Functional expression cloning identifies COX-2 as a suppressor of antigen-specific cancer immunity.

The efficacy of immune surveillance and antigen-specific cancer immunotherapy equally depends on the activation of a sustained immune response targeting cancer antigens and the susceptibility of cancer cells to immune effector mechanisms. Using functional expression cloning and T-cell receptor (TCR) transgenic mice, we have identified cyclooxygenase 2/prostaglandin-endoperoxide synthase 2 (COX-2) as resistance factor against the cytotoxicity induced by activated, antigen-specific T cells. Expressing COX-2, but not a catalytically inactive COX-2 mutant, increased the clonogenic survival of E1A-transformed murine cancer cells when cocultured with lymphocytes from St42Rag2(-/-) mice harboring a transgenic TCR directed against an E1A epitope. COX-2 expressing tumors established in immune-deficient mice were less susceptible to adoptive immunotherapy with TCR transgenic lymphocytes in vivo. Also, immune surveillance of COX-2-positive tumor cells in TCR transgenic mice was less efficient. The growth of murine MC-GP tumors, which show high endogenous COX-2 expression, in immunocompetent mice was effectively suppressed by treatment with a selective COX-2 inhibitor, celecoxib. Mechanistically, COX-2 expression blunted the interferon-gamma release of antigen-specific T cells exposed to their respective cellular targets, and increased the expression of interleukin-4 and indoleamine 2,3-dioxygenase by tumor cells. Addition of interferon-gamma sensitized COX-2 expressing cancer cells to tumor suppression by antigen-specific T cells. In conclusion, COX-2, which is frequently induced in colorectal cancer, contributes to immune evasion and resistance to antigen-specific cancer immunotherapy by local suppression of T-cell effector functions.

Impact of human papilloma virus infection on the response of head and neck cancers to anti-epidermal growth factor receptor antibody therapy.

Infection with human papillomaviruses (HPVs) characterizes a distinct subset of head and neck squamous cell cancers (HNSCCs). HPV-positive HNSCC preferentially affect the oropharynx and tonsils. Localized HPV-positive HNSCCs have a favorable prognosis and treatment outcome. However, the impact of HPV in advanced or metastatic HNSCC remains to be defined. In particular, it is unclear whether HPV modulates the response to cetuximab, an antibody targeting the epidermal growth factor receptor (EGFR), which is a mainstay of treatment of advanced HNSCC. To this end, we have examined the sensitivity of HPV-positive and -negative HNSCC models to cetuximab and cytotoxic drugs in vitro and in vivo. In addition, we have stably expressed the HPV oncogenes E6 and E7 in cetuximab-sensitive cancer cell lines to specifically investigate their role in the antibody response. The endogenous HPV status or the expression of HPV oncogenes had no significant impact on cetuximab-mediated suppression of EGFR signaling and proliferation in vitro. Cetuximab effectively inhibited the growth of E6- and E7-expressing tumors grafted in NOD/SCID mice. In support, formalin-fixed, paraffin-embedded tumor samples from cetuximab-treated patients with recurrent or metastatic HNSCC were probed for p16(INK4a) expression, an established biomarker of HPV infection. Response rates (45.5% versus 45.5%) and median progression-free survival (97 versus 92 days) following cetuximab-based therapy were similar in patients with p16(INK4A)-positive and p16(INK4A)-negative tumors. In conclusion, HPV oncogenes do not modulate the anti-EGFR antibody response in HSNCC. Cetuximab treatment should be administered independently of HPV status.

Targeting MCL-1 sensitizes FLT3-ITD-positive leukemias to cytotoxic therapies.

Patients suffering from acute myeloid leukemias (AML) bearing FMS-like tyrosine kinase-3-internal tandem duplications (FLT3-ITD) have poor outcomes following cytarabine- and anthracyclin-based induction therapy. To a major part this is attributed to drug resistance of FLT3-ITD-positive leukemic cells. Against this background, we have devised an antibody array approach to identify proteins, which are differentially expressed by hematopoietic cells in relation to activated FLT3 signaling. Selective upregulation of antiapoptotic myeloid cell leukemia-1 (MCL-1) was found in FLT3-ITD-positive cell lines and primary mononuclear cells from AML patients as compared with FLT3-wild-type controls. Upregulation of MCL-1 was dependent on FLT3 signaling as confirmed by its reversion upon pharmacological inhibition of FLT3 activity by the kinase inhibitor PKC412 as well as siRNA-mediated suppression of FLT3. Heterologously expressed MCL-1 substituted for FLT3 signaling by conferring resistance of hematopoietic cells to antileukemia drugs such as cytarabine and daunorubicin, and to the proapoptotic BH3 mimetic ABT-737. Conversely, suppression of endogenous MCL-1 by siRNA or by flavopiridol treatment sensitized FLT3-ITD-expressing hematopoietic cells to cytotoxic and targeted therapeutics. In conclusion, MCL-1 is an essential effector of FLT3-ITD-mediated drug resistance. Therapeutic targeting of MCL-1 is a promising strategy to overcome drug resistance in FLT3-ITD-positive AML.

pp32/PHAPI determines the apoptosis response of non-small-cell lung cancer.

During malignant transformation, cancer cells have to evade cell-intrinsic tumor suppressor mechanisms including apoptosis, thus acquiring a phenotype that is relatively resistant to clinically applied anticancer therapies. Molecular characterization of apoptotic signal transduction defects may help to identify prognostic markers and to develop novel therapeutic strategies. To this end we have undertaken functional analyses of drug-induced apoptosis in human non-small cell-lung cancer (NSCLC) cells. We found that primary drug resistance correlated with defects in apoptosome-dependent caspase activation in vitro. While cytochrome c-induced apoptosome formation was maintained, the subsequent activation of caspase-9 and -3 was abolished in resistant NSCLC. The addition of recombinant pp32/putative human HLA class II-associated protein (pp32/PHAPI), described as a putative tumor suppressor in prostate cancer, successfully restored defective cytochrome c-induced caspase activation in vitro. Conditional expression of pp32/PHAPI sensitized NSCLC cells to apoptosis in vitro and in a murine tumor model in vivo. Immunohistochemical analyses of tumor samples from NSCLC patients revealed that the expression of pp32/PHAPI correlated with an improved outcome following chemotherapy. These results identify pp32/PHAPI as regulator of the apoptosis response of cancer cells in vitro and in vivo, and as a predictor of survival following chemotherapy for advanced NSCLC.

Inhibitors of apoptosis confer resistance to tumour suppression by adoptively transplanted cytotoxic T-lymphocytes in vitro and in vivo.

Deregulation of apoptosis signalling is commonly found in cancer and results in resistance to cytotoxic therapies. Immunotherapy is a promising strategy to eliminate resistant cancer cells. The transfer of T-lymphocytes during allogeneic stem cell transplantation is clinically explored to induce a 'graft-versus-tumor' effect (GvT). Cytotoxic T-lymphocytes (CTL), which are major effectors of GvT, eliminate cancer cells by inducing apoptosis via multiple parallel pathways. Here, we study in vitro and in vivo the susceptibility of murine cancer cells engineered to express single antiapoptotic genes to CTL-mediated cytotoxicity. Interestingly, we find that single inhibitors of caspase activation, such as BCL-XL or dominant-negative mutants of FADD and caspase-9, protect cancer cells against antigen-specific CTL in vitro. Moreover, expression of BCL-XL impairs the growth suppression by adoptively transplanted CTL of established tumours in vivo. Hence, apoptosis defects that provide protection to cytotoxic cancer therapies can confer crossresistance to immunotherapy by tumour-reactive CTL.

p53 triggers apoptosis in oncogene-expressing fibroblasts by the induction of Noxa and mitochondrial Bax translocation.

The mechanism of p53-dependent apoptosis is still only partly defined. Using early-passage embryonic fibroblasts (MEF) from wild-type (wt), p53(-/-) and bax(-/-) mice, we observe a p53-dependent translocation of Bax to the mitochondria and a release of mitochondrial Cytochrome c during stress-induced apoptosis. These events proceed independent of zVAD-inhibitable caspase activation, are not prevented by dominant negative FADD (DN-FADD), but are negatively regulated by Mdm-2. Bcl-x(L) expression prevents the release of mitochondrial Cytochrome c and apoptosis, but not Bax translocation. At a single-cell level, enforced expression of p53 is sufficient to induce Bax translocation and Cytochrome c release. Real-time RT-PCR analysis reveals a significant induction of RNA expression of Noxa and Bax in p53(+/+), but not in p53(-/-) MEF. Noxa protein expression becomes detectable prior to Bax translocation, and downregulation of endogenous Noxa by RNA interference protects wt MEF against p53-dependent apoptosis. Hence, in oncogene-expressing MEF p53 induces apoptosis by BH3 protein-dependent caspase activation.

Preventable mortality in Missouri: excess deaths from nine chronic diseases, 1979-1991.

For the period 1979-1991, 54% of the 644,045 deaths in Missouri were attributed to nine chronic diseases--cancers of the breast, uterine cervix, lung, colon/rectum; coronary heart disease; stroke; diabetes; chronic obstructive pulmonary disease; hepatic diseases/cirrhosis. Elimination of risk factors and screening have been shown to reduce the mortality caused by these diseases. To evaluate the range in excess mortality in the state, we calculated excess mortality by county and correlated these rates with three sociodemographic variables. Based on these analyses, an estimated 100,000 deaths may have been prevented through prevention and early detection activities.