Natalie Donin

Obstacles to cancer immunotherapy: expression of membrane complement regulatory proteins (mCRPs) in tumors.

Monoclonal antibodies (mAbs) are being increasingly used in cancer therapy owing to their ability to recognize specifically cancer cells and to activate complement- and cell-mediated cytotoxicity and/or to induce growth arrest or apoptosis. The therapeutic potential of anticancer antibodies is significantly limited due to the ability of cancer cells to block killing by complement. Of the multiple resistance strategies exploited by cancer cells, the expression of membrane complement regulatory proteins (mCRPs), such as CD46 (membrane cofactor protein (MCP)), CD55 (decay-accelerating factor (DAF)), CD35 (complement receptor type-1 (CR1)) and CD59, has received most attention. CD46, CD55 and CD35 block the complement cascade at the C3 activation stage and CD59 prevents assembly of the membrane attack complex of complement (MAC). These proteins protect normal tissues from accidental injury by activated complement, but also confer resistance on cancer cells, thereby limiting the effect of complement-fixing monoclonal antibodies. Expression of mCRPs on malignant cells is highly variable, yet there is clear indication that certain tumors express higher mCRP levels than the normal tissue from which they have evolved. mCRP level of expression and cellular location may also vary during malignant transformation and between differentiated and undifferentiated tumors. Neutralizing anti-mCRP mAbs have been used in vitro to elucidate the significance of mCRP expression to the tumor complement resistance phenotype. In general, CD59 appears to be the most effective mCRP protecting tumor cells from complement-mediated lysis. Nevertheless, it acts additively, and in certain tumors even synergistically, with CD55 and CD46. It is envisaged that treatment of cancer patients with mCRP blocking antibodies targeted specifically to cancer cells in combination with anticancer complement-fixing antibodies will improve the therapeutic efficacy.

Complement resistance of human carcinoma cells depends on membrane regulatory proteins, protein kinases and sialic acid

Nucleated cells employ several strategies to evade killing by homologous complement. We studied complement resistance in the human carcinoma cell lines (CA) T47D (mammary), SKOV3 (ovarian), and PC‐3 (prostate) with emphasis on the following mechanisms of defense: 1. Expression and shedding of the membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59; 2. Resistance based on protein phosphorylation; 3. Cell surface expression of sialic acid residues; 4. Desensitization to complement upon exposure to sublytic complement doses. Anti‐mCRP antibody blocking experiments demonstrated that CD59 is the main mCRP protecting these CA from complement. Soluble CD59 was also found in supernates of PC‐3> SKOV3 > T47D cells. Second, inhibitors of PKC, PKA and MEK sensitized the CA to lysis, thus implicating these protein kinases in CA complement resistance. Third, removal of sialic acid residues with neuraminidase also sensitized CA to lysis. Finally, exposure of CA to sublytic doses of complement conferred on them enhanced resistance to lytic complement doses in a PKC‐dependent process. Combined treatment of CA with anti‐CD59 antibodies, PD98059 (a MEK inhibitor) and neuraminidase produced a large enhancement in CA sensitivity to complement. Our results show that CD59 and sialic acid residues present on the cell surface, and intracellular processes involving protein phosphorylation act additively to secure CA resistance to complement‐mediated lysis. Therefore, the effectiveness of antibody‐ and complement‐based cancer immunotherapy will markedly improve by suppression of the various complement resistance mechanisms.

K562 erythroleukemic cells are equipped with multiple mechanisms of resistance to lysis by complement.

Resistance of tumor cells to lysis by complement is generally attributed to several protective mechanisms. The relative impact of these mechanisms in the same tumor cell, however, has not been assessed yet. We have analyzed the interaction of the human erythroleukemia tumor cell line K562 with human complement. K562 cells express the membrane complement regulatory proteins CD59, CD55 and CD46. As shown here for the first time, K562 also spontaneously release the soluble regulators C1 inhibitor, factor H, and soluble CD59. Complement resistance of K562 cells is augmented upon treatment with PMA, TNF or even with sublytic complement. Unlike TNF and sublytic complement, PMA enhanced the expression of membrane‐bound CD55 and CD59 and led to increased secretion of soluble CD59. In addition, we show that complement‐resistant K562 cells express a membrane‐associated proteolytic activity, higher than the complement‐sensitive K562/S cells. Treatment of complement‐resistant K562 cells with serine protease inhibitors enhance their sensitivity to complement‐mediated lysis. Inhibitors of protein kinase C (PKC) also sensitize K562 cells to complement lysis, implicating PKC‐mediated signaling in cell resistance to complement. Neutralization of the CD55 and CD59 but not of CD46 regulatory activity with specific antibodies significantly increases complement‐mediated K562 cell lysis. Treatment of K562 cells with a mixture of inhibitory reagents results in a significant additive enhancing effect on complement‐mediated lysis of K562. In conclusion, K562 cells resist a complement attack by concomitantly using multiple molecular evasion strategies. Future attempts in antibody‐based tumor therapy should include strategies to interfere with those resistance mechanisms.

Programmed Necrotic Cell Death Induced by Complement Involves a Bid-Dependent Pathway

The membrane attack complex (MAC) of the complement system induces a necrotic-type cell death. Earlier findings suggested that Bcl-2 protects cells from MAC-induced necrosis. Here we examined the involvement of Bid, a proapoptotic protein, in MAC-induced cytotoxicity. Bid knockout (Bid−/−) mouse embryonic fibroblasts (MEF) and primary fibroblasts were damaged by complement but to a significantly lower extent than wild-type (WT) fibroblasts. Bid silencing with small interfering RNA duplexes led to elevated resistance of mouse fibroblasts, human K562, and Jurkat cells to lysis by complement. Bid−/− MEF were also resistant to toxic doses of streptolysin O, melittin, and A23187. Analysis of complement protein deposition on fibroblasts demonstrated that less complement C3 and C9 bound to Bid−/− than to WT cells, even though expression of the membrane complement inhibitors Crry and CD59 was relatively reduced on Bid−/− cells. Bid was rapidly cleaved in WT MEF subjected to lytic doses of MAC. Pretreatment of the cells with the pan-caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone reduced Bid cleavage and cell lysis. These results indicate that complement MAC activates two cell death pathways, one involving caspases and Bid and one that is Bid-independent.

Involvement of the c-jun N-terminal kinases JNK1 and JNK2 in complement-mediated cell death

Cell death and survival signals activated by the complement membrane attack complex C5b-9 play important roles in complement-associated diseases and in antibody-based cancer therapy. Here, we investigated the involvement of the JNK mitogen-activated protein kinase in C5b-9-induced cell lysis. Necrotic-type cell death regulation by JNK1 and JNK2 was selectively studied in mouse fibroblasts and human K562, HeLa and 293T cells. C5b-9 induced higher JNK activation than C5b-8. Pretreatment with a JNK inhibitor reduced cell sensitivity to complement-mediated lysis. KO cells deficient in either JNK1 or JNK2 were less sensitive to lysis than WT cells. This correlated with lower C3 and C5b-9 deposition on KO cells. Furthermore, silencing of JNK1 or JNK2 expression by RNA interference decreased cell lysis by complement. Reconstitution of JNK2 into JNK2-/- cells and over expression of JNK2 in WT cells increased C3 and C5b-9 deposition as well as cell sensitivity to complement-mediated lysis. Pretreatment of cells with the phosphotyrosine phosphatase inhibitor phenylarsine oxide increased JNK activation and JNK-dependent complement-mediated necrotic death of WT and JNK2-/- KO cells but not of JNK1-/- KO cells. The JNK inhibitor and PAO had no effect on complement-mediated lysis in cells lacking Bid, suggesting involvement of Bid in the JNK lytic pathway. Our results demonstrate that complement C5b-9 induce a JNK/Bid-dependent and JNK-independent necrotic cell death. Both JNK1 and JNK2 have cytotoxic potential, however JNK2 is the primary signal transducer.

Comparison of growth rate of two B16 melanomas differing in metastatic potential in young versus middle-aged mice

The rise of cancer frequency as a function of age is a well-established fact. The aspect of the host age-tumor progression relationship, namely the slower metastatic spread in aged patients, has been investigated to a lesser extent. In the present study, we examined whether host-age-dependent growth rate varies with metastatic capacity of the tumor. The parental B16 and the B16/Col/R, a highly metastatic variant, were employed. A more pronounced growth of both tumors in young as compared to middle-aged mice was found. However, the differential growth in middle-aged versus young mice was more evident in the highly metastatic variant. According to the tumor size data, a sixfold growth reduction in middle-aged mice was observed with B16/Col/R and an only twofold growth reduction was seen with the B16 melanoma. The data might eventually contribute to the finding of more appropriate treatment modalities for the middle-aged cancer patient.

Role of immune response as determinant of tumor progression in function of host age in the B16 melanoma

Aging constitutes the major cause for the development of most neoplastic diseases. However, tumors in aged people present with a lower degree of aggressiveness than in young patients. The reasons for this paradoxical behavior are not clear. We attempted to verify whether the immune system has a role in the relation between host age, immune response and tumor progression. We compared the growth rate of B16 melanoma and a highly malignant variant, the B16/Col/R, in young and aged mice that have or have not undergone splenectomy. The following results were obtained: (1) Splenectomy stimulated growth in the parental melanoma in both young and aged mice, indicating a protective role of the spleen against this tumor at all ages; (2) Spleen ablation provoked inhibition of the highly-metastatic variant growth in young mice, suggesting a stimulatory role of the spleen in this case; (3) By contrast, in aged mice inoculated with the B16/Col/R variant, splenectomy enhanced tumor growth, indicating a defensive role of the spleen. Age favors a positive host response against the aggressive clone of the melanoma. Differential host response in young versus aged mice can explain, in this tumor system, the difference in tumor progression rate as a function of age.

Biological behavior and cell properties of new AKR lymphoma malignancy variants

The AKR lymphoma-leukemia is a T lymphocyte neoplasm, most suitable as a model for human T cell malignancies. We have been interested in the process of tumor progression in the AKR lymphoma system. In the present study, two newly isolated variants, the TAU-42 and TAU-44, were characterized with respect to their biological behavior, by comparing them to a previously studied low-malignancy variant, the TAU-39. While the TAU-44 variant formed large s.c. local tumors, the TAU-42 variant formed only small growths or none at all. The TAU-42 lymphoma was found to have the highest malignant potential: it displayed very marked dissemination to spleen, lymph nodes, liver and lungs. The TAU-44 variant had an intermediate degree of metastatic potential but presented a predilection for spread to lymph nodes and spleen and was sometimes found to metastasize to peculiar organs, such as heart and pancreas. Cells derived from the different lymphoma variants varied in their immunophenotype: the highly malignant variant cells expressed more CD4 antigen than the low-malignancy one. The opposite was observed with regard to CD8. The variant cells also differed in their migrating capacity, the more malignant one exhibiting a higher motile activity. Studies on the tumor progression model of AKR lymphoma might contribute to the elucidation of the features determining the aggressiveness of T lymphocytic malignancies.

Apoptosis, cell proliferation and in vivo biological behaviour of primary and metastatic tumour cells of an AKR lymphoma variant.

The possibility that apoptosis and/or cell proliferation have a role in tumour progression in a murine T cell lymphoma was tested. The model consisted of the comparison of primary (PT) and metastatic tumour (MT) cells. The PT cells, but not the MT cells displayed a very pronounced tendency for spontaneous apoptosis. Proliferative capacity of MT cells was lower than that of PT cells, suggesting that it does not contribute to the metastatic phenotype in this system. Release from apoptosis does however, probably, play a role in the aggressiveness of the lymphoma.

Release from apoptosis correlates with tumor progression in the AKR lymphoma.

Disturbance of apoptosis is an established factor in tumorigenesis. The role of apoptosis in tumor progression is not yet clear. In the present study we compared the tendency to spontaneous apoptosis (and the proliferative capacity) of tumor cells derived from primary (PT) and metastatic tumor (MT) cells of several AKR lymphoma variants. Apoptosis-related gene expression was also compared. Our results indicate that release from apoptosis has a role in the tumor progression of this T cell lymphoma. At the cellular level, a markedly lower apoptotic tendency was observed in MT than in PT cells. The existence of macrophages only in PT also supports the presence of apoptotic cells in local but not in MTs. By contrast, proliferative capacity does not determine tumor aggressiveness in this system. At the molecular level, we found a higher staining intensity for bcl-2 in MT than in PT cells, suggesting that bcl-2 might be responsible for the reduced apoptosis in MT compared to PT cells. Evidence for p53 overexpression was found in the MT cells of one of the variants but in none of the PT. Comparison of Fas receptor, unexpectedly showed an increased expression in MT versus PT cells, possibly indicating resistance to Fas-induced apoptosis in the MT cells.