also contribute. These results provide novel information regarding the role of activin A in the malignant transformation process of neuroblastomas and the molecular mechanisms involved in regulating angiogenesis thereof.

Twelve colorectal cancer cell lines exhibit highly variable growth and metastatic capacities in an orthotopic model in nude mice.

Orthotopic tumour models for colorectal cancer are a complementary tool for the study of tumours in vivo. They are more closely related to human cancer than are subcutaneous tumour models, since evaluation of spontaneous metastasis formation is possible. In the present study, fragments of subcutaneous xenografts established from 12 well-described and generally available colorectal cancer cell lines were implanted in the caecum of nude mice and tumour growth and metastatic events registered. The results showed considerable differences between the cell lines with respect to take rate, tumour growth and metastatic ability. This resulted in variable disease progression that seemingly reflects clinically relevant heterogeneity. The most common metastatic findings were mesenteric lymph-node metastases, occurring at variable frequency in tumour-bearing mice with 10 out of 12 cell lines, whereas only one line gave rise to liver metastases, in two of 10 animals. The study provides useful background information on the 12 colorectal cancer cell lines in a clinically relevant orthotopic tumour model.

Immunofluorometric assay for the metastasis-related protein S100A4: release of S100A4 from normal blood cells prohibits the use of S100A4 as a tumor marker in plasma and serum.

The metastasis-related protein S100A4 is released from tumor cells, and since it is highly expressed in colorectal cancer (CRC), it could be a potential tumor marker in plasma or serum. Monoclonal antibodies (MAbs) were raised against human recombinant S100A4 and shown to detect native and recombinant antigen with high sensitivity and specificity. Using two MAbs, an immunofluorometric assay (IFMA) was established to detect S100A4 in clinical samples with high sensitivity and precision. S100A4 in plasma and serum from patients with CRC was highly influenced by sample hemolysis. Both red blood cells and mononuclear cells were found to contain S100A4, possibly contributing to the measured levels in serum and plasma. Since even very low-level hemolysis influenced the results, a potential contribution from an S100A4-expressing tumor could not be discerned, indicating that S100A4 is not suitable as a plasma or serum tumor marker for CRC. The antibodies and the IFMA may still be useful for research purposes.

Nuclear localization of the metastasis-related protein S100A4 correlates with tumour stage in colorectal cancer.

A large number of experimental studies have linked the S100A4 gene product to the metastatic phenotype of cancer cells and clinical evidence indicates a correlation between S100A4 expression and poor prognosis in several cancer types. The aim of the present study was to analyse the expression of the S100A4 protein in colorectal cancer. Paraffin-embedded samples from 277 colorectal cancer patients were immunostained with anti-S100A4 antibody. Cytoplasmic staining was observed in 178 of 277 samples (64%), whereas, unexpectedly, nuclear expression of S100A4 was found in 88 of 277 of the samples (32%). This novel finding was confirmed by western blot analysis of nuclear fractions isolated from frozen tumour tissue. Statistical analysis revealed a significant correlation between nuclear expression of S100A4 and tumour stage at diagnosis, while there was no such correlation between cytoplasmic staining and tumour stage. The nuclear localization of S100A4 in colorectal cancer and its relationship to tumour stage suggest that this protein may be involved in gene regulatory pathways of relevance to the metastatic phenotype of cancer cells.

N-myc oncogene overexpression down-regulates IL-6; evidence that IL-6 inhibits angiogenesis and suppresses neuroblastoma tumor growth.

Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies. Tumor angiogenesis appears to be governed by alterations of tumor suppressor or oncogenes operant in a broad range of tumors. We have addressed this issue in neuroblastoma, a malignancy characterized by the near-exclusive amplification and overexpression of the N-Myc oncogene. Here, we report that N-Myc overexpression results in down-regulation of interleukin-6 (IL-6) and that IL-6 is an inhibitor of endothelial cell proliferation and VEGF-induced rabbit corneal angiogenesis. STAT3 is instrumental for IL-6 activity as infection with adenoviruses expressing a phosphorylation deficient STAT3 mutant renders endothelial cells insensitive to the antiproliferative action of IL-6. Finally, though IL-6 does not influence neuroblastoma cell growth, IL-6-expressing xenograft tumors in mice exhibit reduced neovascularization and suppressed growth. Our data shed new light on the mechanisms by which N-myc oncogene amplification enhances the malignant phenotype in neuroblastomas.

Hypoxia promotes lymph node metastasis in human melanoma xenografts by up-regulating the urokinase-type plasminogen activator receptor.

Clinical studies have shown that metastatic spread is associated with hypoxia in the primary tumor. The mechanism behind this association has not been identified and, in fact, it has not been established whether hypoxia induces metastasis or whether the most metastatic cell phenotypes develop the most hypoxic tumors. The present study demonstrates that hypoxia promotes spontaneous lymph node metastasis in R-18 human melanoma xenografts by up-regulating the urokinase-type plasminogen activator receptor (uPAR). Pimonidazole was used as a hypoxia marker, and hypoxia and uPAR expression were detected by immunohistochemistry. R-18 cells were capable of up-regulating uPAR under hypoxic conditions in vitro, as revealed by Western and Northern blot analyses, and uPAR-positive regions showed a high degree of colocalization with hypoxic regions in R-18 tumors. There was a strong correlation between uPAR-positive fraction and hypoxic fraction in individual tumors (P < 0.00001). Incidence of metastases, hypoxic fraction, and uPAR-positive fraction increased with the size of the primary tumor with similar kinetics. Metastatic tumors showed approximately 1.5-fold higher hypoxic fraction (P = 0.00004) and approximately 1.4-fold higher uPAR-positive fraction (P = 0.0003) than nonmetastatic tumors of the same size. Moreover, treatment with neutralizing antibody against uPAR prevented metastasis almost completely. Only 1 of 30 treated mice developed metastases, whereas 14 of 30 control mice were metastasis positive, suggesting that functional uPAR is a prerequisite for lymph node metastasis in R-18 tumors. The study reported here suggests that metastatic spread may be promoted by hypoxia in the primary tumor and identifies the plasminogen activation system as an important target for the treatment of malignant melanoma.