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MCL-1, a member of the antiapoptotic BCL-2 family, is a prosurvival protein with an essential DNA repair function. This study aims to test whether inhibition of protein synthesis by mTOR complex (mTORC) inhibitors depletes MCL-1, suppresses homologous recombination (HR) repair, and sensitizes cancer cells to PARP inhibitors. Treatment with everolimus decreases MCL-1 in colorectal carcinomas and small cell lung cancer (SCLC) cells but not glioblastoma multiforme (GBM) cells with a PTEN mutational background. However, AZD2014, a dual mTORC inhibitor, depletes MCL-1 in GBMs. Further, we show that everolimus decreases 4EBP1 phosphorylation only in colorectal carcinoma, whereas AZD2014 decreases 4EBP1 phosphorylation in both colorectal carcinoma and GBM cells. Combination therapy using everolimus or AZD2014 with olaparib inhibits the growth of clone A and U87-MG xenografts in in vivo and decreases clonogenic survival in in vitro compared with monotherapy. Reintroduction of MCL-1 rescues the survival of cancer cells in response to combination of everolimus or AZD2014 with olaparib. Treatment of cells with mTORC inhibitors and olaparib increases γ-H2AX and 53BP1 foci, decreases BRCA1, RPA, and Rad51 foci, impairs phosphorylation of ATR/Chk1 kinases, and induces necroptosis. In summary, mTORC inhibitors deplete MCL-1 to suppress HR repair and increase sensitivity to olaparib both in in vitro and in xenografts. IMPLICATIONS: Targeting the DNA repair activity of MCL-1 in in vivo for cancer therapy has not been tested. This study demonstrates that depleting MCL-1 sensitizes cancer cells to PARP inhibitors besides eliciting necroptosis, which could stimulate antitumor immunity to improve the therapeutic intervention of cancers., (©2018 American Association for Cancer Research.)

As human colorectal cancer (CRC) cells metastasize to distant sites, they are susceptible to detachment-induced cell death or anoikis - a form of apoptosis that occurs when anchorage-dependent CRC cells go into suspension. Our goal was to identify whether tumor necrosis factor receptor apoptosis-inducing ligand (TRAIL) receptors mediate anoikis in human CRC cells. First, we assessed whether caspases of the extrinsic (caspase-8) or intrinsic (caspase-9) death pathways were involved. Caspase-8 was cleaved during exposure to suspension culture in four CRC lines, and cell death was inhibited by caspase-3 and caspase-8 inhibitors but not by a caspase-9 inhibitor. Gene transcripts in macrophage inflammatory protein-101 (MIP-110), a weakly metastatic human CRC, were increased at least 2-fold for TRAIL-R2 (DR5) and TRAIL after 24 h of suspension culture compared with cells in monolayer culture. The increased expression of DR5 was confirmed at the protein level at 24 h, and exposure of MIP-101 cells to an antagonistic antibody to DR5 decreased caspase-8 activation. The antagonistic antibody to DR5 inhibited anoikis in four human CRC lines. Treatment with an antagonistic DR4 antibody or a neutralizing antibody to TRAIL ligand did not reduce anoikis consistently. Knockdown of DR5 or TRAIL also inhibited anoikis, whereas exogenous TRAIL or FasL did not consistently increase anoikis. In summary, DR5 receptor mediates death signals for anoikis in human CRC cells through the extrinsic apoptotic pathway.

Carcinoembryonic antigen (CEA) is a tumor marker that is associated with metastasis, poor response to chemotherapy of colorectal cancer (CRC), and anoikis, a form of apoptosis caused by cell detachment from matrix that is dependent on TRAIL-R2 (DR5) and caspase-8 activation in CRC. Although CEA is a homophilic binding protein that may provide survival signals through homotypical cell aggregation, we now report that CEA binds TRAIL-R2 (DR5) directly in two-hybrid assays to decrease anoikis through the extrinsic pathway. Deletion of the PELPK sequence (delPELPK) of CEA (delPELPK CEA) restores sensitivity to anoikis while it maintains its cell aggregation function. Wild-type (WT) CEA also increases experimental hepatic metastasis, whereas the delPELPK CEA does not. Thus, membrane CEA interacts with DR5 to inhibit anoikis and increase metastatic potential in CRC.

Malignant cells undergo anoikis as they encounter fluid shear stress during transit to a metastatic site. We postulated that intracellular nitric oxide (NO) contributes to this cell death by comparing the growth of human colorectal carcinoma cells in low fluid shear stress rotated three-dimensional (Rotated 3-D) cultures with growth in static three-dimensional (Static 3-D) cultures on nonadherent surfaces and with two-dimensional monolayer (Monolayer 2-D) cultures. NO, loss of microtubules, and apoptosis increased significantly in Rotated 3-D cultures within 10 min and persisted at 24 h, whereas inhibition of NO synthase decreased apoptosis and intracellular NO and prevented tubulin degradation. Thus, fluid shear stress and three-dimensional growth increases NO synthase and NO to cause tubulin breakdown and induce anoikis. Intracellular NO in malignant cells entering the circulation may be a novel target for metastasis by colorectal carcinoma.

Microscopic infarcts develop within the livers of athymic nude mice during the first 24 h after human colorectal carcinoma (CRC) cells arrest within hepatic sinusoids. Because these regions are reperfused, essentially all weakly metastatic clone A and MIP-101 CRC cells die, whereas many highly metastatic CX-1 CRC cells survive. Because hepatic sinusoidal endothelial cells kill tumor cells in vitro by producing nitric oxide, superoxide anion, and other reactive oxygen and nitrogen species, our purpose was to determine whether reoxygenation of ischemic hepatic cultures in vitro forms toxic oxygen and nitrogen radicals that kill weakly but not highly metastatic CRC cells. CRC cells (10(7)) were labeled with rhodamine-dextran and calcein AM, cultured with cells from one mouse liver in a rotating suspension culture system for up to 24 h, and the metabolic activity of the CRC cells was determined. Liver fragments oxygenated normally before harvest were not toxic to either CRC cell line, but coculture with liver made ischemic by a 3-min ligation of the portal vein and hepatic artery in vivo before harvest and then cultured in oxygenated medium killed 50-70% of weakly metastatic clone A and MIP-101 cells at 24 h but <15% of highly metastatic CX-1 cells. Inhibition of nitric oxide synthase, addition of exogenous superoxide dismutase, but not catalase or hypoxia, during coculture blocked the killing of weakly metastatic CRC cells. Thus, reoxygenation of hepatic parenchymal and nonparenchymal cells after ischemia may form toxic species that eliminate weakly metastatic CRCs within 24 h of their arrest in the liver.

Human colorectal carcinoma (CRC) cell survival for the first 24 h after implantation in the hepatic sinusoid determines its potential to colonize the liver. Nearly 10-fold more highly metastatic CX-1 cells survive within the livers of nude mice 24 h after intrasplenic injection than weakly metastatic clone A cells. Because CRCs contact sinusoidal endothelial cells (SECs) during implantation, we sought to determine whether SECs were more toxic to clone A than to CX-1 cells. When 2 x 10(4) vital dye-labeled CRC cells were added to murine SEC monolayers, more than 30% of clone A cells lost calcein AM fluorescence compared to fewer than 5% of CX-1 cells after 24 h of coculture with SECs. Kupffer cells did not mediate this effect, because neither enriched Kupffer cells nor SECs treated with a Kupffer cell inhibitor altered the SEC-mediated toxic effect to clone A cells. Pretreatment with a nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine, superoxide dismutase, or dexamethasone, blocked SEC-mediated toxicity to clone A cells, whereas calcium chelation and catalase did not. In addition, clone A cells were more sensitive to a superoxide donor, 3-morpholinosydnonimine N-ethylcarbamide, than were CX-1 cells, and neither cell line was sensitive to sodium nitroprusside, a nitric oxide donor. Thus, unstimulated murine SECs produce reactive oxygen species that are selectively toxic to weakly metastatic clone A cells. This may be a mechanism by which host liver cells eliminate weakly metastatic neoplastic cells.

Carcinoembryonic antigen (CEA) may promote experimental metastasis through production of cytokines. The effect of systemic CEA on the production of proinflammatory cytokines was investigated in mice and compared to levels induced by lipopolysaccharide (LPS). Serum concentrations of interleukin (IL)-6 peaked 1 h after an i.v. CEA injection of 40 microg/mouse to 37-54% of the maximal level induced by a 1 microg/mouse injection of LPS in both normal and immunoincompetent mice. The CEA induction of IL-6 was a specific response, because the peptide PELPK (the pentapeptide on CEA that is the ligand for the CEA receptor on Kupffer cells) conjugated to albumin induced 30% of the maximal CEA response for IL-6, whereas the specificity control PELGK-conjugated albumin did not. IL-1alpha and tumor necrosis factor (TNF)-alpha levels after i.v. injection of CEA were only 3-5% of those induced by LPS. The IL-6 responses of mice pretreated with 100 microg/kg genistein were decreased by more than 40%. However, genistein inhibited the TNF-alpha response to LPS by 46% but increased the CEA-induced response by 300%. When murine Kupffer cells were stimulated with LPS or CEA in vitro, LPS increased tyrosine phosphorylation of a Mr 30,000 protein, whereas CEA decreased phosphorylation of a Mr 60,000 protein and did not increase phosphorylation of the Mr 30,000 protein. Thus, i.v. CEA stimulates production of IL-6 and TNF-alpha after binding to Kupffer cells through signal transduction pathways that appear to be different from those stimulated by LPS.

Somatic mutations in DNA mismatch repair genes have been observed in sporadic tumors as well as cell lines and xenografts derived from such tumors implicating genetic defects of mismatch repair genes in the development of such tumors. However, the proportion of sporadic tumors in which mismatch repair genes have been inactivated has not been determined accurately. We have analyzed 66 sporadic colorectal tumors for the expression of hMLH1 by immunohistochemistry and identified 4 tumors that do not express hMLH1. These four colorectal tumors, a colon tumor cell line (SW48) and an endometrial tumor cell line (AN3CA), did not express hMLH1, despite the absence of mutations in its coding sequence. Cytosine methylation of the hMLH1 promoter region was found in these four colorectal tumors, whereas cytosine methylation of the hMLH1 promoter region was absent in adjacent normal tissue or in nine tumors that expressed hMLH1. In addition, cytosine methylation of the hMLH1 promoter region was observed in the SW48 and AN3CA cell lines that do not express hMLH1 but not in four tumor cell lines known to express hMLH1 mRNA. Our data indicate that DNA methylation is likely to be a common mode of mismatch repair gene inactivation in sporadic tumors.

Galectin-1 and galectin-3, galactoside-binding lectins with molecular weights of M(r) 14,500 and 31,000, respectively, are expressed in normal and malignant cells and have been implicated in regulation of cell growth, adhesion, and metastasis. We analyzed the expression of galectins in 21 cultured human colon carcinoma cell lines by immunoblotting. Galectin-1 was detected in only 7, whereas galectin-3 was found in 20 of the cell lines. KM12 cells, which express only galectin-3, were used to isolate this lectin by affinity chromatography, and the purified lectin was used to identify complementary glycoconjugates by blotting. Galectin-3 was shown to bind to human laminin, carcinoembryonic antigen, and lysosome-associated membrane glycoproteins, which are involved in cell adhesion. Galectin-3 was localized on the KM12 cell surface and colocalized with carcinoembryonic antigen. Several endogenous glycoproteins and cell surface proteins of molecular weights in the range M(r) 58,000 to > 200,000, including carcinoembryonic antigen and lysosome-associated membrane glycoproteins, were identified as galectin-3 ligands by coimmunoprecipitation with and affinity chromatography on immobilized galectin-3. These data demonstrate that galectin-3 interacts with several adhesion molecules and suggest that this lectin may have a role in human colon carcinoma cell adhesion.

Coordinate loss of one copy of the p53 gene and mutation of the remaining copy occur in colorectal carcinomas and in many other human malignancies. However, the prevalence of p53 gene mutations in carcinomas which maintain both parental copies of p53 has not previously been evaluated. Moreover, it is not known whether p53 gene mutations are limited to malignant tumors or whether they can also occur in benign neoplasms. To answer these questions, a total of 58 colorectal tumors have been examined; in each tumor, allelic losses were assessed using restriction fragment length polymorphisms and p53 gene mutations were assessed by sequencing cloned polymerase chain reaction products. The following conclusions emerged: (a) p53 gene mutations occurred but were relatively rare in adenomas, regardless of size and whether the adenomas were derived from patients with familial adenomatous polyposis; (b) In carcinomas as well as in adenomas, p53 gene mutations were infrequently observed in tumors which contain both copies of chromosome 17p (17% of 30 tumors), while tumors which lost one copy of chromosome 17p usually had a mutation in the remaining p53 allele (86% of 28 tumors); (c) p53 gene mutations were found at similar frequencies in primary tumor samples and in cell lines derived from tumors. These and other data suggest that the rate limiting step in p53 inactivation is point mutation and that once a mutation occurs, loss of the remaining wild-type allele rapidly follows. Both mutations and allelic losses generally occur near the transition from benign to malignant growth, and the p53 gene may play a causal role in this progression.

The purpose of this study was to compare the expression of O-acetylated sialic acids on normal colonic epithelial cells to that on primary and metastatic human adenocarcinoma of the colon and rectum. In 24 cases, the relative percentages of biosynthetically labeled non-, mono-, di-, and tri-O-acetylated sialic acids were measured after hydrolytic release, separation, and identification by paper chromatography. In one case, the presence of di- and tri-O-acetylated sialic acids was confirmed by fast atom bombardment-mass spectral analysis. Differences were observed in the expression of sialic acids between normal colonic epithelium, "uninvolved" colon mucosa remote to a colonic adenocarcinoma, and colonic adenocarcinoma. The levels of mono- and tri-O-acetylated sialic acids accounted for the difference in the ratios of sialic acids expressed between normal and "uninvolved" colonic mucosa, while the total amount of O-acetylation was unchanged. However, no difference was observed in the relative amounts of non- and O-acetylated sialic acids between either fresh and tissue culture-established colon carcinomas, or fresh and tissue culture-established liver metastasis derived from carcinoma of the colon. The relative expression of these O-acetylated sialic acids molecules appears to vary according to tissue type. This study suggests that individuals with adenocarcinoma of the colon express a field defect resulting in abnormal ratios of O-acetylated sialic acids.

The purpose of these studies was to select and isolate cells with increased liver-metastasizing potential from heterogeneous primary human colon carcinomas (HCCs). Cells derived from a primary HCC classified as Dukes' stage B2 were directly established in culture or were injected into the subcutis, cecum, or spleen of nude mice. Progressively growing tumors were excised, dissociated, and established in culture. Subsequent to implantation into the cecum or spleen of nude mice, cells from all four lines produced only a few liver tumor foci. HCC cells from the few liver metastases were expanded in culture and then injected into the spleen of nude mice to provide a source for further cycles of selection. With each successive in vivo selection cycle, the metastatic ability of the isolated propagated cells increased. Four cycles of selection yielded cell lines with a very high metastatic efficiency in nude mice. In parallel studies using another primary HCC classified as Dukes' stage D, we isolated cell lines that were highly metastatic in nude mice. Successive selection cycles for growth in the liver increased the metastatic properties of the HCC cells, albeit to a lesser extent than it did those of the Dukes' B2 stage HCC. The ability of the HCC cells to produce liver metastases was not due to simple trapping in the liver. In vivo distribution studies using [125I] iododeoxyuridine-labeled tumor cells revealed that, shortly after injection into the spleen, a comparable number of cells with either low or high metastatic properties arrested in the liver. The differences between the low- and high-degree metastatic cells became apparent by 24 h after injection and, by 72 h, only highly metastatic cells survived in the liver. These results demonstrate that hepatic metastasis by HCC cells is a selective process and that the nude mouse model can be useful for isolating highly metastatic HCC cells and for studying the relevant host organ factors that regulate the pathogenesis of metastasis.

The purpose of these studies was to determine whether blood monocytes of patients with different stages of colorectal carcinoma could be activated by various immunomodulators to become tumor cytolytic. Monocytes obtained from 12 colorectal carcinoma patients and 8 normal donors were incubated in vitro with free or liposome-encapsulated agents. The cytotoxic properties of the monocytes were determined subsequent to interaction with radioactively labeled allogeneic colon carcinoma cells, melanoma cells, glioblastoma cells, and allogeneic nontumorigenic skin cells. Blood monocytes from normal donors and all colorectal carcinoma patients were activated in vitro to become tumoricidal by immunomodulators in free form or entrapped within liposomes; i.e., the monocytes recognized and lysed tumorigenic cells but not nontumorigenic cells. The tumoricidal activity of monocytes was observed in blood monocytes obtained from patients even after multiple doses of radiotherapy and chemotherapy, and that fact suggests that the in vivo activation of macrophages may be feasible.

To determine whether the production of experimental hepatic metastases in athymic nude mice by human colorectal carcinomas (HCC) correlated with the clinical outcome in patients, we harvested colorectal carcinomas from 82 patients, dissociated the tumors with collagenase and DNase, and injected them into groups of nude mice, either in the flank to assess experimental tumorigenicity or into the spleen to produce experimental metastasis in the liver. Growth in mice was then associated with clinicopathological factors and clinical outcome. Growth of HCC in either the flanks or the livers of nude mice was associated with the time to recurrence in a Wilcoxon analysis. Analysis of the outcome data in a Cox proportional hazards model suggested that there was an interaction between tumorigenicity and metastatic potential of HCC in nude mice and serum CEA concentration in the patient and stage of disease. A univariate analysis indicated that both tumorigenicity and metastatic potential of HCC in nude mice were significantly associated with the serum CEA concentration of the patient but not with the other variables of stage of disease, mucin production, local tissue invasion, state of differentiation, or sex. A subset of 57 patients was operated upon for cure and followed prospectively for up to 61 months. Tumorigenicity and, to a lesser extent, experimental metastatic potential were associated with disease recurrence in 23 of these patients. Seventy-eight % of the subset of patients who were operated upon for cure developed liver metastasis as one site of their progressive disease. Thus, the ability of HCC cells isolated from surgical specimens to grow in athymic nude mice correlates with the development of advanced disease in patients.

The purpose of these studies was to examine the growth characteristics and metastatic behavior of freshly isolated human colorectal carcinomas implanted into athymic nude mice. Four tumor lines were derived from primary colorectal carcinomas, three lines from hepatic metastases, and one line from a metastasis to a mesenteric lymph node. Subsequent to implantation into the subcutis or the muscularis, tumor lines derived from metastases grew faster in the nude mice than did cells isolated from the primary neoplasms. Regardless of the source of the cells, however, little or no visceral organ metastasis was found. Subsequent to i.v. injection, experimental lung colonies could be produced by some of the cells, but there was no correlation between lung tumor colony formation and the origin of the human colorectal cells. The intrasplenic injection of colorectal carcinoma cells provided a useful procedure to identify human colorectal carcinoma cells with metastatic potential to liver. Extensive tumor burdens in the liver were observed as early as 30 days after injection with two of the three liver metastasis-derived tumor lines. No liver metastases were found after the intrasplenic injection of cells isolated from the lymph node-derived tumor line. Ninety days after the intrasplenic injection of cells from the four primary colorectal carcinomas, limited liver metastases were observed. We conclude that metastasis of human colorectal carcinomas can be studied in nude mice, and its outcome depends upon both the intrinsic metastatic capacity of the human tumor cells and the organ environment of implantation.

The purpose of these studies was to determine whether the biological and metastatic behaviors of tumor cells isolated from fresh surgical specimens of human colon carcinomas are influenced by the isolation method and the organ site of implantation and growth in nude mice. Three surgical specimens were obtained from three different patients. Two tumors were primary human colorectal carcinomas (HCC) classified as Dukes' B2 (KM12) and Dukes' D stages (KM20), and the third was from a liver metastasis (KM23). The tumors were enzymatically dissociated, and viable cells were implanted into the subcutis or spleen of different nude mice or were established in culture. Tumors developed in both sites of implantation, but hepatic metastases were found only in those nude mice that received splenic implantations of HCC cells. Cells from Dukes' D stage tumors produced more hepatic disease than cells from the Dukes' B tumor. Cells of the parental KM12C (culture) were injected into the spleen or cecum of nude mice to produce experimental and spontaneous hepatic metastases, respectively. HCC lesions were harvested from livers of nude mice and established as individual cell lines in culture. This procedure yielded cell lines KM12SM (spontaneous metastasis) and KM12L1 (experimental metastasis). The selection cycle for cells implanted into the spleen was repeated three more times to produce the cell line designated KM12L4. Cells of the parental KM12C and the three selected variants were injected into nude mice by different routes: i.v., s.c. into the cecum, and into the spleen. Subsequent to implantation into the spleen, all cell lines were shown to be tumorigenic. Cells from the selected KM12L4 and KM12SM lines produced a significantly higher number of experimental liver metastases than the parental cells. Moreover, subsequent to the injection into the cecum, cells of the once-selected KM12SM (for spontaneous metastasis) produced a higher incidence of spontaneous liver metastasis than all other lines. The human origin of all the lines was confirmed by isoenzyme and karyotype analyses. The two highly metastatic lines (KM12L4 and KM12SM) were tetraploid and produced elevated levels of type IV collagenolytic activity. Collectively, the results demonstrate that the orthotopic implantation of HCC cells into the appropriate organ environment can be used for efficient isolation and for study of metastatic subpopulations of cells from human colon carcinoma.

A preoperative serum carcinoembryonic antigen (CEA) concentration greater than 5 ng/ml portends a poor prognosis for patients with colorectal carcinoma. The purpose of this study was to determine if the tumorigenicity of colorectal carcinomas in nude mice was associated with the preoperative serum CEA concentration. Neoplasms from 53 patients were either implanted as fragments or dissociated with collagenase and DNase, and 3 x 10(6) viable cells were injected into the flanks of BALB/c nude mice. The growth potential of tumors resected from patients with CEA levels exceeding 5 ng/ml was greater than that of tumors from patients with normal serum CEA: 26 of 33 carcinomas from patients with CEA greater than or equal to 5 ng/ml were tumorigenic in nude mice, whereas only 8 of 22 neoplasms from patients with normal serum CEA were tumorigenic in nude mice (P less than 0.001). Primary colorectal cancers, not metastases, were the basis for the association between tumorigenicity and preoperative CEA. Tumorigenicity was also associated with stage of disease, since Dukes' D primary tumors and metastases were more tumorigenic than Dukes' A to C primary tumors. Growth in nude mice was not associated with other prognostic factors such as tumor site, mucin production, local invasion, or stage of histological differentiation. The tumorigenic capability of human colorectal carcinomas may be associated with the preoperative serum CEA concentration and may reflect an increased potential to develop clinical metastases.