ISSN 00062979, Biochemistry (Moscow), 2014, Vol. 79, No. 8, pp. 733739. © Pleiades Publishing, Ltd., 2014. Original Russian Text © M. O. Golovastova A. V. Bazhin, P. P. Philippov, 2014, published in Biokhimiya, 2014, Vol. 79, No. 8, pp. 923931.
REVIEW
CancerRetina Antigens – A New Group of Tumor Antigens M. O. Golovastova1,2*, A. V. Bazhin1,3, and P. P. Philippov1 1
Lomonosov Moscow State University, Belozersky Institute of PhysicoChemical Biology, 119991 Moscow, Russia 2 Lomonosov Moscow State University, Faculty of Bioengineering and Bioinformatics, 119991 Moscow, Russia; fax: +7 (495) 9393181; Email: [email protected] 3 Department of General, Visceral, and Transplant Surgery, LudwigMaximiliansUniversity, 81377 Munich, Germany; fax: +49 (89) 440076433; Email: [email protected]muenchen.de Received April 8, 2014 Abstract—Some photoreceptor proteins normally specific for the eye retina are aberrantly expressed in malignant tumors. These proteins include recoverin, visual rhodopsin, transducin, cGMPphosphodiesterase 6 (PDE 6), cGMPdependent cationic channels, guanylyl cyclase 1, rhodopsin kinase, and arrestin. By analogy with cancertestis antigens, these pho toreceptor proteins form the group of cancerretina antigens. It is shown that an aberrant demethylation of the promoter region of recoverin is involved in the aberrant expression of this protein. The cascade Wnt5a → Frizzled2 → transducin → PDE 6 is shown to function in skin melanoma cells, and this suggests that these cancerretina antigens can play a function al role. The events accompanying the signal transduction in this cascade, including those involving calcium ions and cGMPdependent protein kinase (protein kinase G), are discussed. DOI: 10.1134/S000629791408001X Key words: visual transduction, retina, cancerretina antigens, aberrant expression, cancer
Among numerous human diseases, malignant tumors are one of major causes of death in both developed and developing countries. Thus, 12.4 million new cancer cases and 7.6 million cancercaused deaths were record ed throughout the world in 2008, and 28 million patients continued to live with this diagnosis for five years. The number of new cancer cases by 2030 in the world is pre dicted to be 2126 million with deaths of 1317 million people per year. Such predictions are significantly pro moted by global industrialization and urbanization and the associated ecological and social problems. Because the risk of oncological diseases increases with age, the increase in lifespan due to improvement of life conditions and public health quality, especially noticeable in some developing countries, also contributes to this prognosis [1, 2]. Current therapeutic approaches allow clinicians to cure a significant number of patients with oncological diseases. Under other similar conditions, a positive result strongly depends on the stage of starting the treatment. Abbreviations: ADC, 5aza2′deoxycytidine; CAR, cancer associated retinopathy; MAR, melanomaassociated retinopa thy; NSCLC, nonsmall cell lung carcinoma; PDE 6, cGMP phosphodiesterase 6; SCLC, small cell lung carcinoma. * To whom correspondence should be addressed.
Thus, in the case of a localized tumor the fiveyear sur vival is more than 90% in patients with cancer of mam mary gland, colon, urinary bladder, kidney, uterine body, etc. In some cancers this value decreases manifold if the treatment is started at the stage of distant metastases and is about 6 and 8%, respectively, for urinary bladder and renal cancers [3]. These figures obviously indicate the importance of the development and use of methods for diagnosis of cancer at its earliest stage. Malignant transformation is accompanied by dra matic changes in features of the affected cells. In particu lar, the expression levels of various proteins of the cells can increase or decrease – and this concerns tumorasso ciated antigens. More rarely, healthy cells upon malig nant transformation begin to synthesize a protein or even a set of proteins that are completely absent in normal cells – this is a group of tumorspecific antigens [4, 5]. Both types of tumor antigens are promising for diagnosis of cancer and/or as targets for immunotherapy of cancer. From this standpoint, tumorspecific antigens as mole cules unique for a tumor seem more attractive than tumorassociated antigens. Based on data available by 2011, more than 20 anti gens have been characterized as tumor markers detectable in blood, urine, or tissue of oncological patients and are already used in clinical practice [6]. However, the number
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of antigens described in the literature is much higher, and some of them are considered as potential targets for immunotherapy of cancer. Such antigens are exemplified by proteins of cancertestis antigens [79]. Normally these antigens are expressed only in testicles, which are an immunologically privileged zone of the organism. However, in some types of cancer such antigens can be aberrantly expressed in tumors located beyond this zone. The nervous system is another immunologically privileged zone of the organism. The eye retina, which is a member of this system, contains several types of neu rons including photoreceptor cells, rods and cones, responsible for phototransduction. This process is trig gered by a light quantum, which activates the visual pig ment rhodopsin and thus initiates signal transduction in the cascade of rhodopsin → transducin → cGMPphos phodiesterase 6 (PDE 6) that leads to hydrolysis of the photoreceptor secondary messenger cGMP. A decrease in the intracellular concentration of cGMP is accompanied by closing cGMPsensitive cationic channels located in the plasma membrane and by its hyperpolarization. Such is a pathway of the light quantum energy transformation into an electrophysiological signal, which is then trans mitted to the brain through the higher order neurons [10]. Normally, the abovementioned participants of the phototransduction and some other photoreceptor pro teins involved in its regulation are specific for the normal retina. However, some recent data have shown that all these participants can be aberrantly expressed in malig nant tumors located beyond the nervous system. By anal ogy with the cancertestis antigens, such photoreceptor proteins were designated as a group of cancerretina anti gens [11, 12]. The present review will consider and dis cuss the currently available data on the aberrant expres sion of cancerretina antigens in malignant tumors.
PHOTORECEPTOR PROTEINS AS CANCERRETINA ANTIGENS Recoverin [13, 14] was the first photoreceptor pro tein proved to be capable of aberrant expression in malig nant tumors [15]. Later a number of other photoreceptor proteins were added to recoverin, such as visual rhodopsin, transducin, PDE 6, cGMPdependent cationic channels, guanylyl cyclase 1, rhodopsin kinase, and arrestin [12]. Similarly to cancertestis antigens [7 9], all these photoreceptor proteins are characterized, first, by their normal expression limited to the immuno logically privileged zone (the testicles and retina, respec tively), second, by their ability to be expressed in trans formed cells, and third, by the ability of at least some of them to cause an immune response of the organism. Cancerretina antigens in lung carcinoma. As men tioned above, recoverin was the first photoreceptor pro tein proved to be aberrantly expressed in malignant
tumors. This was found as a result of studies for many years on a paraneoplastic neurological syndrome, para neoplastic retina degeneration, or cancerassociated retinopathy (CAR). Mechanisms of the CAR syndrome are considered in detail in some reviews [16], and here we shall note that it is a result of the immune system response to the aberrant expression of recoverin in a malignant tumor located beyond the nervous system. The produced autoantibodies interact with recoverin located in the reti na photoreceptor cells, rods and cones [15], and trigger their apoptosis [17]. The αwave amplitude is suppressed in the electroretinogram of patients with the CAR syn drome, which indicates a dysfunction of the photorecep tors [18]. It should be added that the CAR syndrome is associated with dispersed vision, the loss of peripheral and color vision, nocturnal blindness, and finally with total blindness [19]. It is important that in some cases signs of this syndrome are manifested months and some times even years before the malignant tumor, which is its initial source, can be detected [20]. The first indications of the involvement of an unknown antigen in the CAR syndrome pathogenesis were obtained in works [2123]: autoantibodies of blood sera from patients with this syndrome revealed certain cells in retina sections. The target for these autoantibod ies occurred to be a protein with an apparent molecular weight of 23 kDa that was named CARantigen [24] and later identified as recoverin [15, 25]. In blood sera from patients with small cell lung car cinoma (SCLC) [15, 2430] and nonsmall cell lung car cinoma (NSCLC) [31], antirecoverin antibodies were found in high titers. However, such autoantibodies, although in low titer, could also be found in sera from patients with SCLC and NSCLC but in the absence of the signs of CAR syndrome [32]. The authors used recombi nant recoverin as an antigen to analyze blood sera from 143 patients with SCLC and NSCLC and found that fre quencies of the recoverinpositive samples were, respec tively, 15 and 20%. And signs of CAR syndrome were absent in all patients studied, thus its frequency in this study may be assessed to be lower than 1%. Moreover, the recoverin expression frequencies were also determined in tumor tissues using affinity purified antirecoverin anti bodies, and frequencies of 68 and 85% were obtained for the SCLC and NSCLC, respectively. Thus, the frequency of recoverin expression in lung tumors was higher than the frequency of autoantibodies against this protein in the blood sera from the oncological patients and much high er than the CAR syndrome frequency [32]. Only two of nine analyzed cell lines of lung carcino ma occurred to be recoverinpositive [33], i.e. the fre quency of recoverin expression was only about 20%, which was much lower than the abovepresented values for the tumor tissues. Certainly, the number of cell lines analyzed in this work was insufficient for statistically sig nificant assessment of the frequency of recoverin expres BIOCHEMISTRY (Moscow) Vol. 79 No. 8 2014
CANCERRETINA ANTIGENS sion in them. Moreover, our unpublished data have shown that cell lines of SCLC during their isolation and cultiva tion can lose the ability to express recoverin. Thus, the absence of its expression in any cell line does not prove that it is really absent in the initial tumor tissue. It is rea sonable to suppose that this conclusion is true also for tumor antigens others than recoverin. For today the aberrant expression of cancerretina antigens in tumors has been studied only for recoverin, whereas for skin melanoma there are data on a number of these antigens. Cancerretina antigens in skin melanoma. A number of cell lines and tissue specimens of skin melanoma have been analyzed for the presence of aberrant expression of genes of photoreceptor proteins [12]. These proteins included visual rhodopsin, transducin, PDE 6, cGMP dependent cationic channels, guanylyl cyclase 1, rhodopsin kinase, recoverin, and arrestin. In preparations of skin melanoma transcripts of all the abovelisted genes, they were present with different frequencies, whereas rhodopsin and transducin occurred only as their cone variants and PDE 6 and guanylyl cyclase 1 were only as their rod variants. Transcripts of all these genes except for the transducin gene were also found in melanocytes iso lated from healthy skin and nevi, but they were absent in keratinocytes of the HaCat line. The majority of the abovementioned cancerretina antigens were found in cell lines and tissue specimens of skin melanoma also on the level of proteins, but they were absent in healthy skin, nevi, and cultured melanocytes and keratinocytes. Note that mRNAs of rhodopsin, transducin, PDE 6, cGMP dependent cationic channels, guanylyl cyclase 1, rhodopsin kinase, recoverin, and arrestin, but not of transducin and rhodopsin kinase, were also found in var ious normal tissues different from skin. However, these antigens were not expressed at the protein level in all studied normal tissues except for the retina. It has been long known that expression of mRNA encoding some proteins and of the proteins themselves related with phototransduction is modulated by light in photoreceptor cells [3436]. This was also true for some cancerretina antigens expressed in melanoma cell lines [37]. Thus, light suppressed in these cells the expression of rhodopsin and transducin but stimulated the expres sion of guanylyl cyclase 1, recoverin, and arrestin both on the mRNA and protein levels. However, in the case of PDE 6 the expression was present only on the level of mRNA but not of the protein. Expression of the cancer retina antigens similarly depended on illumination also in retinoblastoma cells [37]. This effect was shown to be functional for photoreceptor cells [36]. But yet it is unclear whether this effect plays a role in tumor cells. In patients with skin melanoma a paraneoplastic syndrome of melanomaassociated retinopathy (MAR) is sometimes observed. The MAR syndrome is character ized by night blindness, photopsia, and disturbance of the BIOCHEMISTRY (Moscow) Vol. 79 No. 8 2014
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vision fields. Immunocytochemical staining of the retina with blood serum or immunoglobulin fraction taken from patients with this syndrome reveals an intense reaction in the layer of bipolar cells [38, 39]. And the βwave ampli tude in the electroretinogram of such patients is decreased, which suggests a dysfunction of the bipolar cells [39]. In blood sera of patients with the MAR syndrome, autoantibodies are sometimes found against rhodopsin [12], recoverin [12], arrestin [12, 40], and transducin [40, 41], as well as against some other proteins different from those of the cancerretina antigens, including the 32kDa protein of Muller’s glia, 22kDa neuronal antigen, mitofilin, titin, and cyclooxygenase [42]. However, the presence of such autoantibodies in blood serum only sug gests but does not prove their involvement in the develop ment of MAR syndrome. A direct dependence between this syndrome and autoantibodies against one of the cancerretina antigens was shown in work [40] on transgenic mice ret with spon taneous malignant skin melanoma, which were used as a model of MAR. Tumorbearing and tumorfree mice of this line were examined including ophthalmoscopy, elec troretinography, and histological study of the retina. The tumorbearing mice were shown to have sharply pro nounced pathological changes specific for the MAR syn drome, and some of the mice displayed signs of this syn drome even in the absence of detectable neoplasms. In tumors of the ret mice there were transcripts of the rhodopsin, transducin, PDE 6, guanylyl cyclase 1, recov erin, and arrestin genes. On the protein level rhodopsin, transducin, PDE 6, recoverin, and arrestin were found with different frequencies (the presence of guanylyl cyclase 1 was not analyzed in this case). It is extremely important that injection of wildtype mice with spleen immune cells or blood serum from melanomabearing transgenic ret mice caused pronounced signs of the MAR syndrome in four animals (of 16) three weeks after the injection. Autoantibodies against arrestin (but not against rhodopsin, transducin, and recoverin) were found in blood sera of two from 15 transgenic ret mice with melanomas, whereas no such autoantibodies were detect ed in the tumorfree ret mice and in wildtype mice. In total, data of works [12, 40] have confirmed that the MAR syndrome, similarly to the CAR syndrome, is a consequence of the immune response of the organism to tumor antigens, which in this case are presented by one or several cancerretina antigens. Thus, these antigens, at least some of them, can act as paraneoplastic antigens. As mentioned in the beginning of this section, the term “cancerretina antigens” was given to photoreceptor proteins aberrantly expressed by tumor cells, by analogy with cancertestis antigens. The comparison of the MAGEA1 antigen as a representative of cancertestis antigens and recoverin as a representative of cancerreti na antigens has shown that in the case of skin melanoma
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this analogy is both qualitative and quantitative [12]. Thus, on the mRNA level the expression frequencies of MAGEA1 and recoverin in tumor cells are 80 and 70%, respectively. The expression frequencies of these antigens on the protein level are, respectively, 1975 and 45%. The frequencies of the presence of specific autoantibodies and/or Tcells in the blood sera of patients with melanoma are, respectively, 6 and 7%. By now aberrant expression of recoverin has been shown for such oncological diseases as lung carcinoma, gynecologic cancers, gastrointestinal tract cancers, etc. [43]. The aberrant expression of cancerretina antigens has been found only in the case of skin melanoma [12]. The degree of distribution of these antigens in other type tumors is to be shown by further studies.
MOLECULAR MECHANISMS OF ABERRANT EXPRESSION OF CANCERRETINA ANTIGENS The question arises why photoreceptor proteins nor mally specific for the retina are expressed in tumor cells different from retina cells. It is known that malignant transformation of different cells can be caused by changes in the methylation pattern of their DNA. These changes can be hypermethylation in the promoter region of tumor suppressor genes leading to their silencing or by hypomethylation in this region of other genes leading to their activation [4447]. Thus, such activation is exempli fied by the hypomethylation of the cancertestis genes [48, 49] and of the gene encoding recoverin [50]. The human recoverin gene is located at chromosome 17p13.1, which is a neighbor of the tumor suppressor p53 gene [51]. This gene with length of 910 kb is present in one copy and contains three exons and two introns [52]. According to calculations, the 5′sequence of this gene is characterized by a middle level of the density of CpG nucleotides with CpG ratio of 0.58 between positions –7 and +524 with respect to the transcription start. According to the data of bisulfite sequencing of DNA iso lated from different tissues, the majority of CpGdinu cleotides in the recoverin gene are located in the region of the first promoter before the first exon and in the first exon itself. In different tissues the average level of methy lation of CpGdinucleotides in the recoverin gene varies between 77 and 94% of their total number [50]. In total, the methylation level of the recoverin gene in different tissues and cell lines of skin melanoma is 49 87%, which is lower than in normal skin where it is 94%. And the expression level of recoverin mRNA in melanoma specimens is within the limits of 1.520.4 mRNA copies per cell, which is higher than the value for normal skin (1.0). The correlation coefficient between the average level of the recoverin gene methylation in the promoter region before the first exon and in the exon itself, on one hand, and the expression of recoverin
mRNA, on the other hand, is –0.9508. This means that in the skin melanoma cells the expression of recoverin mRNA inversely depends on the methylation level of CpGdinucleotides in the recoverin gene region under consideration. The main contributors are CpGdinu cleotides –7, –1, 44, 120, and 154, whereas methylation of some other CpGdinucleotides has weaker influence on the expression of recoverin mRNA [50]. With an inhibitor of DNA methyl transferase, 5 aza2′deoxycytidine (ADC), acting as a demethylating agent, it was shown in the same work that the conclusion about the inverse dependence between the expression of recoverin mRNA and the methylation level of CpGdi nucleotides in the recoverin gene region under consider ation was true also for lung tumors. The treatment of SCLC cells of the NCIN82 line with this agent signifi cantly decreased the methylation level of the recoverin gene concurrently with an increase in the number of recoverin mRNA transcripts from 1.3 to 5.7 copies per cell. On the contrary, the inhibitor of histone deacetylase trichostatin A displayed only a slight influence on the expression of recoverin mRNA in the SCLC cells, whereas the effect of trichostatin A combined with ADC was the same as the effect of ADC alone. Consequently, the methylation of the recoverin gene in the cells of lung tumors is involved in the control of the expression of recoverin mRNA, as also observed in the melanoma cells. The final conclusion of work [50] is as follows: a) the methylation of CpGdinucleotides in the recoverin gene leads to inhibition of synthesis of recoverin mRNA in normal tissues, and b) the demethylation of definite nucleotides in the recoverin gene promoter region before the first exon and in the exon itself contributes to the aberrant expression of this protein in transformed cells.
IS THERE A “SENSE” IN THE ABERRANT EXPRESSION OF CANCERRETINA ANTIGENS FOR TUMOR CELLS? As already stated, the illumination of photoreceptor cells initiates signal transduction in the cascade of rhodopsin → transducin → PDE 6. This transduction is accompanied by a decrease in the concentration of the photoreceptor secondary messenger cGMP and an increase in the level of calcium ions in the cytoplasm of the photoreceptor. The regulation of this signal is also contributed to by rhodopsin kinase, which phosphory lates and thus desensitizes the photoexcited rhodopsin. In turn, the activity of rhodopsin kinase is controlled by the Ca2+binding protein recoverin [10]. In tumor cells, all these participants of the phototransduction are cancer retina antigens [12]. Therefore, the question arises if the aberrant expression of the cancerretina antigens has a “sense” for tumor cells. BIOCHEMISTRY (Moscow) Vol. 79 No. 8 2014
CANCERRETINA ANTIGENS In work [33], initially recoverinnegative lung ade nocarcinoma cells of A549 line were transfected with recoverin cDNA. The transfection noticeably suppressed proliferation of the adenocarcinoma cells during their cultivation. In such transfected cells, GRK kinases and caveolin1 could be possible targets for recoverin [53]. But there was a problem: the tumor cells used were given with the capability of expressing recoverin which was “alien” for them and could readily produce complexes with hydrophobic targets represented by proteins or membranes. Therefore, the question remains whether the model used in this work adequately presented events occurring in tumor cells, which could spontaneously express this cancerretina antigen. As stated in the section “Cancerretina antigens in skin melanoma”, skin melanoma cells can express a num ber of cancerretina antigens [12]. Two of them, trans ducin and PDE 6, were directly shown to be involved in the metabolism of melanoma cells with the functioning cascade of Frizzled2 → transducin → PDE 6 [54]. In this cascade, the protein Frizzled2, a structural homolog of visual rhodopsin, acts as a receptor [55]. As differenti ated from rhodopsin, Frizzled2 is activated not by light but by protein Wnt5a [56] from a large family of regulato ry Wnt proteins [57]. Then a signal from the receptor Frizzled2 is transmitted to transducin and then to PDE 6, activating this effector enzyme. The activated PDE 6 hydrolyzes cGMP, decreasing its intracellular concentra tion, and this, in turn, results in mobilization of calcium ions and increase in their intracellular level. Thus, as shown in work [54], transducin and PDE 6 as members of the Frizzled2 → transducin → PDE 6 cascade are involved in the control of cGMP metabolism and homeo stasis of calcium ions in skin melanoma cells. There are a number of intracellular targets for cGMP, including cGMPdependent protein kinases or protein kinases G (PKG) represented by two families, PKG I and PKG II, and also cGMPdependent cationic channels and some phosphodiesterases [58]. In such con text, PKG I is interesting for us because this enzyme is important for functioning of tumor cells. Thus, inhibition of PKG I had a cytotoxic effect on cells of pancreatic ade nocarcinoma and of ovary carcinoma [5961] and also suppressed the proliferation and migration of pancreatic adenocarcinoma cells [59]. The signal transduction in the Frizzled2 → transducin → PDE 6 cascade leads to a decrease in the intracellular concentration of cGMP and as a result to a decrease in the activity of PKG I capable of phosphorylating different calcium channels and changing their conductivity [58]. The increase in intra cellular level of calcium ions triggers various downstream events including those that modulate the transcription of some genes. Thus, in teratocarcinoma cells, calcium ions activate transcription regulated by the NFAT factor with involvement of Ca/calmodulindependent protein phos phatase calcineurin [62]. It is supposed that in resting BIOCHEMISTRY (Moscow) Vol. 79 No. 8 2014
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tumor cells a highly phosphorylated inactive factor NF AT is located in the cytoplasm. The calcineurinmediat ed signal transduction is accompanied by dephosphoryla tion of NFAT and demasking a sequence of the nuclear localization that leads to translocation of this factor into the nucleus, where it binds to DNA and participates in the regulation of transcription [63]. Thus, we assert that the aberrant expression of at least two cancerretina antigens, transducin and PDE 6, is functionally significant for tumor cells. As to recoverin, so far there are only indirect data on a possible function ality of its aberrant expression. The question remains of the “sense” of aberrant expression of other cancerretina antigens for tumor cells. The diagnostic value of cancer retina antigens, first of all of recoverin, or more strictly of serum autoantibodies against this protein, is also unclear. These autoantibodies with high specificity of 98% display only 20% sensitivity [32]. However, the CAR syndrome caused by them can manifest itself months or even years before a malignant tumor, which is its initial source, can be detected [20], and this makes the question of their diagnostic value an open question. This work was supported by the Russian Foundation for Basic Research (project 120400922) and by the Research Institute of Mitoengineering, Lomonosov Moscow State University.
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25. Polans, A. S., Witkowska, D., Haley, T. L., Amundson, D., Baizer, L., and Adamus, G. (1995) Recoverin, a photore ceptorspecific calciumbinding protein, is expressed by the tumor of a patient with cancerassociated retinopathy, Proc. Natl. Acad. Sci. USA, 92, 91769180. 26. Adamus, G., Amundson, D., and MacKay, P. G. (1998) Longterm persistence of antirecoverin antibodies in endometrial cancerassociated retinopathy, Arch. Ophthalmol., 116, 251253. 27. Klingele, T. G., Burde, R. M., Rappazzo, J. A., Isserman, M. J., Burgess, D., and Kantor, O. (1984) Paraneoplastic retinopathy, J. Clin. Neuroophthalmol., 4, 239245. 28. Eltabbakh, G. H., and Hoogereland, D. L. (1995) Paraneoplastic retinopathy associated with uterine sarco ma, Gynecol. Oncol., 58, 120123. 29. Matsubara, S., Yamaji, Y., Fujita, T., Kanayama, T., Yamadori, I., Sato, M., Fujita, J., Shiotani, T., and Takahara, J. (1996) Cancerassociated retinopathy syn drome: a case of small cell lung cancer expressing recoverin immunoreactivity, Lung Cancer, 14, 265271. 30. Thirkill, C. E., Keltner, J. L., Tyler, N. K., and Roth, A. M. (1993) Antibody reactions with retina and cancerassociat ed antigens in 10 patients with cancerassociated retinopa thy, Arch. Ophthalmol., 111, 931937. 31. Salgia, R., Hedges, T. R., Rizk, M., Reimerm, R. H., and Skarin, A. T. (1998) Cancerassociated retinopathy in a patient with nonsmallcell lung carcinoma, Lung Cancer, 22, 149152. 32. Bazhin, A. V., Savchenko, M. S., Shifrina, O. N., Demoura, S. A., Chikina, S. Yu., Jaques, G., Kogan, E. A., Chuchalin, A. G., and Philippov, P. P. (2004) Recoverin as a paraneoplastic antigen in lung cancer: the occurrence of antirecoverin autoantibodies in sera and recoverin in tumors, Lung Cancer, 44, 193198. 33. Maeda, A., Ohguro, H., Maeda, T., Wada, I., Sato, N., Kuroki, Y., and Nakagawa, T. (2000) Aberrant expression of photoreceptorspecific calciumbinding protein (recov erin) in cancer cell lines, Cancer Res., 60, 19141920. 34. Farber, D. B., Danciger, J. S., and Organisciak, D. T. (1991) Levels of mRNA encoding proteins of the cGMP cascade as a function of light environment, Exp. Eye Res., 53, 781786. 35. McGinnis, J. F., Whelan, J. P., and Donoso, L. A. (1992) Transient, cyclic changes in mouse visual cell gene products during the lightdark cycle, J. Neurosci. Res., 31, 584 590. 36. Wiechmann, A. F., and Sinacola, M. K. (1997) Diurnal expression of recoverin in the rat retina, Brain Res. Mol. Brain Res., 45, 321324. 37. Bazhin, A. V., Schadendorf, D., Owen, R. W., Zernii, E. Yu., Philippov, P. P., and Eichmuller, S. B. (2008) Visible light modulates the expression of cancerretina antigens, Mol. Cancer Res., 6, 110118. 38. Milam, A. H., Saari, J. C., Jacobson, S. G., Lubinski, W. P., Feun, L. G., and Alexander, K. R. (1993) Autoantibodies against retinal bipolar cells in cutaneous melanomaassociated retinopathy, Invest. Ophthalmol. Vis. Sci., 34, 91100. 39. Potter, M. J., Thirkill, C. E., Dam, O. M., Lee, A. S., and Milam, A. H. (1999) Clinical and immunocytochemical findings in a case of melanomaassociated retinopathy, Ophthalmology, 106, 21212125. BIOCHEMISTRY (Moscow) Vol. 79 No. 8 2014
CANCERRETINA ANTIGENS 40. Bazhin, A., Dalke, C., Willner, N., Abschutz, O., Wildberger, H., Philippov, P., Dummer, R., Graw, J., Hrabe de Angelis, M., Schadendorf, D., Umansky, V., and Eichmьller, S. (2009) Cancerretina antigens as potential paraneoplastic antigens in melanomaassociated retinopa thy, Int. J. Cancer, 124, 140149. 41. Potter, M. J., Adamus, G., Szabo, S. M., Lee, R., Mohaseb, K., and Behn, D. (2002) Autoantibodies to transducin in a patient with melanomaassociated retinopathy, Am. J. Ophthalmol., 134, 128130. 42. Pfohler, C., Preuss, K. D., Tilgen, W., Stark, A., Regitz, E., Fadle, N., and Pfreundschuh, M. (2007) Mitofilin and titin as target antigens in melanomaassociated retinopathy, Int. J. Cancer, 120, 788795. 43. Matsuo, S., Ohguro, H., Ohguro, I., and Nakazawa, M. (2010) Clinicopathological roles of aberrantly expressed recoverin in malignant tumor cells, Ophthalmic Res., 43, 139144. 44. Esteller, M. (2007) Epigenetic gene silencing in cancer: the DNA hypermethylome, Hum. Mol. Genet., 16, R50R59. 45. Jones, P. A., and Baylin, S. B. (2007) The epigenomics of cancer, Cell, 128, 683692. 46. Gronbeek, K., Hother, C., and Jones, P. A. (2007) Epigenetic changes in cancer, APMIS, 115, 10391059. 47. Li, Y., Liu, Z. L., Zhang, K. L., Chen, X. Y., Kong, Q. Y., Wu, M. L., Sun, Y., Liu, J., and Li, H. (2009) Methylation associated silencing of S100A4 expression in human epi dermal cancers, Exp. Dermatol., 18, 842848. 48. De Smet, C., De Backer, O., Faraoni, F., Lurquin, C., Brasseur, F., and Boon, T. (1996) The activation of human gene MAGE1 in tumor cells is correlated with genomewide demethylation, Proc. Natl. Acad. Sci. USA, 93, 71497153. 49. De Smet, C., Lurquin, C., Lethe, B., Martelange, V., and Boon, T. (1999) DNA methylation is the primary silencing mechanism for a set of germ line and tumorspecific genes with a CpGrich promoter, Mol. Cell. Biol., 19, 73277335. 50. Bazhin, A. V., De Smet, Ch., Golovastova, M. O., Schmidt, J., and Philippov, P. P. (2010) Aberrant demethy lation of the recoverin gene is involved in the aberrant expression of recoverin in cancer cells, Exp. Dermatol., 19, 10231025. 51. McGinnis, J. F., Austin, B., Klisak, I., Heinzmann, C., Kojis, T., Sparkes, R. S., Bateman, J. B., and Lerious, V. (1995) Chromosomal assignment of the human gene for the cancerassociated retinopathy protein (recoverin) to chro mosome 17p13.1, J. Neurosci. Res., 40, 165168.
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REVIEW
CancerRetina Antigens – A New Group of Tumor Antigens M. O. Golovastova1,2*, A. V. Bazhin1,3, and P. P. Philippov1 1
Lomonosov Moscow State University, Belozersky Institute of PhysicoChemical Biology, 119991 Moscow, Russia 2 Lomonosov Moscow State University, Faculty of Bioengineering and Bioinformatics, 119991 Moscow, Russia; fax: +7 (495) 9393181; Email: [email protected] 3 Department of General, Visceral, and Transplant Surgery, LudwigMaximiliansUniversity, 81377 Munich, Germany; fax: +49 (89) 440076433; Email: [email protected]muenchen.de Received April 8, 2014 Abstract—Some photoreceptor proteins normally specific for the eye retina are aberrantly expressed in malignant tumors. These proteins include recoverin, visual rhodopsin, transducin, cGMPphosphodiesterase 6 (PDE 6), cGMPdependent cationic channels, guanylyl cyclase 1, rhodopsin kinase, and arrestin. By analogy with cancertestis antigens, these pho toreceptor proteins form the group of cancerretina antigens. It is shown that an aberrant demethylation of the promoter region of recoverin is involved in the aberrant expression of this protein. The cascade Wnt5a → Frizzled2 → transducin → PDE 6 is shown to function in skin melanoma cells, and this suggests that these cancerretina antigens can play a function al role. The events accompanying the signal transduction in this cascade, including those involving calcium ions and cGMPdependent protein kinase (protein kinase G), are discussed. DOI: 10.1134/S000629791408001X Key words: visual transduction, retina, cancerretina antigens, aberrant expression, cancer
Among numerous human diseases, malignant tumors are one of major causes of death in both developed and developing countries. Thus, 12.4 million new cancer cases and 7.6 million cancercaused deaths were record ed throughout the world in 2008, and 28 million patients continued to live with this diagnosis for five years. The number of new cancer cases by 2030 in the world is pre dicted to be 2126 million with deaths of 1317 million people per year. Such predictions are significantly pro moted by global industrialization and urbanization and the associated ecological and social problems. Because the risk of oncological diseases increases with age, the increase in lifespan due to improvement of life conditions and public health quality, especially noticeable in some developing countries, also contributes to this prognosis [1, 2]. Current therapeutic approaches allow clinicians to cure a significant number of patients with oncological diseases. Under other similar conditions, a positive result strongly depends on the stage of starting the treatment. Abbreviations: ADC, 5aza2′deoxycytidine; CAR, cancer associated retinopathy; MAR, melanomaassociated retinopa thy; NSCLC, nonsmall cell lung carcinoma; PDE 6, cGMP phosphodiesterase 6; SCLC, small cell lung carcinoma. * To whom correspondence should be addressed.
Thus, in the case of a localized tumor the fiveyear sur vival is more than 90% in patients with cancer of mam mary gland, colon, urinary bladder, kidney, uterine body, etc. In some cancers this value decreases manifold if the treatment is started at the stage of distant metastases and is about 6 and 8%, respectively, for urinary bladder and renal cancers [3]. These figures obviously indicate the importance of the development and use of methods for diagnosis of cancer at its earliest stage. Malignant transformation is accompanied by dra matic changes in features of the affected cells. In particu lar, the expression levels of various proteins of the cells can increase or decrease – and this concerns tumorasso ciated antigens. More rarely, healthy cells upon malig nant transformation begin to synthesize a protein or even a set of proteins that are completely absent in normal cells – this is a group of tumorspecific antigens [4, 5]. Both types of tumor antigens are promising for diagnosis of cancer and/or as targets for immunotherapy of cancer. From this standpoint, tumorspecific antigens as mole cules unique for a tumor seem more attractive than tumorassociated antigens. Based on data available by 2011, more than 20 anti gens have been characterized as tumor markers detectable in blood, urine, or tissue of oncological patients and are already used in clinical practice [6]. However, the number
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of antigens described in the literature is much higher, and some of them are considered as potential targets for immunotherapy of cancer. Such antigens are exemplified by proteins of cancertestis antigens [79]. Normally these antigens are expressed only in testicles, which are an immunologically privileged zone of the organism. However, in some types of cancer such antigens can be aberrantly expressed in tumors located beyond this zone. The nervous system is another immunologically privileged zone of the organism. The eye retina, which is a member of this system, contains several types of neu rons including photoreceptor cells, rods and cones, responsible for phototransduction. This process is trig gered by a light quantum, which activates the visual pig ment rhodopsin and thus initiates signal transduction in the cascade of rhodopsin → transducin → cGMPphos phodiesterase 6 (PDE 6) that leads to hydrolysis of the photoreceptor secondary messenger cGMP. A decrease in the intracellular concentration of cGMP is accompanied by closing cGMPsensitive cationic channels located in the plasma membrane and by its hyperpolarization. Such is a pathway of the light quantum energy transformation into an electrophysiological signal, which is then trans mitted to the brain through the higher order neurons [10]. Normally, the abovementioned participants of the phototransduction and some other photoreceptor pro teins involved in its regulation are specific for the normal retina. However, some recent data have shown that all these participants can be aberrantly expressed in malig nant tumors located beyond the nervous system. By anal ogy with the cancertestis antigens, such photoreceptor proteins were designated as a group of cancerretina anti gens [11, 12]. The present review will consider and dis cuss the currently available data on the aberrant expres sion of cancerretina antigens in malignant tumors.
PHOTORECEPTOR PROTEINS AS CANCERRETINA ANTIGENS Recoverin [13, 14] was the first photoreceptor pro tein proved to be capable of aberrant expression in malig nant tumors [15]. Later a number of other photoreceptor proteins were added to recoverin, such as visual rhodopsin, transducin, PDE 6, cGMPdependent cationic channels, guanylyl cyclase 1, rhodopsin kinase, and arrestin [12]. Similarly to cancertestis antigens [7 9], all these photoreceptor proteins are characterized, first, by their normal expression limited to the immuno logically privileged zone (the testicles and retina, respec tively), second, by their ability to be expressed in trans formed cells, and third, by the ability of at least some of them to cause an immune response of the organism. Cancerretina antigens in lung carcinoma. As men tioned above, recoverin was the first photoreceptor pro tein proved to be aberrantly expressed in malignant
tumors. This was found as a result of studies for many years on a paraneoplastic neurological syndrome, para neoplastic retina degeneration, or cancerassociated retinopathy (CAR). Mechanisms of the CAR syndrome are considered in detail in some reviews [16], and here we shall note that it is a result of the immune system response to the aberrant expression of recoverin in a malignant tumor located beyond the nervous system. The produced autoantibodies interact with recoverin located in the reti na photoreceptor cells, rods and cones [15], and trigger their apoptosis [17]. The αwave amplitude is suppressed in the electroretinogram of patients with the CAR syn drome, which indicates a dysfunction of the photorecep tors [18]. It should be added that the CAR syndrome is associated with dispersed vision, the loss of peripheral and color vision, nocturnal blindness, and finally with total blindness [19]. It is important that in some cases signs of this syndrome are manifested months and some times even years before the malignant tumor, which is its initial source, can be detected [20]. The first indications of the involvement of an unknown antigen in the CAR syndrome pathogenesis were obtained in works [2123]: autoantibodies of blood sera from patients with this syndrome revealed certain cells in retina sections. The target for these autoantibod ies occurred to be a protein with an apparent molecular weight of 23 kDa that was named CARantigen [24] and later identified as recoverin [15, 25]. In blood sera from patients with small cell lung car cinoma (SCLC) [15, 2430] and nonsmall cell lung car cinoma (NSCLC) [31], antirecoverin antibodies were found in high titers. However, such autoantibodies, although in low titer, could also be found in sera from patients with SCLC and NSCLC but in the absence of the signs of CAR syndrome [32]. The authors used recombi nant recoverin as an antigen to analyze blood sera from 143 patients with SCLC and NSCLC and found that fre quencies of the recoverinpositive samples were, respec tively, 15 and 20%. And signs of CAR syndrome were absent in all patients studied, thus its frequency in this study may be assessed to be lower than 1%. Moreover, the recoverin expression frequencies were also determined in tumor tissues using affinity purified antirecoverin anti bodies, and frequencies of 68 and 85% were obtained for the SCLC and NSCLC, respectively. Thus, the frequency of recoverin expression in lung tumors was higher than the frequency of autoantibodies against this protein in the blood sera from the oncological patients and much high er than the CAR syndrome frequency [32]. Only two of nine analyzed cell lines of lung carcino ma occurred to be recoverinpositive [33], i.e. the fre quency of recoverin expression was only about 20%, which was much lower than the abovepresented values for the tumor tissues. Certainly, the number of cell lines analyzed in this work was insufficient for statistically sig nificant assessment of the frequency of recoverin expres BIOCHEMISTRY (Moscow) Vol. 79 No. 8 2014
CANCERRETINA ANTIGENS sion in them. Moreover, our unpublished data have shown that cell lines of SCLC during their isolation and cultiva tion can lose the ability to express recoverin. Thus, the absence of its expression in any cell line does not prove that it is really absent in the initial tumor tissue. It is rea sonable to suppose that this conclusion is true also for tumor antigens others than recoverin. For today the aberrant expression of cancerretina antigens in tumors has been studied only for recoverin, whereas for skin melanoma there are data on a number of these antigens. Cancerretina antigens in skin melanoma. A number of cell lines and tissue specimens of skin melanoma have been analyzed for the presence of aberrant expression of genes of photoreceptor proteins [12]. These proteins included visual rhodopsin, transducin, PDE 6, cGMP dependent cationic channels, guanylyl cyclase 1, rhodopsin kinase, recoverin, and arrestin. In preparations of skin melanoma transcripts of all the abovelisted genes, they were present with different frequencies, whereas rhodopsin and transducin occurred only as their cone variants and PDE 6 and guanylyl cyclase 1 were only as their rod variants. Transcripts of all these genes except for the transducin gene were also found in melanocytes iso lated from healthy skin and nevi, but they were absent in keratinocytes of the HaCat line. The majority of the abovementioned cancerretina antigens were found in cell lines and tissue specimens of skin melanoma also on the level of proteins, but they were absent in healthy skin, nevi, and cultured melanocytes and keratinocytes. Note that mRNAs of rhodopsin, transducin, PDE 6, cGMP dependent cationic channels, guanylyl cyclase 1, rhodopsin kinase, recoverin, and arrestin, but not of transducin and rhodopsin kinase, were also found in var ious normal tissues different from skin. However, these antigens were not expressed at the protein level in all studied normal tissues except for the retina. It has been long known that expression of mRNA encoding some proteins and of the proteins themselves related with phototransduction is modulated by light in photoreceptor cells [3436]. This was also true for some cancerretina antigens expressed in melanoma cell lines [37]. Thus, light suppressed in these cells the expression of rhodopsin and transducin but stimulated the expres sion of guanylyl cyclase 1, recoverin, and arrestin both on the mRNA and protein levels. However, in the case of PDE 6 the expression was present only on the level of mRNA but not of the protein. Expression of the cancer retina antigens similarly depended on illumination also in retinoblastoma cells [37]. This effect was shown to be functional for photoreceptor cells [36]. But yet it is unclear whether this effect plays a role in tumor cells. In patients with skin melanoma a paraneoplastic syndrome of melanomaassociated retinopathy (MAR) is sometimes observed. The MAR syndrome is character ized by night blindness, photopsia, and disturbance of the BIOCHEMISTRY (Moscow) Vol. 79 No. 8 2014
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vision fields. Immunocytochemical staining of the retina with blood serum or immunoglobulin fraction taken from patients with this syndrome reveals an intense reaction in the layer of bipolar cells [38, 39]. And the βwave ampli tude in the electroretinogram of such patients is decreased, which suggests a dysfunction of the bipolar cells [39]. In blood sera of patients with the MAR syndrome, autoantibodies are sometimes found against rhodopsin [12], recoverin [12], arrestin [12, 40], and transducin [40, 41], as well as against some other proteins different from those of the cancerretina antigens, including the 32kDa protein of Muller’s glia, 22kDa neuronal antigen, mitofilin, titin, and cyclooxygenase [42]. However, the presence of such autoantibodies in blood serum only sug gests but does not prove their involvement in the develop ment of MAR syndrome. A direct dependence between this syndrome and autoantibodies against one of the cancerretina antigens was shown in work [40] on transgenic mice ret with spon taneous malignant skin melanoma, which were used as a model of MAR. Tumorbearing and tumorfree mice of this line were examined including ophthalmoscopy, elec troretinography, and histological study of the retina. The tumorbearing mice were shown to have sharply pro nounced pathological changes specific for the MAR syn drome, and some of the mice displayed signs of this syn drome even in the absence of detectable neoplasms. In tumors of the ret mice there were transcripts of the rhodopsin, transducin, PDE 6, guanylyl cyclase 1, recov erin, and arrestin genes. On the protein level rhodopsin, transducin, PDE 6, recoverin, and arrestin were found with different frequencies (the presence of guanylyl cyclase 1 was not analyzed in this case). It is extremely important that injection of wildtype mice with spleen immune cells or blood serum from melanomabearing transgenic ret mice caused pronounced signs of the MAR syndrome in four animals (of 16) three weeks after the injection. Autoantibodies against arrestin (but not against rhodopsin, transducin, and recoverin) were found in blood sera of two from 15 transgenic ret mice with melanomas, whereas no such autoantibodies were detect ed in the tumorfree ret mice and in wildtype mice. In total, data of works [12, 40] have confirmed that the MAR syndrome, similarly to the CAR syndrome, is a consequence of the immune response of the organism to tumor antigens, which in this case are presented by one or several cancerretina antigens. Thus, these antigens, at least some of them, can act as paraneoplastic antigens. As mentioned in the beginning of this section, the term “cancerretina antigens” was given to photoreceptor proteins aberrantly expressed by tumor cells, by analogy with cancertestis antigens. The comparison of the MAGEA1 antigen as a representative of cancertestis antigens and recoverin as a representative of cancerreti na antigens has shown that in the case of skin melanoma
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this analogy is both qualitative and quantitative [12]. Thus, on the mRNA level the expression frequencies of MAGEA1 and recoverin in tumor cells are 80 and 70%, respectively. The expression frequencies of these antigens on the protein level are, respectively, 1975 and 45%. The frequencies of the presence of specific autoantibodies and/or Tcells in the blood sera of patients with melanoma are, respectively, 6 and 7%. By now aberrant expression of recoverin has been shown for such oncological diseases as lung carcinoma, gynecologic cancers, gastrointestinal tract cancers, etc. [43]. The aberrant expression of cancerretina antigens has been found only in the case of skin melanoma [12]. The degree of distribution of these antigens in other type tumors is to be shown by further studies.
MOLECULAR MECHANISMS OF ABERRANT EXPRESSION OF CANCERRETINA ANTIGENS The question arises why photoreceptor proteins nor mally specific for the retina are expressed in tumor cells different from retina cells. It is known that malignant transformation of different cells can be caused by changes in the methylation pattern of their DNA. These changes can be hypermethylation in the promoter region of tumor suppressor genes leading to their silencing or by hypomethylation in this region of other genes leading to their activation [4447]. Thus, such activation is exempli fied by the hypomethylation of the cancertestis genes [48, 49] and of the gene encoding recoverin [50]. The human recoverin gene is located at chromosome 17p13.1, which is a neighbor of the tumor suppressor p53 gene [51]. This gene with length of 910 kb is present in one copy and contains three exons and two introns [52]. According to calculations, the 5′sequence of this gene is characterized by a middle level of the density of CpG nucleotides with CpG ratio of 0.58 between positions –7 and +524 with respect to the transcription start. According to the data of bisulfite sequencing of DNA iso lated from different tissues, the majority of CpGdinu cleotides in the recoverin gene are located in the region of the first promoter before the first exon and in the first exon itself. In different tissues the average level of methy lation of CpGdinucleotides in the recoverin gene varies between 77 and 94% of their total number [50]. In total, the methylation level of the recoverin gene in different tissues and cell lines of skin melanoma is 49 87%, which is lower than in normal skin where it is 94%. And the expression level of recoverin mRNA in melanoma specimens is within the limits of 1.520.4 mRNA copies per cell, which is higher than the value for normal skin (1.0). The correlation coefficient between the average level of the recoverin gene methylation in the promoter region before the first exon and in the exon itself, on one hand, and the expression of recoverin
mRNA, on the other hand, is –0.9508. This means that in the skin melanoma cells the expression of recoverin mRNA inversely depends on the methylation level of CpGdinucleotides in the recoverin gene region under consideration. The main contributors are CpGdinu cleotides –7, –1, 44, 120, and 154, whereas methylation of some other CpGdinucleotides has weaker influence on the expression of recoverin mRNA [50]. With an inhibitor of DNA methyl transferase, 5 aza2′deoxycytidine (ADC), acting as a demethylating agent, it was shown in the same work that the conclusion about the inverse dependence between the expression of recoverin mRNA and the methylation level of CpGdi nucleotides in the recoverin gene region under consider ation was true also for lung tumors. The treatment of SCLC cells of the NCIN82 line with this agent signifi cantly decreased the methylation level of the recoverin gene concurrently with an increase in the number of recoverin mRNA transcripts from 1.3 to 5.7 copies per cell. On the contrary, the inhibitor of histone deacetylase trichostatin A displayed only a slight influence on the expression of recoverin mRNA in the SCLC cells, whereas the effect of trichostatin A combined with ADC was the same as the effect of ADC alone. Consequently, the methylation of the recoverin gene in the cells of lung tumors is involved in the control of the expression of recoverin mRNA, as also observed in the melanoma cells. The final conclusion of work [50] is as follows: a) the methylation of CpGdinucleotides in the recoverin gene leads to inhibition of synthesis of recoverin mRNA in normal tissues, and b) the demethylation of definite nucleotides in the recoverin gene promoter region before the first exon and in the exon itself contributes to the aberrant expression of this protein in transformed cells.
IS THERE A “SENSE” IN THE ABERRANT EXPRESSION OF CANCERRETINA ANTIGENS FOR TUMOR CELLS? As already stated, the illumination of photoreceptor cells initiates signal transduction in the cascade of rhodopsin → transducin → PDE 6. This transduction is accompanied by a decrease in the concentration of the photoreceptor secondary messenger cGMP and an increase in the level of calcium ions in the cytoplasm of the photoreceptor. The regulation of this signal is also contributed to by rhodopsin kinase, which phosphory lates and thus desensitizes the photoexcited rhodopsin. In turn, the activity of rhodopsin kinase is controlled by the Ca2+binding protein recoverin [10]. In tumor cells, all these participants of the phototransduction are cancer retina antigens [12]. Therefore, the question arises if the aberrant expression of the cancerretina antigens has a “sense” for tumor cells. BIOCHEMISTRY (Moscow) Vol. 79 No. 8 2014
CANCERRETINA ANTIGENS In work [33], initially recoverinnegative lung ade nocarcinoma cells of A549 line were transfected with recoverin cDNA. The transfection noticeably suppressed proliferation of the adenocarcinoma cells during their cultivation. In such transfected cells, GRK kinases and caveolin1 could be possible targets for recoverin [53]. But there was a problem: the tumor cells used were given with the capability of expressing recoverin which was “alien” for them and could readily produce complexes with hydrophobic targets represented by proteins or membranes. Therefore, the question remains whether the model used in this work adequately presented events occurring in tumor cells, which could spontaneously express this cancerretina antigen. As stated in the section “Cancerretina antigens in skin melanoma”, skin melanoma cells can express a num ber of cancerretina antigens [12]. Two of them, trans ducin and PDE 6, were directly shown to be involved in the metabolism of melanoma cells with the functioning cascade of Frizzled2 → transducin → PDE 6 [54]. In this cascade, the protein Frizzled2, a structural homolog of visual rhodopsin, acts as a receptor [55]. As differenti ated from rhodopsin, Frizzled2 is activated not by light but by protein Wnt5a [56] from a large family of regulato ry Wnt proteins [57]. Then a signal from the receptor Frizzled2 is transmitted to transducin and then to PDE 6, activating this effector enzyme. The activated PDE 6 hydrolyzes cGMP, decreasing its intracellular concentra tion, and this, in turn, results in mobilization of calcium ions and increase in their intracellular level. Thus, as shown in work [54], transducin and PDE 6 as members of the Frizzled2 → transducin → PDE 6 cascade are involved in the control of cGMP metabolism and homeo stasis of calcium ions in skin melanoma cells. There are a number of intracellular targets for cGMP, including cGMPdependent protein kinases or protein kinases G (PKG) represented by two families, PKG I and PKG II, and also cGMPdependent cationic channels and some phosphodiesterases [58]. In such con text, PKG I is interesting for us because this enzyme is important for functioning of tumor cells. Thus, inhibition of PKG I had a cytotoxic effect on cells of pancreatic ade nocarcinoma and of ovary carcinoma [5961] and also suppressed the proliferation and migration of pancreatic adenocarcinoma cells [59]. The signal transduction in the Frizzled2 → transducin → PDE 6 cascade leads to a decrease in the intracellular concentration of cGMP and as a result to a decrease in the activity of PKG I capable of phosphorylating different calcium channels and changing their conductivity [58]. The increase in intra cellular level of calcium ions triggers various downstream events including those that modulate the transcription of some genes. Thus, in teratocarcinoma cells, calcium ions activate transcription regulated by the NFAT factor with involvement of Ca/calmodulindependent protein phos phatase calcineurin [62]. It is supposed that in resting BIOCHEMISTRY (Moscow) Vol. 79 No. 8 2014
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tumor cells a highly phosphorylated inactive factor NF AT is located in the cytoplasm. The calcineurinmediat ed signal transduction is accompanied by dephosphoryla tion of NFAT and demasking a sequence of the nuclear localization that leads to translocation of this factor into the nucleus, where it binds to DNA and participates in the regulation of transcription [63]. Thus, we assert that the aberrant expression of at least two cancerretina antigens, transducin and PDE 6, is functionally significant for tumor cells. As to recoverin, so far there are only indirect data on a possible function ality of its aberrant expression. The question remains of the “sense” of aberrant expression of other cancerretina antigens for tumor cells. The diagnostic value of cancer retina antigens, first of all of recoverin, or more strictly of serum autoantibodies against this protein, is also unclear. These autoantibodies with high specificity of 98% display only 20% sensitivity [32]. However, the CAR syndrome caused by them can manifest itself months or even years before a malignant tumor, which is its initial source, can be detected [20], and this makes the question of their diagnostic value an open question. This work was supported by the Russian Foundation for Basic Research (project 120400922) and by the Research Institute of Mitoengineering, Lomonosov Moscow State University.
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