Human tankyrases are aberrantly expressed in colon tumors and contain multiple epitopes that induce humoral and cellular immune responses in cancer patients - PubMed (original) (raw)

doi: 10.1007/s00262-007-0423-z. Epub 2007 Nov 20.

Inna N Lavrik, Julia Karbach, Svetlana V Khlgatian, Ekaterina P Koroleva, Pavel V Belousov, Kirill N Kashkin, Alexander Knuth, Elke Jager, Nai-Wen Chi, Dmitry V Kuprash, Sergei A Nedospasov

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Human tankyrases are aberrantly expressed in colon tumors and contain multiple epitopes that induce humoral and cellular immune responses in cancer patients

Yuriy V Shebzukhov et al. Cancer Immunol Immunother. 2008 Jun.

Abstract

Purpose: Tankyrases 1 and 2 are telomere-associated poly(ADP-ribose) polymerases (PARP) that can positively regulate telomere elongation and interact with multiple cellular proteins. Recent reports implicated tankyrases as tumor antigens and potential targets of anticancer treatment. We examined expression of tankyrases in colon tumors and immune response to these enzymes in patients with different types of cancer.

Methods: mRNA and protein expression was evaluated by quantitative real-time RT-PCR and Western blotting, respectively. Humoral immune response to recombinant tankyrases was investigated by modified enzyme-linked immunoassays. Cellular immune response was analysed by ELISPOT and (51)Cr release assays.

Results: We found that both mRNA and protein levels of tankyrase 2 (TNKL) are upregulated in colon tumors. In contrast, protein level of tankyrase 1 (TNKS) is downregulated, while mRNA level shows variable changes. More than a quarter of colon cancer patients develop humoral immune response to at least one of the two tankyrases. In this study we mapped common and unique B-cell epitopes located in different domains of the two proteins. Additionally, we present evidence for T-cell responses both to epitopes that are unique for TNKL and to those shared between TNKL and TNKS.

Conclusion: Our study favors a biomarker usage of antibody response to tankyrases. Spontaneous CD8(+) T-cell responses to these enzymes are rare and further investigation is needed to evaluate tankyrases as potential targets for cancer immunotherapy.

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Figures

Fig. 1

Fig. 1

Immunogenic human poly(ADP-ribose)polymerases. a Domain structure of tankyrases and PARP-1. Tankyrases: HPS histidine-proline-serine rich domain of TNKS, NTD N-terminal domain of TNKL, ANK ankyrin-repeat domain, SAM sterile α motif, PARP poly(ADP-ribose) polymerase domain [6]. PARP-1 F1 and F2 zinc fingers, NLS nuclear localization signal motif, AM auto modification domain, PARP poly(ADP-ribose)polymerase domain [10]. b Alignment of catalytic domains of human tankyrases and PARP-1

Fig. 2

Fig. 2

Expression levels of human tankyrases in colon tumors. a Quantitative real-time RT-PCR. TNKS and TNKL mRNA levels ratios (tumor vs. normal tissue) in patients with different stages of colorectal cancer are shown. *P < 0.005 according Student’s t test for paired samples. b Western blotting. Upper rows Immunoblotting with anti-TNKS and anti-TNKL antibodies, N normal colon, T tumor tissue. AP (only for TNKL) affinity precipitation. −GST (negative control), _+_GST-IRAP. Lower rows Normalization of loading by silver staining (representative major protein bands of approx. molecular weight 70 kDa are shown). The results of one of two representative experiments are shown

Fig. 3

Fig. 3

Serological cross-reactivity between TNKS and TNKL. Axes: relative (normalized to positive controls, see Table 2) intensities of serological reactions of tankyrases. Serological reactions to TNKS and TNKL demonstrate correlation designated as linear regression trend. Threshold levels (designated by dashed lines) separating serologically positive and negative samples: average value for sera from normal donors + 2 standard deviations

Fig. 4

Fig. 4

Sera of cancer patients recognize various protein domains of tankyrases. a Tankyrase fragments tested for serological reactivity. HPS histidine–proline–serine rich domain of TNKS, NTD N-terminal domain of TNKL, ANK ankyrin-repeat domain, SAM sterile α motif, PARP poly(ADP-ribose) polymerase domain [6]. TNKS-A to -E and TNKL-A to -E: tested tankyrases fragments. b Mini-array of recombinant protein domains and full-length tankyrases. Loading BSA (bovine serum albumin) −negative control, CC colon cancer patients, BC01 breast cancer patient. c Frequencies of humoral immune response to different tankyrases domains. Total of 38 pre-selected TNKL and/or TNKS positive sera tested (colon cancer19 patients, breast cancer 5 patients, ovarian cancer and head and neck cancer: in groups of 3 patients, lung cancer, renal cancer and melanoma: in groups of 2 patients, cervical cancer and prostate cancer: one patient)

Fig. 5

Fig. 5

Cellular immune response to TNKL. a CD8+ reactivity to HLA-A2-restricted peptides in ELISPOT IFN-γ assay. TNKL peptides DLADPSAKAV (P2, aa 162–170), LLSYGADPTL (P8, aa 296–305), LLAHGADPTL (P23, aa 764–773), ALPSCYKPQV (P24, aa 802–811) and GMFGAGIYFA (P31, aa 1053–1062). ATADALFQV (C1): control irrelevant peptide. b Cytotoxicity test with CD8+ T cells from HLA-A2+ patient. Cytotoxicity was measured by 51Cr release assay

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