Upregulation of complement proteins in lung cancer cells mediates tumor progression

IntroductionIn vivo, cancer cells respond to signals from the tumor microenvironment resulting in changes in expression of proteins that promote tumor progression and suppress anti-tumor immunity. This study employed an orthotopic immunocompetent model of lung cancer to define pathways that are altered in cancer cells recovered from tumors compared to cells grown in culture.MethodsStudies used four murine cell lines implanted into the lungs of syngeneic mice. Cancer cells were recovered using FACS, and transcriptional changes compared to cells grown in culture were determined by RNA-seq.ResultsChanges in interferon response, antigen presentation and cytokine signaling were observed in all tumors. In addition, we observed induction of the complement pathway. We previously demonstrated that activation of complement is critical for tumor progression in this model. Complement can play both a pro-tumorigenic role through production of anaphylatoxins, and an anti-tumorigenic role by promoting complement-mediated cell killing of cancer cells. While complement proteins are produced by the liver, expression of complement proteins by cancer cells has been described. Silencing cancer cell-specific C3 inhibited tumor growth In vivo. We hypothesized that induction of complement regulatory proteins was critical for blocking the anti-tumor effects of complement activation. Silencing complement regulatory proteins also inhibited tumor growth, with different regulatory proteins acting in a cell-specific manner.DiscussionBased on these data we propose that localized induction of complement in cancer cells is a common feature of lung tumors that promotes tumor progression, with induction of complement regulatory proteins protecting cells from complement mediated-cell killing

Identification and functional characterization of a novel UDP-glucuronosyltransferase 2A1 splice variant: potential importance in tobacco-related cancer susceptibility

UDP-glucuronosyltransferase (UGT) 2A1 is a respiratory and aerodigestive tract-expressing phase II detoxifying enzyme that metabolizes various xenobiotics including polycyclic aromatic hydrocarbons (PAHs). In the present study, a novel exon 3 deletion splice variant was identified for UGT2A1 (UGT2A1Δexon3). As determined by reverse transcription-polymerase chain reaction (PCR), UGT2A1Δexon3 was shown to be expressed in various tissues including lung, trachea, larynx, tonsil, and colon. The ratio of UGT2A1Δexon3/wild-type UGT2A1 expression was highest in colon (0.79 ± 0.08) and lung (0.42 ± 0.12) as determined by real-time PCR; an antibody specific to UGT2A1 showed splice variant protein (UGT2A1_i2) to wild-type protein (UGT2A1_i1) ratios in the range of 0.5 to 0.9 in these tissues. Using ultra-pressure liquid chromatography, we found that homogenates prepared from UGT2A1_i2-overexpressing human embryonic kidney 293 cells exhibited no glucuronidation activity against PAHs, including benzo[a]pyrene-7,8-dihydrodiol (B[a]P-7,8-diol). An inducible in vitro system was created to determine the effect of UGT2A1_i2 expression on UGT2A1_i1 activity. Increasing UGT2A1_i2 levels resulted in a significant (p < 0.01) decrease in the UGT2A1_i1 V(max) against 1-hydroxy (OH)-pyrene, 3-OH-benzo[a]pyrene, and B[a]P-7,8-diol; no significant changes in K(M) were observed for any of the three substrates. Coimmunoprecipitation experiments suggested the formation of UGT2A1_i1 and UGT2A1_i2 hetero-oligomers and UGT2A1_i1 homo-oligomers; coexpression of UGT2A1_i1 or UGT2A1_i2 with other UGT1A or UGT2B enzymes caused no change in UGT1A or UGT2B glucuronidation activity. These data suggest that a novel UGT2A1 splice variant regulates UGT2A1-mediated glucuronidation activity via UGT2A1-specific protein-protein interactions, and expression of this variant could play an important role in the detoxification of carcinogens within target tissues for tobacco carcinogenesis

Identification and functional characterization of a novel UDP-glucuronosyltransferase 2A1 splice variant: potential importance in tobacco-related cancer susceptibility

UDP-glucuronosyltransferase (UGT) 2A1 is a respiratory and aerodigestive tract-expressing phase II detoxifying enzyme that metabolizes various xenobiotics including polycyclic aromatic hydrocarbons (PAHs). In the present study, a novel exon 3 deletion splice variant was identified for UGT2A1 (UGT2A1Δexon3). As determined by reverse transcription-polymerase chain reaction (PCR), UGT2A1Δexon3 was shown to be expressed in various tissues including lung, trachea, larynx, tonsil, and colon. The ratio of UGT2A1Δexon3/wild-type UGT2A1 expression was highest in colon (0.79 ± 0.08) and lung (0.42 ± 0.12) as determined by real-time PCR; an antibody specific to UGT2A1 showed splice variant protein (UGT2A1_i2) to wild-type protein (UGT2A1_i1) ratios in the range of 0.5 to 0.9 in these tissues. Using ultra-pressure liquid chromatography, we found that homogenates prepared from UGT2A1_i2-overexpressing human embryonic kidney 293 cells exhibited no glucuronidation activity against PAHs, including benzo[a]pyrene-7,8-dihydrodiol (B[a]P-7,8-diol). An inducible in vitro system was created to determine the effect of UGT2A1_i2 expression on UGT2A1_i1 activity. Increasing UGT2A1_i2 levels resulted in a significant (p < 0.01) decrease in the UGT2A1_i1 V(max) against 1-hydroxy (OH)-pyrene, 3-OH-benzo[a]pyrene, and B[a]P-7,8-diol; no significant changes in K(M) were observed for any of the three substrates. Coimmunoprecipitation experiments suggested the formation of UGT2A1_i1 and UGT2A1_i2 hetero-oligomers and UGT2A1_i1 homo-oligomers; coexpression of UGT2A1_i1 or UGT2A1_i2 with other UGT1A or UGT2B enzymes caused no change in UGT1A or UGT2B glucuronidation activity. These data suggest that a novel UGT2A1 splice variant regulates UGT2A1-mediated glucuronidation activity via UGT2A1-specific protein-protein interactions, and expression of this variant could play an important role in the detoxification of carcinogens within target tissues for tobacco carcinogenesis

Importance of UDP-glucuronosyltransferases 2A2 and 2A3 in tobacco carcinogen metabolism

UDP-glucuronosyltransferase A1 (UGT2A1) is expressed in the lung and exhibits activity against polycyclic aromatic hydrocarbons (PAHs), suggesting UGT2A1 involvement in the local metabolism of PAH tobacco carcinogens. The goal of the present study was to investigate the importance of two additional UGT2A enzymes, UGT2A2 and UGT2A3, in tobacco carcinogen metabolism. Real-time polymerase chain reaction suggested that wild-type UGT2A2 had the highest expression in the breast, followed by trachea > larynx > kidney. A novel splice variant of UGT2A2 lacking exon 3 (termed UGT2A2Δexon3) was investigated, with UGT2A2Δexon3 expression determined to be 25-50% that of wild-type UGT2A2 in all tissues examined. UGT2A3 was determined to be well expressed in the liver and colon, followed by pancreas > kidney > lung > tonsil > trachea > larynx. Cell homogenates prepared from human embryonic kidney (HEK)293 cells overexpressing wild-type UGT2A2 (termed UGT2A2_i1) exhibited glucuronidation activity, as observed by reverse-phase ultra-pressure liquid chromatography, against 1-hydroxy-(OH)-pyrene, 1-naphthol, and hydroxylated benzo(a)pyrene metabolites, whereas homogenates prepared from HEK293 cells overexpressing UGT2A3 only showed activity against simple PAHs like 1-OH-pyrene and 1-naphthol. Activity assays showed the UGT2A2Δexon3 protein (termed UGT2A2_i2) exhibited no detectable glucuronidation activity against all substrates examined; however, coexpression studies suggested that UGT2A2_i2 negatively modulates UGT2A2_i1 activity. Both UGT2A2 and UGT2A3 exhibited no detectable activity against complex PAH proximate carcinogens, tobacco-specific nitrosamines, or heterocyclic amines. These data suggest that, although UGT2A1 is the only UGT2A enzyme active against PAH proximate carcinogens (including PAH diols), both UGTs 2A1 and 2A2 play an important role in the local detoxification of procarcinogenic monohydroxylated PAH metabolites

Characterization of UDP-glucuronosyltransferase 2A1 (UGT2A1) variants and their potential role in tobacco carcinogenesis

UDP-glucuronosyltransferases (UGTs) play an important role in the metabolism and excretion of various endogenous and xenobiotic compounds, including carcinogens and chemotherapeutic agents. The goal of the present study was to examine UGT2A1 expression in human tissues, determine its glucuronidation activity against tobacco carcinogens, and assess the potential functional role of UGT2A1 missense single nucleotide polymorphisms on UGT2A1 enzyme activity. As determined by reverse-transcription polymerase chain reaction, UGT2A1 was expressed in aerodigestive tract tissues including trachea, larynx and tonsil, and was also expressed in lung and colon; no expression was observed in breast, whole brain, pancreas, prostate, kidney, liver or esophagus. Real-time PCR suggested that UGT2A1 exhibited highest expression in the lung, followed by trachea > tonsil > larynx > colon > olfactory tissue. Cell homogenates prepared from wild-type UGT2A1 75Lys308Gly -over-expressing HEK293 cells showed significant glucuronidation activity, as observed by reverse-phase UPLC, against a variety of polycyclic aromatic hydrocarbons (PAHs) including, 1-hydroxy-benzo(a)pyrene, benzo(a)pyrene-7,8-diol, and 5-methylchrysene-1,2-diol. No activity was observed in UGT2A1-over-expressing cell homogenate against substrates that form N -glucuronides, such as NNAL, nicotine, or N -OH-PhIP. A significant (p<0.05) ~25% decrease in glucuronidation activity (V max /K M ) was observed against all PAH substrates for the UGT2A1 75Arg308Gly variant as compared to homogenates from wild-type UGT2A1 75Lys308Gly ; no activity was observed for cell homogenates over-expressing the UGT2A1 75Lys308Arg variant for all substrates tested. These data suggest that UGT2A1 is an important detoxification enzyme in the metabolism of PAHs within target tissues for tobacco carcinogens, and functional polymorphisms in UGT2A1 may play a role in tobacco-related cancer risk

Enhanced CDC of B cell chronic lymphocytic leukemia cells mediated by rituximab combined with a novel anti-complement factor H antibody

<div><p>Rituximab therapy for B cell chronic lymphocytic leukemia (B-CLL) has met with mixed success. Among several factors to which resistance can be attributed is failure to activate complement dependent cytotoxicity (CDC) due to protective complement regulatory proteins, including the soluble regulator complement factor H (CFH). We hypothesized that rituximab killing of non-responsive B-CLL cells could be augmented by a novel human monoclonal antibody against CFH. The B cells from 11 patients with B-CLL were tested <i>ex vivo</i> in CDC assays with combinations of CFH monoclonal antibody, rituximab, and a negative control antibody. CDC of rituximab non-responsive malignant B cells from CLL patients could in some cases be augmented by the CFH monoclonal antibody. Antibody-mediated cytotoxicity of cells was dependent upon functional complement. In one case where B-CLL cells were refractory to CDC by the combination of rituximab plus CFH monoclonal antibody, additionally neutralizing the membrane complement regulatory protein CD59 allowed CDC to occur. Inhibiting CDC regulatory proteins such as CFH holds promise for overcoming resistance to rituximab therapy in B-CLL.</p></div

A Therapeutic Antibody for Cancer, Derived from Single Human B Cells

Summary: Some patients with cancer never develop metastasis, and their host response might provide cues for innovative treatment strategies. We previously reported an association between autoantibodies against complement factor H (CFH) and early-stage lung cancer. CFH prevents complement-mediated cytotoxicity (CDC) by inhibiting formation of cell-lytic membrane attack complexes on self-surfaces. In an effort to translate these findings into a biologic therapy for cancer, we isolated and expressed DNA sequences encoding high-affinity human CFH antibodies directly from single, sorted B cells obtained from patients with the antibody. The co-crystal structure of a CFH antibody-target complex shows a conformational change in the target relative to the native structure. This recombinant CFH antibody causes complement activation and release of anaphylatoxins, promotes CDC of tumor cell lines, and inhibits tumor growth in vivo. The isolation of anti-tumor antibodies derived from single human B cells represents an alternative paradigm in antibody drug discovery. : Bushey et al. clone antibodies against complement factor H (CFH) from single human B cells. CFH protects tumor cells from complement-dependent cytotoxicity (CDC). The authors demonstrate that a recombinant CFH antibody induces CDC of tumor cells, inhibits tumor growth in vivo, and stimulates infiltration of the tumor by lymphocytes

Patient treatment history.

<p>Patient treatment history.</p

CDC of B-CLL cells.

<p>(A) CDC of B-CLL cells in the presence and absence of rituximab (RTX). B-CLL cells from each of 11 CLL patients were treated with RTX or left untreated, with NHS added as a source of complement. Mean percent CDC in the presence of RTX was divided by the mean percent CDC in the absence of RTX to obtain "fold cytotoxicity" for each patient. (B) CDC of B-CLL cells from patient 11 treated with a CFH mAb or control mAb in the presence or absence of RTX. B-CLL cells from this patient were treated with CFH mAb7968 or isotype-matched negative control antibody 7B2 with or without RTX, with NHS added as a source of complement. Mean percent CDC was divided by the mean percent CDC of the “no antibody” control to obtain "fold cytotoxicity" for each condition.</p