CONICET | Buscador de Institutos y Recursos Humanos

DISCURSO DE LA PRESENTACIÓN EN SEATTLE 2015Good afternoon to all. Thank you for your invitation. I am Raúl A. Ruggiero and I am working in the Laboratory of Experimental Oncology at the National Academy of Medicine in Buenos Aires, Argentina.For the last 25 years Dr. Prehn helped us a lot in our work with his wise comments, his constructive criticism and his always provoking ideas. In fact, I met Dr Prehn and his wife, during a Symposium of Cancer Research held in Buenos Aires, Argentina, 19 years ago.Today, I will talk briefly about 3 lines of investigation in which we collaborated with Dr. Prehn or he collaborated with us. These lines are:1) The immunostimulatory theory of cancer.2) The hypothesis of the biological sense of cancer3) The phenomenon of Concomitant Tumor Resistance1) The immunostimulatory theory of cancer was proposed by Prehn many years ago. He suggested, using Winn test, that the immune response (IR) evoked by chemically-induced strongly immunogenic tumors was not monotonic ?as the orthodoxy predicted ? but biphasic, with weak IR inducing stimulation and strong IR inducing inhibition of tumor growth. This year, we have finished a long paper in which we have confirmed and extended those previous observations to weakly antigenic murine tumors, most of spontaneous origin. We have also suggested a putative mechanistic explanation for the phenomenon of tumor-immunostimulation, based on the fact that it is observed in conventional mice but not observed neither in vitro nor in vivo in mice displaying a low inflammatory response. Our experiments suggested that the interaction of specifically immune T cells with target tumor cells at low stimulatory ratios enhanced the production of chemokines (as compared with that produced by tumor cells alone or mixed with normal T cells or cells immune to another tumor) that recruit resident macrophages at the tumor site. In turn, these macrophages, upon activation of Toll-like receptor 4 and some pro-inflammatory signaling pathways, wouls stimulate the production of more cytokines that would recruit more macrophages and other inflammatory cells (for example B cells) at the tumor site that would produce more growth-stimulating signals leading to an accelerated tumor growth. The concept of Immunostimulatory has not merely theoretic value but also therapeutic implications because, upon certain circumstances, in other cases, they may run a real risk of doing harm if the vaccine-induced immunity is too weak to move the reaction beyond the stimulatory part of the IRC. The experiments of this work were carried out at the National Academy of Medicine of Buenos Aires, but most of them were suggested by Dr. Prehn and therefore it appears as the last author of the paper. The final manuscript was sent to Oncoimmunology by suggestion of Dr. parmiani and I hope the reviewers find it suitable for publication. In our laboratory we are now performing experiments aimed to extend the concet of immunostimulation to normal antigens using the C57/Bl model. 2) The hypothesis of the biological sense of cancer was based , in part, on a former paper of Prehn called ?Regeneration versus neoplastic growth?. The hypothesis, that was published some years ago in a theoretical journal and also as a chapter of a book, states that cancer would be an attempt (although spurious) to restore or repair an organ or tissue that has lost or diminished its reparative ability. This statement is, in turn, based on the observation that throughout the animal kingdom, cancer is rarely ? if ever ? induced in healthy organ displaing an efficient reparative mechanism. I have not enough time to explain all the details of this hypothesis, but I want to indicate some of their features that are very different from those of conventional theories of cancer. For example, in the context of this hypothesis, cancer would have a profound biological sense since it would be an attempt (perhaps an ultimate attempt) to restore a damaged organ and the tumor cell would not be an autonomic entity that evades the rules that control normal cell proliferation because the tumor cell would always be responding to a reparative signal in the context of a damaged organ that has lost its reparative ability. Further, in the context of this hypothesis, the organ (or part of the organ) and not the cell would be the anatomical and physiological unit of cancer. In consequence, the neoplastic behavior could not be predicted even by the most exhaustive molecular analysis of the cell and this hypothesis might explain one of the most intriguing riddles of cancer biology: the riddle of the blue whale and the mouse that asks: Why don?t extremely larger animals develop neoplasms with a much higher incidence than very small ones since the cell population at risk is greater for several orders of magnitude?? Although this line of investigation is up to date highly theoretic, we are now performing in our laboratory experiments aimed to test some predictions of this hypothesis. 3) The phenomenon of Concomitant Tumor Resistance (CR) has been one of the most important lines of investigation in our laboratory for the last 30 years. CR is the phenomenon according to which a tumor bearing host inhibits or retards the growth of secondary tumor implants. In fact, Dr. Prehn in his article ?Two competing influences that may explain concomitant tumor resistance? (Cancer Res 1993) proposed this name instead of the former ?concomitant immunity? based on the observation that CR is induced not only by immunogenic tumors but also by tumors exhibiting undetectable immunogenicity. CR was first described by Paul Ehrlich in 1906, but, apart from a few isolated papers, the phenomenon remained virtually forgotten for the next 60 years until it was rediscovered in the 60? by Gershon, Southam and others. Since that moment on, some laboratories studied the phenomenon especially in tumor models in mice, rats and hamsters. However, even after its rediscovery, CR did not attract much attention by many oncology researchers as compared with other areas of cancer research, despite the fact that CR has been directly described in human beings and despite the fact that it may tell us about putative mechanisms of metastases control taking into account that metastases can be considered natural secondary tumor implants developed spontaneously during the growth of a primary tumor. In this regard, clinical and experimental evidence accumulated throughout the years has demonstrated that the surgical removal or extirpation of a primary tumor may be followed by an abrupt acceleration of metastatic growth, suggesting that a primary tumor may, at least in some cases, exert an inhibitory effect on its own metastases.CR has received many and often contradictory explanations.For the last 30 years, in our laboratory at the National Academy of Buenos Aires, we have studied the phenomenon of CR associated with the growth of 17 murine tumors exhibiting widely different degrees of immunogenicity ? since strongly immunogenic up to non-immunogenic tumors ? in an attempt to integrate the different explanations into a coherent picture. Our results demonstrated that, apart from a relatively small number of tumors that induce CR by immunological mechanisms (mainly associated to small sized strongly immunogenic tumors), the most universal manifestation of CR associated with most immunogenic and non-immunogenic tumors of large size, is not associated with any conventional immunological mechanism. This most universal manifestation of CR (from now on simply CR) proved to be not tumor specific and T-independent since it was observed in both euthymic and nude mice and tumor cells of the secondary implant inhibited by CR remained alive, in a state of dormant like state, at the site of secondary inoculum, without any signs of necrosis and without any infiltration by host cells, contrasting with an immunological reaction of rejection. Further, when we surgically removed the primary tumor, the otherwise inhibited secondary implant, started to grow, indicating a cytostatic mechanism rather than a cytotoxic one. The intensity of CR correlated to the presence of serum factors - different from antibodies and complement - that inhibited the proliferation of tumor cells in both in vitro and in vivo settings by a reversible mechanism. The only two tumors, from our collection of tumors, that did not exhibit CR, were two highly metastatic ones; in turn mice bearing these tumors, did not display any antitumor activity in serum. However, tumors that did not induce CR were very sensitive to the CR induced by other unrelated tumors. In fact lung metastases produced by the former, were strongly inhibited by the contra-lateral inoculation of tumors that do induce CR and by the periodic inoculation of serum from mice bearing these tumors.Although we knew that serum factors were associated with the phenomenon of CR, the origin and chemical nature of these factors remained elusive (or an enigma) for years, as well as the paradoxical question about why such a factor could inhibit the proliferation of a secondary tumor but not of a large primary tumor composed of the same cells type of cells.The task of purification and identification of these elusive serum factors was long and difficult. At each step of purification, in each fraction of each step of purification we had to evaluate the presence of the antitumor inhibitory activity using routinely (although not exclusively) the [3 H] - thymidine uptake assay. One of the most important problems was related to the following fact: when we used the supposedly last step of purification [High-performance liquid chromatography (HPLC) using a gradient of acetonitrile and trifluoroacetic acid (TFA)], we found that all the antitumor activity was concentrated in only one fraction. However, when we analyzed this fraction by amino acid analysis and sequencing and by ion electrospray mass (MS) and tandem mass (MS/MS) spectrometry, only tyrosine was detected. However, that result was impossible because tyrosine is a common amino acid lacking any inhibitory activity on both tumor and normal cell proliferation. We repeated the experiment many times but with identical result. A clue to understand this puzzle was provided by reading a paper of botany or agronomy: the authors, while studying the development of more environmentally friendly weed management system, showed that the unusual ability of many grasses to outcompete or displace other neighboring plants was based on the phytotoxic properties of a factor produced by their roots. To identify this factor they used a very similar procedure of purification than ours and found that the factor had an identical MW of tyrosine: but it was not tyrosine!, it was an isomer of tyrosine called meta-tyrosine. After that our work was faster although some problems remained since in our fraction had actually an excess of tyrosine that masked the presence of other molecules. Finally, using an HPLC that distinguished the isomers of tyrosine, we could demonstrate that the antitumor activity of the fraction and the whole serum could be attributes a low amounts of meta- and ortho-tyrosine, two unnatural isomers of tyrosine, unnatural meaning that they are not usually present in normal proteins.Meta- and ortho-tyrosine proved to be inhibitory on the in vitro proliferation of several tumor cells, and it was also demonstrated that the antitumor power of meta-tyrosine was about ten times stronger than that of ortho-tyrosine. The antitumor activity of both isomers was counteracted by phenylalanine and, but to a lesser extent, by glutamic and aspartic acid, glutamine and histidine but not by tyrosine and the remaining amino acids. When we inoculated phenylalanine at the site of a secondary tumor implant otherwise inhibited by CR, the second implant started to grow and when we inoculated meta-tyrosine at the site of a primary tumor implant, this implant otherwise growing, was inhibited. Taken together, these observations strongly suggested that the phenomenon of CR generated in vivo is, at least in our tumor models, mediated by meta- and ortho tyrosine.Afterwards, we evaluated the effect of meta-tyrosine on the growth of established metastases, the main clinical problem in tumor pathology. In the first series of experiments we tested this effect on tumor-bearing mice using three different models of highly metastatic murine tumors. We divided tumor-bearing mice into two groups at the time when we knew that metastases were already present in lung. In the figure is shown a representative experiment. One group (experimental group) received a daily injection of meta-tyrosine for the intravenous route for the next 21 days. The other group received saline and served as control. Three weeks later, mice were sacrificed and lung metastases counted. As you can see in the Figure, the treatment with meta-tyrosine strongly reduced the number and size of metastatic foci as compared with controls. This anti-metastatic effect was dose dependent but even a dose that was 20 times lower than the maximum dose used (67 mg/Kg/day) was effective to inhibit metastatic growth. In the second series of experiments, we attempted to mimic a clinical situation: tumors from tumor-bearing mice were surgically removed at the time when we knew that metastases were already present in lung and liver and then mice were divided into two groups. One group (experimental group) received a daily injection of meta-tyrosine for the intravenous route for the next 42 days. The other group received saline and served as control. As you can see the Figure, that showed a representative experiment, while the nine controls died relatively rapid after tumor extirpation, displaying a huge number of metastases in both lung and liver, only two out of eight treated-mice died; the remaining 6 remained disease free for the rest of their lives. When they died at about two years old, the autopsy did not reveal any sign of metastases neither in lung nor in liver. These results were rather impressive and more, taking into account that the anti-metastatic effect was achieved without any toxic-side effect.Molecular analysis of the anti-proliferative effects of meta- and ortho-tyrosine demonstrated that they reduced significantly the activation of ERK ½ and MAPK signaling pathway. More experiments are underway in our laboratory to understand more accurately the molecular basis of this inhibition.A last comment: the central paradox of CR, that is, the inhibition of secondary tumor implants together with the progressive growth of the primary tumor, has remained unsolved for more than a century. Our experiments suggested a putative explanation for this riddle: as a primary tumor grows, relatively large amounts of most amino acids, including those that counteract the inhibitory effect of meta- and ortho-tyrosine are accumulated in the tumor microenvironment which could act as a shield to protect the primary tumor from the inhibitory effect of meta- and ortho-tyrosine, while at distant sites, , such as sites of putative secondary tumor implants, the content of amino acids is significantly lower.A challenge in order to improve the therapeutic effects of meta- and ortho-tyrosine would be to achieve a steady state of meta- and ortho-tyrosine in circulation after intravenous inoculation because up to date, in our hands, the concentration of both isomers wanes after one hour and a half after inoculation.What will be the future of these investigations? Will these promissory factors improve the current therapies of cancer against at least one type of human cancer? Really, I don?t know. Only the time new experiments and ingenuity will answer these questions.