Colorectal cancer liver metastatic growth depends on PAD4-driven citrullination of the extracellular matrix.

Citrullination of proteins, a post-translational conversion of arginine residues to citrulline, is recognized in rheumatoid arthritis, but largely undocumented in cancer. Here we show that citrullination of the extracellular matrix by cancer cell derived peptidylarginine deiminase 4 (PAD4) is essential for the growth of liver metastases from colorectal cancer (CRC). Using proteomics, we demonstrate that liver metastases exhibit higher levels of citrullination and PAD4 than unaffected liver, primary CRC or adjacent colonic mucosa. Functional significance for citrullination in metastatic growth is evident in murine models where inhibition of citrullination substantially reduces liver metastatic burden. Additionally, citrullination of a key matrix component collagen type I promotes greater adhesion and decreased migration of CRC cells along with increased expression of characteristic epithelial markers, suggesting a role for citrullination in promoting mesenchymal-to-epithelial transition and liver metastasis. Overall, our study reveals the potential for PAD4-dependant citrullination to drive the progression of CRC liver metastasis.

Tumor-infiltrating monocytes/macrophages promote tumor invasion and migration by upregulating S100A8 and S100A9 expression in cancer cells.

Myeloid cells promote the development of distant metastases, but little is known about the molecular mechanisms underlying this process. Here we have begun to uncover the effects of myeloid cells on cancer cells in a mouse model of liver metastasis. Monocytes/macrophages, but not granulocytes, isolated from experimental liver metastases stimulated migration and invasion of MC38 colon and Lewis lung carcinoma cells. In response to conditioned media from tumor-infiltrating monocytes/macrophages, cancer cells upregulated S100a8 and S100a9 messenger RNA expression through an extracellular signal-related kinase-dependent mechanism. Suppression of S100A8 and S100A9 in cancer cells using short hairpin RNA significantly diminished migration and invasion in culture. Downregulation of S100A8 and S100A9 had no effect on subcutaneous tumor growth. However, colony size was greatly reduced in liver metastases with decreased invasion into adjacent tissue. In tissue culture and in the liver colonies derived from cancer cells with knockdown of S100A8 and S100A9, MMP2 and MMP9 expression was decreased, consistent with the reduction in migration and invasion. Our findings demonstrate that monocytes/macrophages in the metastatic liver microenvironment induce S100A8 and S100A9 in cancer cells, and that these proteins are essential for tumor cell migration and invasion. S100A8 and S100A9, however, are not responsible for stimulation of proliferation. This study implicates S100A8 and S100A9 as important mediators of tumor cell aggressiveness, and highlights the therapeutic potential of S100A8 and S100A9 for interference of metastasis.

Targeting ATR in vivo using the novel inhibitor VE-822 results in selective sensitization of pancreatic tumors to radiation.

Combined radiochemotherapy is the currently used therapy for locally advanced pancreatic ductal adenocarcinoma (PDAC), but normal tissue toxicity limits its application. Here we test the hypothesis that inhibition of ATR (ATM-Rad3-related) could increase the sensitivity of the cancer cells to radiation or chemotherapy without affecting normal cells. We tested VE-822, an ATR inhibitor, for in vitro and in vivo radiosensitization. Chk1 phosphorylation was used to indicate ATR activity, γH2AX and 53BP1 foci as evidence of DNA damage and Rad51 foci for homologous recombination activity. Sensitivity to radiation (XRT) and gemcitabine was measured with clonogenic assays in vitro and tumor growth delay in vivo. Murine intestinal damage was evaluated after abdominal XRT. VE-822 inhibited ATR in vitro and in vivo. VE-822 decreased maintenance of cell-cycle checkpoints, increased persistent DNA damage and decreased homologous recombination in irradiated cancer cells. VE-822 decreased survival of pancreatic cancer cells but not normal cells in response to XRT or gemcitabine. VE-822 markedly prolonged growth delay of pancreatic cancer xenografts after XRT and gemcitabine-based chemoradiation without augmenting normal cell or tissue toxicity. These findings support ATR inhibition as a promising new approach to improve the therapeutic ration of radiochemotherapy for patients with PDAC.

On the dual roles and polarized phenotypes of neutrophils in tumor development and progression.

Inconsistencies plague our understanding of the role of neutrophils in cancer and the literature provides evidence for a duality in neutrophil activity on the outcome of cancer. Here, the different effects of neutrophils during the multiple subprocesses of cancer development and progression are overviewed, in order to gain insight into the features of both antitumoral and protumoral tumor-associated neutrophils (TAN). Neutrophils can counteract the progression of malignancies through tumor cytotoxicity, tumor rejection and enhancement of antitumoral immune memory. These cells have recently been phenotypically denominated N1 neutrophils. Recent studies indicate that cytokines, such as TGF-ß and IFN-ß, are involved in directing neutrophil polarization by the tumor microenvironment. With the opposite polarity, N2 neutrophils may be detrimental for the host and beneficial for tumor growth, invasion and metastasis, e.g. through proteolysis of extracellullar matrix components, promotion of angiogenesis and mediation of immunosuppression.

Farnesyltransferase inhibitors as radiation sensitizers.

PURPOSE: The inhibition of activated Ras combined with radiotherapy was identified as a potential method for radiosensitization. MATERIALS AND METHODS: Immunoblotting was used to control for prenylation inhibition of the respective Ras isoforms and for changes in activity of downstream proteins. Clonogenic assays with human and rodent tumour cell lines and transfected cell lines served for the testing of radiosensitivity. Xenograft tumours were treated with farnesyl transferase inhibitors and radiation and assayed for ex vivo plating efficiency, regrowth of tumours and EF5 staining for detection of hypoxia. Concurrent treatment with L-778,123 and radiotherapy was performed in non-small cell lung cancer (NSCLC) and head and neck cancer (HNC) patients. RESULTS: Blocking the prenylation of Ras proteins in cell lines with Ras activated by mutations or receptor signalling resulted in radiation sensitization in in vitro and in vivo. The PI3 kinase downstream pathway was identified as a contributor to Ras-mediated radiation resistance. Additionally, increased oxygenation of xenograft tumours was observed after FTI treatment. Combined treatment in a phase I study was safe and effective in NSCLC and HNC. CONCLUSIONS: Tumour cells with activated Ras were sensitized to radiation. Unravelling the underlying mechanisms promises to lead to even more specific drugs with higher potency and safety.

The Ras radiation resistance pathway.

The critical pathways determining the resistance of tumor cells to ionizing radiation are poorly defined. Because the ras oncogene, a gene activated in many human cancers treated with radiotherapy, can induce increased radioresistance, we have asked which Ras effector pathways are significant in conferring a survival advantage to tumor cells. The phosphoinositide-3-kinase (PI3K) inhibitor LY294002 radiosensitized cells bearing mutant ras oncogenes, but the survival of cells with wild-type ras was not affected. Inhibition of the PI3K downstream target p70S6K by rapamycin, the Raf-MEK-MAPK pathway with PD98059, or the Ras-MEK kinase-p38 pathway with SB203580 had no effect on radiation survival in cells with oncogenic ras. Expression of active PI3K in cells with wild-type ras resulted in increased radiation resistance that could be inhibited by LY294002. These experiments have indicated the importance of PI3K in mediating enhanced radioresistance and have implicated PI3K as a potential target for specific radiosensitization of tumors.

Decreased cyclin B1 expression contributes to G2 delay in human brain tumor cells after treatment with camptothecin.

DNA damage produces delayed mitosis (G2/M delay) in proliferating cells, and shortening the delay sensitizes human malignant glioma and medulloblastoma cells to cytotoxic chemotherapy. Although activation of the cyclin-dependent kinase CDC2 mediates G2/M transition in all tumor cells studied to date, regulation of CDC2 varies between tumor types. Persistent hyperphosphorylation of kinase and reduced cyclin expression have been implicated as mediators of treatment-induced G2 delay in different tumor models. To evaluate regulation of G2/M transition in human brain tumors, we studied the expression and/or activity of CDC2 kinase and cyclins A and B1 in U-251 MG and DAOY medulloblastoma cells after their treatment with camptothecin (CPT). Synchronized cells were treated during S phase, then harvested at predetermined intervals for evaluation of cell cycle kinetics, kinase activity mRNA, and protein expression. CPT produced G2 delay associated with decreased CDC2 kinase activity and cyclin B1 expression. Kinase activity was associated with CDC2 bound to cyclin B1, not cyclin A, in both cell lines. Cyclin A mRNA and protein expression were reduced after CPT treatment; however, decreased protein expression was short lived and moderate in the glioma and primitive neuroectodermal tumor/medulloblastoma cells, respectively. We conclude that G2 delay is a common response of brain tumor cells to chemotherapy with topoisomerase I inhibitors and that a mechanism of this delay may be reduced expression of cyclin B1.

Paradoxical effects of trichostatin A: inhibition of NF-Y-associated histone acetyltransferase activity, phosphorylation of hGCN5 and downregulation of cyclin A and B1 mRNA.

Trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), is widely used to study the role of histone acetylation in gene expression, since genes that use histone acetylation as a means of regulating expression may be up regulated when TSA is added. In this study, however, we show that TSA has an unexpected paradoxical effect leading to inhibition of NF-Y-associated histone acetyl transferase (HAT) activity and phosphorylation of the HAT, hGCN5. TSA treatment of cells resulted in diminished levels of NF-Y-associated HAT activity without changes in NF-Y(A) amount. hGCN5 is one of the HATs known to associate with NF-Y. The association of hGCN5 with NF-Y was not altered by TSA treatment. The enzymatic activity of hGCN5 is known to be inhibited by phosphorylation. TSA treatment of Hela cells resulted in phosphorylation of hGCN5. Exposure of the NF-Y immunoprecipitates from TSA-treated cells to a phosphatase resulted in enhanced HAT activity. We have also shown that the mRNA levels of several genes, cyclin B1 and cyclin A, are downregulated by TSA; these effects do not require protein synthesis and the downregulation of cyclin B1 by TSA occurs through transcription. These results suggest that TSA can have contradictory effects, on one hand stimulating HAT activity in general by inhibition of HDACs, but also resulting in inhibition of NF-Y-associated HAT activity and phosphorylation of hGCN5.

The farnesyltransferase inhibitor L744,832 reduces hypoxia in tumors expressing activated H-ras.

Many tumors contain extensive regions of hypoxia. Because hypoxic cells are markedly more resistant to killing by radiation, repeated attempts have been made to improve the oxygenation of tumors to enhance radiotherapy. We have studied the oxygenation of tumor xenografts in nude mice after treatment with the farnesyltransferase inhibitor L744,832. Hypoxia was assessed by measuring the binding of the hypoxic cell marker pentafluorinated 2-nitroimidazole. We show that xenografts from two tumor cell lines with mutations in H-ras had markedly improved oxygenation after farnesyltransferase treatment. In contrast, xenografts from two tumors without ras mutations had equivalent hypoxia regardless of treatment. The effect on tumor oxygenation could be detected at 3 days and remained after 7 days of treatment. These results indicate that treatment with farnesyltransferase inhibitors can alter the oxygenation of certain tumors and suggest that such treatment might be useful in the radiosensitization of these tumors.

Apoptosis: an early event in metastatic inefficiency.

Whereas large numbers of cells from a primary tumor may gain access to the circulation, few of them will give rise to metastases. The mechanism of elimination of these tumor cells, often termed "metastatic inefficiency," is poorly understood. In this study, we show that apoptosis in the lungs within 1-2 days of introduction of the cells is an important component of metastatic inefficiency. First, we show that death of transformed, metastatic rat embryo cells occurred via apoptosis in the lungs 24-48 h after injection into the circulation. Second, we show that Bcl-2 overexpression in these cells inhibited apoptosis in culture and also conferred resistance to apoptosis in vivo in the lungs 24-48 h after injection. This inhibition of apoptosis led to significantly more macroscopic metastases. Third, comparison between the extent of apoptosis by a poorly metastatic cell line to that by a highly metastatic cell line 24 h after injection in the lungs revealed more apoptosis by the poorly metastatic cell line. These results indicate that apoptosis, which occurs at 24-48 h after hematogenous dissemination in the lungs is an important determinant of metastatic inefficiency. Although prior work has shown an association between apoptosis in culture and metastasis in vivo, this work shows that apoptosis in vivo corresponds to decreased metastasis in vivo.

GeneTransfection for Metastasis Research Using Animal Models.

One means of testing a candidate gene for involvement in the metastatic process is to alter the expression of that gene in a tumor cell and then to test the metastatic potential of the altered cells. In designing such experiments, it is crucial to take into account the factor of tumor heterogeneity (1). Some cell lines or cultures contain highly heterogeneous populations in regard to metastasis. Upon cloning these cells, some clones will be highly metastatic, but others will not. Thus, selection of a clone itself might skew the results if only a few clones are evaluated. Furthermore, the use of pooled populations after transfection must be considered with caution because the population may contain cells with a variety of metastatic potentials. To avoid these problems, it is necessary to do a preliminary experiment in which the chosen parental cell is subcloned and the subclones are tested for metastatic behavior. If the subclones have a similar metastatic potential to the parental line, then it should be a suitable recipient for metastasis studies. If, however, there is considerable variability in the subclones, either the subclones can be tested for stability or another more stable cell line should be sought.

Direct evidence for the contribution of activated N-ras and K-ras oncogenes to increased intrinsic radiation resistance in human tumor cell lines.

Transformation with ras oncogenes results in increased radiation sur vival in many but not all cells. In addition, prenyltransferase inhibitors which inhibit ras proteins by blocking posttranslational modification radiosensitize cells with oncogenic ras. These findings suggest that oncogenic ras contributes to intrinsic radiation resistance. However, because introduction of ras oncogenes does not increase radiation survival in all cells and because prenyltransferase inhibitors target molecules other than ras, these studies left the conclusion that ras increases the intrinsic radi ation resistance of tumor cells in doubt. Here we show that genetic inactivation of K- or N-ras oncogenes in human tumor cells (DLD-1 and HT1080, respectively) leads to increased radiosensitivity. Reintroduction of the activated N-ras gene into the HT1080 line, having lost its mutant allele, resulted in increased radiation resistance. This study lends further support to the hypothesis that expression of activated ras can contribute to intrinsic radiation resistance in human tumor cells and extends this finding to the K- and N- members of the ras family. These findings support the development of strategies that target ras for inactivation in the treatment of cancer.

Matrix metalloproteinase 9 promoter activity is induced coincident with invasion during tumor progression.

Matrix metalloproteinase 9 (MMP-9, also known as gelatinase B or 92-kd Type IV collagenase) is overexpressed in many human and murine cancers. We induced carcinomas in mice carrying a transgene that links the MMP-9 promoter to the reporter ss-galactosidase so that activation of the MMP-9 promoter would be indicated by ss-galactosidase. Mammary carcinomas were induced by mating the MMP-9 promoter reporter transgenic mice with mice carrying a transgene for murine mammary tumor virus promoter linked to polyoma middle T antigen, a transgene that leads to rapid development of mammary tumors in female mice. None of the hyperplastic mammary glands and none of the carcinomas in situ expressed ss-galactosidase. However, all invasive tumors had evidence of ss-galactosidase expression. In addition to the breast carcinomas, a malignant teratoma in a female and a papillary adenocarcinoma in the pelvic region of a male arose and were also ss-galactosidase positive. We also induced skin tumors in the mice with the MMP-9 reporter transgene with 7, 12-dimethylbenz[a]anthracene (DMBA) treatment followed by phorbol 12 myristate 13-acetate (TPA). None of the papillomas or in situ carcinomas showed any ss-galactosidase expression, but expression was seen in invasive carcinoma. Although normal skin epithelial cells did not express ss-galactosidase, we did find staining in a few cells at the duct of the sebaceous gland at the base of the hair follicles. The MMP-9 reporter transgene did not lead to expression in the alveolar macrophages, confirming that additional upstream sequences are required for expression in macrophages. These experiments have revealed that MMP-9 promoter activity is induced coincident with invasion during tumor progression. Furthermore, this indicates that the more proximal upstream elements of the promoter are sufficient for MMP-9 transcription during tumor progression.

Farnesyltransferase inhibitors potentiate the antitumor effect of radiation on a human tumor xenograft expressing activated HRAS.

Successful radiosensitization requires that tumor cells become more radiosensitive without causing an equivalent reduction in the survival of cells of the surrounding normal tissues. Since tumor cell radiosensitivity can be influenced by RAS oncogene activation, we have hypothesized that inhibition of oncogenic RAS activity would lead to radiosensitization of tumors with activated RAS. We previously showed in tissue culture that prenyltransferase treatment of cells with activated RAS resulted in radiosensitization, whereas treatment of cells with wild-type RAS had no effect on radiation survival. Here we ask whether the findings obtained in vitro have applicability in vivo. We found that treatment of nude mice bearing T24 tumor cell xenografts with farnesyltransferase inhibitors resulted in a significant and synergistic reduction in tumor cell survival after irradiation. The regrowth of T24 tumors expressing activated RAS was also significantly prolonged by the addition of treatment with farnesyltransferase inhibitors compared to the regrowth after irradiation alone. In contrast, there was no effect on the radiosensitivity of HT-29 tumors expressing wild-type RAS. These results demonstrate that specific radiosensitization of tumors expressing activated RAS oncogenes can be obtained in vivo.

RAS-Mediated radiation resistance is not linked to MAP kinase activation in two bladder carcinoma cell lines.

The expression of activated RAS oncogenes has been shown to increase radioresistance in a number of cell lines. The pathways by which RAS leads to radioresistance, however, are unknown. RAS activates several signal transduction pathways, with the RAF-MAP2K-MAP kinase pathway perhaps the best studied. MAP kinase has also been shown to be activated by radiation through this pathway. Given the important role of MAP kinase in multiple signaling events, we asked if radioresistance induced by RAS was mediated through the activation of MAPK. Cells of two human bladder carcinoma cell lines were used, one with a mutated oncogenic HRAS (T24) and other with a wild-type HRAS (RT4). The surviving fraction after exposure to 2 Gy of radiation (SF2) for the T24 cell lines was found to be 0.62, whereas that for RT4 cells was 0.40. Treatment with the farnesyl transferase inhibitor (FTI) L744,832, which inhibits RAS processing and activity, decreased the SF2 of T24 cells to 0.29, whereas the SF2 of RT4 cells remained unchanged after FTI treatment, thus demonstrating the importance of RAS activation to the radiosensitivity of cells with mutated RAS. MAP kinase activation was found to be constitutive and dependent on RAS in T24 cells, while it was inducible by radiation and was independent of RAS in RT4 cells. Treatment of both cell lines with the MAP2K inhibitor PD98059 inhibited MAPK activation; however, inhibiting MAPK activation had no effect on radiation survival of T24 or RT4 cells. These data indicate that MAPK activation does not contribute to RAS-induced radioresistance in this system.

DNA as an important target in radiation-induced apoptosis of MYC and MYC plus RAS transfected rat embryo fibroblasts.

PURPOSE: This study uses a radiation chemistry approach to determine if DNA is an important target for radiation-induced apoptosis of myc (MR4) and myc plus ras (3.7) transfected rat embryo fibroblast cell lines. MATERIALS AND METHODS: The radiation protection efficiency of four thiols was compared with net molecular charge ranging from -1 to +2: mercaptopropionic acid (Z= -1), mercaptoethanol (Z=0), cysteamine (Z= +1), N(2-mercaptoethyl)-1,3-diaminopropane (Z= +2). Protection factors were determined for these thiols against radiation-induced apoptosis (Apoalert assay), mitotic cell death (clonogenic assay) and double-strand break (dsb) induction (pulse field gel electrophoresis) in MR4 and 3.7 cells. Theoretical protection factors for these thiols against dsb induction were also calculated from second-order chemical repair constants for single-strand breaks (ssb) and the concentration of added thiols in MR4 and 3.7 cell lines. RESULTS: The charge-dependent increases observed for measured protection factors against radiation-induced apoptosis did not differ significantly between the two cell lines, nor did they differ significantly from the corresponding increases observed for radiation-induced mitotic cell killing and for induction of dsb. The calculated protection factor for dsb also showed a thiol charge-dependent increase similar to the measured protection factors for all of the other parameters studied. CONCLUSIONS: These results are consistent with the hypothesis that DNA is an important target for radiation-induced apoptosis.

BRCA1 effects on the cell cycle and the DNA damage response are linked to altered gene expression.

The breast and ovarian cancer susceptibility gene product BRCA1 has been reported to be expressed in a cell cycle-dependent manner; possess transcriptional activity; associate with several proteins, including the p53 tumor suppressor; and play an integral role in certain types of DNA repair. We show here that ectopic expression of BRCA1 using an adenovirus vector (Ad-BRCA1) leads to dephosphorylation of the retinoblastoma protein accompanied by a decrease in cyclin-dependent kinase activity. Flow cytometric analysis on Ad-BRCA1-infected cells revealed a G(1) or G(2) phase accumulation. High density cDNA array screening of colon, lung, and breast cancer cells identified several genes affected by BRCA1 expression in a p53-independent manner, including DNA damage response genes and genes involved in cell cycle control. Notable changes included induction of the GADD45 and GADD153 genes and a reduction in cyclin B1 expression. Therefore, BRCA1 has the potential to modulate the expression of genes and function of proteins involved in cell cycle control and DNA damage response pathways.

Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis.

Metastasis is a frequent complication of cancer, yet the process through which circulating tumor cells form distant colonies is poorly understood. We have been able to observe the steps in early hematogenous metastasis by epifluorescence microscopy of tumor cells expressing green fluorescent protein in subpleural microvessels in intact, perfused mouse and rat lungs. Metastatic tumor cells attached to the endothelia of pulmonary pre-capillary arterioles and capillaries. Extravasation of tumor cells was rare, and it seemed that the transmigrated cells were cleared quickly by the lung, leaving only the endothelium-attached cells as the seeds of secondary tumors. Early colonies were entirely within the blood vessels. Although most models of metastasis include an extravasation step early in the process, here we show that in the lung, metastasis is initiated by attachment of tumor cells to the vascular endothelium and that hematogenous metastasis originates from the proliferation of attached intravascular tumor cells rather than from extravasated ones. Intravascular metastasis formation would make early colonies especially vulnerable to intravascular drugs, and this possibility has potential for the prevention of tumor cell attachment to the endothelium.