CONICET | Buscador de Institutos y Recursos Humanos

CONCOMITANT TUMOR RESISTANCE: A PUTATIVE MECHANISM TO CONTROL METASTATIC GROWTHToday, I am talking about a not very well- known phenomenon of tumor biology called Concomitant Tumor Resistance or simply Concomitant Resistance (CR).CR is the phenomenon according to which a primary tumor bearing host inhibits or retards the growth of secondary tumor implants.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), and also apart from an even smaller number of mid-sized tumors that induce CR by antiangiogenic mechanisms associated with angiostatin, the most universal manifestation of CR associated with most immunogenic and non-immunogenic tumors of relatively 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.We carried out the purification of these elusive factors starting from the serum of mice bearing tumors that induce CR. The task of purification and identification of these factors was long and difficult because of the low concentration of them and because, the fraction that concentrated all the antitumor activity (at the supposedly last step of purification), seemed to contain tyrosine only. 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 agronomy in which the authors showed that the ability of many grasses to outcompete or displace other neighboring plants was based on the phytotoxic properties of a factor produced by their roots that had an identical MW of tyrosine: but it was not tyrosine!, it was an isomer of tyrosine called meta-tyrosine. This paper encouraged us to continue our work and finally, we could demonstrate that our fraction contained an excess of tyrosine that had been masked the presence of 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.The inhibitory effect of m- and o-tyr was associated with an arrest of tumor cells in G0 and S. Molecular studies revealed that the inhibitory effect induced by both tyrosine isomers was associated with a reduced expression of survivin, Ki-67 and Hes-1 and with a down regulation of the NFKBeta/NOTCH/STAT3 axis and the induction of autophagy probably as a consequence of the production of anomalous proteins generated by the mis-incorporation of m- and o-tyr instead of phenylalanine. More experiments are underway to evaluate the relative importance of the different pathways affected in the whole inhibitory effect.Finally, we evaluated the effect of m-tyr and o-tyr 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 m-tyr 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. O-Tyr also exhibited anti-metastatic effects although they were weaker than that of m-tyr. These results were rather impressive and more, taking into account that the anti-metastatic effect was achieved without any toxic-side effect.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.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? I hope so but really, I don?t know. Only the time, new experiments and ingenuity will answer these questions.