Antibodies to HSP70 and HSP90 in serum in non-small cell lung cancer patients
Introduction
Heat shock proteins (HSPs) comprise of a group of highly conserved and stress-related proteins with regulatory, homeostatic and chaperoning activities [1], [2], [3]. HSP70 and HSP90 are over-expressed by a number of solid tumors, including non-small cell lung cancer (NSCLC) suggesting a role in tumor cell growth and survival [4], [5], [6]. Recently, HSP70 over-expression, has been identified as a marker of poor prognosis in adenocarcinomas of the lung [7]. Notably, HSPs are immunogenic [8], [9], [10] and antibodies to HSP70 and HSP90, have been identified in sera of individuals with a variety of cancers [11], [12], [13], [14], [15], [16], a number of nonmalignant conditions [17], [18], [19], [20], [21], [22], [23], and in a percentage of normals [20], [24], [25], [26]. In breast cancer and osteosarcoma, elevated levels of antibodies to HSP90 have been shown to have independent prognostic significance [11], [12]. In osteosarcoma, HSP70 antibodies have also been shown to correlate with metastastic spread [13]. Antibodies to neither the protein have been reported in NSCLC.
During library screening of a T7 phage-expressed NSCLC cDNA library for immunogenic tumor proteins using preferential affinity of antibodies in NSCLC patient versus normal sera, we isolated phages expressing HSP70 and HSP90 proteins. The selection of these proteins using this approach indicated the presence of HSP70 and HSP90 antibodies in our NSCLC patient sera. Since HSP70 and HSP90 antibodies are potentially novel markers in NSCLC with putative diagnostic and prognostic importance, we further investigated these findings, including an assessment of sensitivity and specificity of antibody levels in NSCLC compared to normal controls.
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Human subjects
Following informed consent, peripheral blood was obtained from individuals with histologically confirmed NSCLC. The majority of patients were stage 3a/b (stages 1–2b: n=11; stage 3a/b: n=25; stage 4: n=13). NSCLC was biopsy-proven in all subjects and comprised of a variety of cell types (squamous cell: n=17; adenocarcinoma: n=14; large cell carcinoma: n=1), although specific cell types were not discernable from available biopsies from a number of subjects (undifferentiated or poorly
Isolating HSP70 and HSP90 from a T7 phage library
Following a series of sequential selection and enrichment steps, we isolated phages that had high immunochemical reactivity with patient sera. After PCR amplification and sequencing, by comparison to known cDNA sequences in GeneBank, we identified HSP70 and HSP90 from the T7 NSCLC cDNA phage library. Relative specificity for these two proteins using this approach was confirmed by the comparison of immunochemical reactivity of normal and NSCLC patient sera (Fig. 1).
Quantitative ELISA
To quantify the antibodies to
Discussion
We interrogated a NSCLC cDNA T7 phage-expressed library with antibodies in NSCLC patient sera to identify aberrantly expressed tumor proteins in lung cancer, and extracted phage-expressed HSP70 and HSP90 proteins. Since the selection technique uses the preferential affinity of antibodies found in NSCLC patient sera but not in normal sera, the isolation of these proteins suggested corresponding antibodies could be specifically elevated in NSCLC. Notably, HSP70 and HSP90 are abundantly expressed
Acknowledgements
These studies were supported by the Veterans Administration Career Development Award project #596-416905007-0001 and from a generous donation from the McDowell Foundation—Papa Johns/Valvano fund for cancer research. We would like to thank Basil Hirschowitz for his help in writing this manuscript.
References (42)
Escaping cell death: survival proteins in cancer
Exp Cell Res.
(1999)- et al.
Heat shock proteins: endogenous modulators of apoptotic cell death
Biochem Biophys Res Commun.
(2001) - et al.
Heat shock proteins: the ‘Swiss Army Knife’ vaccines against cancers and infectious agents
Vaccine
(2001) - et al.
Serum antibodies against the heat shock protein 60 are elevated in patients with osteosarcoma
Immunobiology
(2000) - et al.
Serum heat shock protein and anti-heat shock protein antibody levels in aging
Exp Gerontol.
(2001) - et al.
Cyfra 21-1, neuron specific enolase and prognosis of non-small cell lung cancer: prospective study in 621 patients
Lung Cancer
(2001) - et al.
Cyfra 21-1 as a biologic marker of non-small cell lung cancer. Evaluation of sensitivity, specificity, and prognostic role
Chest
(1995) - et al.
Diagnostic and prognostic significance of squamous cell carcinoma antigen in non-small cell lung cancer
Lung Cancer
(2001) - et al.
Expression of 70-kDa heat shock protein in oral lesions: marker of biological stress or pathogenicity
Oral Oncol
(1998) - et al.
Expression of resistance-related proteins in tumoral and peritumoral tissues of patients with lung cancer
Cancer Lett
(1996)