After publishing the monograph Evolution by Tumor Neofunctionalization, the author continued developing a theory of the evolutionary role of tumors in subsequent publications, in which the carcino-evo-devo theory assumed its current state. In this part of the article, the author reviews the current state of the carcino-evo-devo theory of the evolutionary role of tumors and its relationship with other biological theories.

The theory of the evolutionary role of hereditary tumors, or carcino-evo-devo theory, may be considered as the next step after A.N. Severtsov’s theory of phylembryogenesis, the theory of evo-devo, and Susumu Ohno’s theory of evolution by gene duplication. The carcino-evo-devo theory pretends to be a unifying biological theory, because it unifies within an integrated consideration three main types of biological development—individual, evolutionary, and neoplastic development. The carcino-evo-devo theory explains a series of unexplained biological phenomena. In the first place, it explains the mechanisms of progressive evolution and biological complexity increase using the concept of relatively unstable transitory forms and autonomous uncontrolled processes. The theory of the evolutionary role of hereditary tumors has formulated several nontrivial predictions in various fields of biology, which have been confirmed in the lab of the author and in other laboratories. The consequences of the carcino-evo-devo theory have implications in medicine and biotechnology. The first part of this article describes the basic principles from which the main hypothesis followed, the progressive developments of the concept, and the first experimental data in support of nontrivial predictions obtained in the laboratory of the author in the period before 2014, when our monograph Evolution by Tumor Neofunctionalization (Kozlov, 2014) was published.

New chapters of carcino-evo-devo theory are devoted to tumor participation in biological computational processes, the principle of increase in biological complexity, and the formula of complexity growth in progressive evolution based on carcino-evo-devo diagrams. The conclusion is made that new chapters establish the basis of a more general theory, that is, the theory of the complexity increase, a special part of which is given in the carcino-evo-devo theory.

In this part of the article, the formation of the carcino-evo-devo theory of the evolutionary role of hereditary tumors in the monograph Evolution by Tumor Neofunctionalization is discussed. In 2014 our monograph Evolution by Tumor Neofunctionalization was published by Elsevier/Academic Press. The monograph generalized the results of 30 years of work. It contained twelve chapters and over 1000 references, an order of magnitude more than in previous theoretical papers. New chapters appeared compared to previous papers. The structure of the book corresponds to the current structure of the carcino-evo-devo theory. Thus, in the monograph the concept of the possible evolutionary role of hereditary tumors acquired the structure of a theory. The monograph was later published in Russian and Chinese.

Orthopedia homeobox ( OTP ) gene encodes a homeodomain-containing transcription factor involved in brain development. OTP is mapped to human chromosome 5q14.1. Earlier we described transcription in the second intron of this gene in wide variety of tumors, but among normal tissues only in testis. In GeneBank these transcripts are presented by several 300-400 nucleotides long AI267901-like ESTs.We assumed that AI267901-like ESTs belong to longer transcript(s). We used the Rapid Amplification of cDNA Ends (RACE) approach and other methods to find the full-length transcript. The found transcript was 2436 nucleotides long polyadenylated sequence in antisense to OTP gene. The corresponding gene consisted of two exons separated by an intron of 2961 bp long. The first exon was found to be 91 bp long and located in the third exon of OTP gene. The second exon was 2345bp long and located in the second intron of OTP gene.The search of possible open reading frames (ORFs) showed the lack of significant ORFs. We have shown the expression of new gene in many human tumors and only in one sampled normal testis. The data suggest that we discovered a new antisense cancer-testis sequence OTP - AS1 ( OTP - antisense RNA 1), which belongs to long noncoding RNAs (lncRNAs). According to our findings we assume that OTP-AS1 and OTP genes may be the CT-coding gene/CT-ncRNA pair involved in regulatory interactions.Author summary Previously, long non-coding RNAs (lncRNAs) were considered as genetic “noise”. However, it was later shown that only 2% of genomic transcripts have a protein-coding ability. Non-coding RNA is divided into short non-coding RNAs (20-200 nucleotides) and long noncoding RNAs (200-100,000 nucleotides). Genes encoding lncRNA often overlap or are adjacent to protein-coding genes, and localization of this kind is beneficial in order to regulate the transcription of neighboring genes. Studies have shown that of lncRNAs play many roles in the regulation of gene expression. New evidence indicates that dysfunctions of lncRNAs are associated with human diseases and cancer.In our study we found a new cancer-testis long noncoding RNA ( OTP-AS1 ), which is an antisense of protein-coding cancer-testis gene ( OTP ). Thus, OTP-AS1 and OTP genes may be the CT-coding gene/CT-ncRNA pair involved in regulatory interactions. This is supported by the similar profile of their expression. OTP-AS1 may be of interest as a potential diagnostic marker of cancer or a potential target for cancer therapy.Part of OTP-AS1 gene (5’-end of the second exon) is evolutionary younger than the rest of gene sequence and is less conservative. This links OTP-AS1 gene with so-called TSEEN (tumor-specifically expressed, evolutionary novel) genes described by the authors in previous papers.

In 1958, Francis Crick 1 formulated the sequence hypothesis and central dogma of molecular biology. 1 The formulation of the sequence hypothesis was, as follows: "The specificity of a piece of nucleic acid is expressed solely by the sequence of its bases, and this sequence is a (simple) code for the amino acid sequence of a particular protein."The formulation for the central dogma was, as follows: "This states that once 'information' has passed into protein, it cannot get out again.In more detail, the transfer of information from nucleic acid to nucleic acid, or from nucleic acid to protein may be possible, but the transfer from protein to protein, or from protein to nucleic acid is impossible.Information means here

A theory of the evolutionary role of hereditary tumors, or the carcino-evo-devo theory, is being developed. The main hypothesis of the theory, the hypothesis of evolution by tumor neofunctionalization, posits that hereditary tumors provided additional cell masses during the evolution of multicellular organisms for the expression of evolutionarily novel genes. The carcino-evo-devo theory has formulated several nontrivial predictions that have been confirmed in the laboratory of the author. It also suggests several nontrivial explanations of biological phenomena previously unexplained by the existing theories or incompletely understood. By considering three major types of biological development-individual, evolutionary, and neoplastic development-within one theoretical framework, the carcino-evo-devo theory has the potential to become a unifying biological theory.

The idea of computational processes, which take place in nature, for example, DNA computation, is discussed in the literature. DNA computation that is going on in the immunoglobulin locus of vertebrates shows how the computations in the biological possibility space could operate during evolution. We suggest that the origin of evolutionarily novel genes and genome evolution constitute the original intrinsic computation of the information about new structures in the space of unrealized biological possibilities. Due to DNA computation, the information about future structures is generated and stored in DNA as genetic information. In evolving ontogenies, search algorithms are necessary, which can search for information about evolutionary innovations and morphological novelties. We believe that such algorithms include stochastic gene expression, gene competition, and compatibility search at different levels of structural organization. We formulate the increase in complexity principle in terms of biological computation and hypothesize the possibility of in silico computing of future functions of evolutionarily novel genes.

Adipose expansion during obesity has remarkable similarities with the growth of solid tumors. These similarities are the following: the capability to unlimited expansion; reversible plasticity; induction of angiogenesis; chronic inflammation; remodeling and disfunction; systemic influence on the organism; hormone production; production of miRNAs that influence other tissues; immunosuppression; DNA damage and resistance to apoptosis; infiltration in other organs and tissues. The existing similarities between adipose and tumors suggest the possible evolutionary origin of mammalian adipose from some ancestral benign mesenchymal hereditary tumors. Transgenic inducible zebrafish tumor model was used to verify the above hypothesis. We described several genes, which originated in fish and were expressed in fish tumors. Their human orthologs LEP, SPRY1, PPARG, ID2, and CIDEA acquired functions connected with human adipose. They are also involved in tumor development in humans. If the hypothesis of the evolutionary origin of mammalian adipose from the ancestral benign hereditary tumors is correct, it may open new opportunities to resolve the oncological problem and the problem of the obesity epidemic. New interventions targeting LEP, SPRY1, PPARG, ID2, and CIDEA gene network, in addition to what already is going on, can be designed for treatment and prevention of both obesity and tumors. Citation Format: Andrei P. Kozlov. Mammalian adipose has many tumor features, which suggests the possible way of its evolutionary origin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6077.

According to the hypothesis of the evolutionary role of tumors, evolutionarily new genes should be specifically expressed in tumors [A.P. Kozlov, 2014]. Indeed, TSEEN (tumor specific evolutionarily novel) genes were described by us [Kozlov, 2016]. 1015 genes were identified by the Protein Historian program as evolutionary novel for humans, that is, originated in humans, and 1392 as evolutionarily young, that is, originated in primates. As a result of analyzing the GTEx database data, we have shown that out of 1015 evolutionarily new human genes and 1392 evolutionarily young human genes, 309 genes and 592 genes are not expressed in any of 33 normal tissues, respectively. The expression level of 101 evolutionarily new human genes originated in humans and 233 evolutionarily young human genes originated in primates is significant in most tumors, according to transcriptome data from the TCGA database. Samples of 101 evolutionarily novel human genes and 233 evolutionarily young human genes were selected, which are present only in the genomes of humans and primates, not expressed in normal tissues, and expressed in one or more tumors in 10-100% of patients.For Theria and Eutheria, the similar distribution was as follows: among 365 and 896 genes novel for these taxons, 71 and 124 genes, respectively, are not expressed in normal human tissues, respectively, but at the same time have tumor-specific expression pattern (inclusion criteria of expression - fpkm >2 in 10-100% of patients).There are also 898 genes, originated in mammalia and not expressed in normal human tissues. 119 of them are expressed in at least 1 or more tumors in 10-100% of patients.The TSEEN genes database allows [us] to select genes according to their evolutionary age and expression pattern in normal and tumor tissues. We can retrieve gene(s), which are expressed tumor-specifically and are evolutionarily young or novel.As far as TSEEN genes have many immunogenic epitopes they could be promising targets for development of universal prophylactic and therapeutic antitumor vaccines, as well as universal markers for cancer laboratory diagnostics.Created TSEEN genes database is available at: https://tseendb.org/#/ Citation Format: Ekaterina Matyunina, Andrei Makashov, Andrei P. Kozlov. TSEEN genes database [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3350.

BACKGROUND:In previous publications, the author developed the theory of carcino-evo-devo, which predicts that evolutionarily novel organs should recapitulate some features of tumors in their development.MAIN TEXT:Mammalian adipose is currently recognized as a multi-depot metabolic and endocrine organ consisting of several adipose tissues. Although lipid-storing cells and proteins are ancient, the adipose organ as a whole is evolutionarily novel to mammals. The adipose expansion has remarkable similarities with the growth of solid tumors. These similarities are the following: (1) The capability to unlimited expansion; (2) Reversible plasticity; (3) Induction of angiogenesis; (4) Chronic inflammation; (5) Remodeling and disfunction; (6) Systemic influence on the organism; (7) Hormone production; (8) Production of miRNAs that influence other tissues; (9) Immunosuppression; (10) DNA damage and resistance to apoptosis; (11) Destructive infiltration in other organs and tissues. These similarities include the majority of "hallmarks of cancer". In addition, lipomas are the most frequent soft tissue tumors, and similar drugs may be used for the treatment of obesity and cancer by preventing infiltration. This raises the possibility that obesity, at least in part, may represent an oncological problem. The existing similarities between adipose and tumors suggest the possible evolutionary origin of mammalian adipose from some ancestral benign mesenchymal hereditary tumors. Indeed, using a transgenic inducible zebrafish tumor model, we described many genes, which originated in fish and were expressed in fish tumors. Their human orthologs LEP, NOTCH1, SPRY1, PPARG, ID2, and CIDEA acquired functions connected with the adipose organ. They are also involved in tumor development in humans.CONCLUSION:If the hypothesis of the evolutionary origin of the adipose organ from the ancestral hereditary tumor is correct, it may open new opportunities to resolve the oncological problem and the problem of the obesity epidemic. New interventions targeting LEP, NOTCH1, SPRY1, PPARG, ID2, and CIDEA gene network, in addition to what already is going on, can be designed for treatment and prevention of both obesity and tumors.

To explain the sources of additional cell masses in the evolution of multicellular organisms, the theory of carcino-evo-devo, or evolution by tumor neofunctionalization, has been developed. The important demand for a new theory in experimental science is the capability to formulate non-trivial predictions which can be experimentally confirmed. Several non-trivial predictions were formulated using carcino-evo-devo theory, four of which are discussed in the present paper: (1) The number of cellular oncogenes should correspond to the number of cell types in the organism. The evolution of oncogenes, tumor suppressor and differentiation gene classes should proceed concurrently. (2) Evolutionarily new and evolving genes should be specifically expressed in tumors (TSEEN genes). (3) Human orthologs of fish TSEEN genes should acquire progressive functions connected with new cell types, tissues and organs. (4) Selection of tumors for new functions in the organism is possible. Evolutionarily novel organs should recapitulate tumor features in their development. As shown in this paper, these predictions have been confirmed by the laboratory of the author. Thus, we have shown that carcino-evo-devo theory has predictive power, fulfilling a fundamental requirement for a new theory.

Since 1981, when AIDS was identified as a disease about 60 million of HIV infected people have been registered and 30 million have died according to WHO. The development of vaccines against HIV/AIDS is an important step in solving this problem. In the absence of an adequate animal model for HIV, vaccine development requires an understanding of immunological correlates of HIV protection. In this study we investigated the immunological correlates of a candidate HIV-1 DNA vaccine. We analyzed the specific immune responses in phase I clinical trial participants and in HIV exposed seronegative individuals. In both groups, an increase in TNFα cytokine by CD4+ T cells in response to specific peptide stimulation was found. This could be an indicator of the possible vaccine efficacy.

The original publication of this article [1] contained 4 errors in column 1 of Table 4. In this correction article the errors and updated table are published.

The hypothesis of evolution by tumor neofunctionalization (the "main hypothesis") describes the possible role of hereditary tumors in evolution. The present article examines the relationship of the main hypothesis to other biological theories. As shown in this paper, the main hypothesis does not contradict to the existing biological theories, but fills the lacunas between them and explains some unexplained (or not completely understood) questions. Common features of embryonic development and tumorigenesis are described by several recognized theories. Similarities between normal development and tumorigenesis suggest that tumors could participate in the evolution of ontogenesis and in the origin of new cell types, tissues and organs. A wide spectrum of non-trivial explanations and non-trivial predictions in different fields of biology, suggested by the main hypothesis, is an indication of its fundamental nature and the potential to become a new biological theory, a theory of the role of tumors in evolution of development, or carcino-evo-devo.

We developed a candidate DNA vaccine called "DNA-4"consisting of 4 plasmid DNAs encoding Nef, Gag, Pol(rt), and gp140 HIV-1 proteins. The vaccine was found to be safe and immunogenic in a phase I clinical trial. Here we present the results of a phase II clinical trial of "DNA-4". This was a multicenter, double-blind, placebo-controlled clinical trial of safety, and dose selection of "DNA-4" in HIV-1 infected people receiving antiretroviral therapy (ART). Fifty-four patients were randomized into 3 groups (17 patients-group DNA-4 0.25 mg, 17 patients-group DNA-4 0.5 mg, 20 patients-the placebo group). All patients were immunized 4 times on days 0, 7, 11, and 15 followed by a 24-week follow-up period. "DNA-4" was found to be safe and well-tolerated at doses of 0.25 mg and 0.5 mg. We found that the amplitudes of the spontaneous viral load increases in three patients immunized with the candidate DNA vaccine were much higher than that in placebo group-2800, 180,000 and 709 copies/mL, suggesting a possible influence of therapeutic DNA vaccination on viral reservoirs in some patients on ART. We hypothesize that this influence was associated with the reactivation of proviral genomes.

Understanding features of the HIV-1 transmission process has the potential to inform biological interventions for prevention. We have examined the transmitted virus in a cohort of people who inject drugs and who are at risk of HIV-1 infection through blood contamination when injecting in a group. This study focused on seven newly infected participants in St. Petersburg, Russia, who were in acute or early infection. We used end-point dilution polymerase chain reaction to amplify single viral genomes to assess the complexity of the transmitted virus. We also used deep sequencing to further assess the complexity of the virus. We interpret the results as indicating that a single viral variant was transmitted in each case, consistent with a model where the exposure to virus during transmission was limited. We also looked at phenotypic properties of the viral Env protein in isolates from acute and chronic infection. Although differences were noted, there was no consistent pattern that distinguished the transmitted variants. Similarly, despite the reduced genetic heterogeneity of the more recent subtype A HIV-1 epidemic in St. Petersburg, we did not see reduced variance in the neutralization properties compared to isolates from the more mature subtype C HIV-1 epidemic. Finally, in looking at members of injecting groups related to the acute HIV-1 infection/early subjects, we found examples of sequence linkage consistent with ongoing and rapid spread of HIV-1 in these groups. These studies emphasize the dynamic nature of this epidemic and reinforce the idea that improved prevention methods are needed.