Species-specific mechanisms of tumor suppression are fundamental drivers of vertebrate speciation: critical implications for the ‘war on cancer’

We recently reported our detection of an anthropoid primate-specific, ‘kill switch’ tumor suppression system that reached its greatest expression in humans, but that is fully functional only during the first twenty-five years of life, corresponding to the primitive human lifespan that has characterized the majority of our species' existence. This tumor suppression system is based upon the kill switch being triggered in cells in which p53 has been inactivated; such kill switch consisting of a rapid, catastrophic increase in ROS caused by the induction of irreversible uncompetitive inhibition of glucose-6- phosphate dehydrogenase (G6PD), which requires high concentrations of both inhibitor (DHEA) and G6P substrate. While high concentrations of intracellular DHEA are readily available in primates from the importation and subsequent de-sulfation of circulating DHEAS into p53-affected cells, both an anthropoid primate-specific sequence motif (GAAT) in the glucose-6-phosphatase (G6PC) promoter, and primate-specific inactivation of de novo synthesis of vitamin C by deletion of gulonolactone oxidase (GLO) were required to enable accumulation of G6P to levels sufficient to enable irreversible uncompetitive inhibition of G6PD. Malignant transformation acts as a counterforce opposing vertebrate speciation, particularly increases in body size and lifespan that enable optimized exploitation of particular niches. Unique mechanisms of tumor suppression that evolved to enable niche exploitation distinguish vertebrate species, and prevent one vertebrate species from serving as a valid model system for another. This here-to-fore unrecognized element of speciation undermines decades of cancer research data, using murine species, which presumed universal mechanisms of tumor suppression, independent of species. Despite this setback, the potential for pharmacological reconstitution of the kill switch tumor suppression system that distinguishes our species suggests that ‘normalization’ of human cancer risk, from its current 40% to the 4% of virtually all other large, long-lived species, represents a realistic near-term goal.

2 Schmitz et al. 2015). They have evolved very large eyes well suited for nocturnal hunting (albeit absent the reflective tapetum lucidem of nocturnal Strepsirrhine primates), and very long limbs designed for leaping at their prey. Basal Haplorrhine primates are thought to have resembled tarsiers, being similarly small, arboreal insectivores. Lending support to this idea, a complete skeleton of Archicebus achilles, a basal tarsier, was discovered in a bed of 55-million-year-old shale in China (Ni et al., 2013). Archicebus was even more diminutive than modern day tarsiers, just 7 cm (2.8 inches) long and weighing an estimated 20-30 grams (1 ounce). Detailed examination of this fossil revealed that the shape of the heel of Archicebus more resembled that of an anthropoid primate than a tarsier, suggesting that ancestral tarsiers and basal Haplorrhines were very similar.
The purpose of speciation is to expand the exploitation of environmental resources beyond those representing the niche of basal species (Supplementary Section 1). A frequent speciation strategy to expand beyond the environmental niche of basal species is to increase body size, which under natural circumstances generally leads to a parallel increase in lifespan as a matter of economy. Increases in body size can transform predator-prey relationships, enhance capacity for locomotion, enable access to new environmental resources that were unavailable to basal species, etc. This certainly appears to have been the case with Haplorrhine primates, which expanded in body mass from the few tens of grams of basal Haplorrhines and tarsiers, to the 220 kg (484 pounds) of the gorilla. Lifespan, too, underwent dramatic increases during primate evolution, with Strepsirrhine primates having lifespans of 12-15 years, compared to Haplorrhine primate lifespans of approximately 25 years. Any increase in body size and lifespan beyond that of basal species requires species-specific mechanisms to suppress the amplified risk of malignant transformation that would otherwise be a consequence of such increases (Supplementary Section 1). As we shall now discuss, primate evolution is characterized by a very clear step-wise upgrading of species-specific tumor suppression mechanisms to enable the eight thousand-fold increase in body size from basal Haplorrhine primates to the largest members of this lineage.

S2.2 Primate-specific components of the kill switch tumor suppression system
We have previously described how the kill switch mechanism-irreversible uncompetitive inhibition of Glucose-6-phosphate Dehydrogenase (G6PD) leading to a catastrophic increase in intercellular ROS-requires high levels of circulating DHEAS, which only occur in primates (Figure 3 in Nyce, 2018). For uncompetitive inhibition of G6PD to reach irreversibility, accumulation of G6P substrate to high intracellular concentrations must also occur. This has been accomplished by selection for an anthropoid primate-specific sequence motif in the Glucose-6-phosphatase (G6PC) promoter (GAAT; Figure 2 in Nyce, 2018) that disables induction of G6PC activity, preventing catabolism of G6P to glucose and inorganic phosphate (P i ). Unlike anthropoid primates, tarsiers and Strepsirrhine primates retained the canonical GCAG G6PC promoter sequence motif that is characteristic of the vast majority of animal species. They therefore did not evolve the kill switch tumor suppression system, at least not to the extent that anthropoid primates did. Either their small size precluded necessity for the kill switch, or, more likely, their small size enabled a much less 4 optimized form of the kill switch to be sufficient, i.e., they required the evolution of only a rudimentary version of the kill switch.

S2.3 The inability to synthesize vitamin C is a further distinguishing feature of anthropoid primates
We now wish to discuss an additional lineage-specific trait of Haplorrhine primates that appears to be critically associated with the evolution of the adrenal androgen-mediated kill switch tumor suppression system; namely, loss of the ability to synthesize vitamin C (ascorbate). Most species, including Strepsirrhine primates, are capable of de novo synthesis of ascorbate (Smirnoff, 2018). However, loss of the capacity for de novo synthesis of vitamin C is a distinguishing feature of Haplorrhine primates, such that anthropoid primates (ibid.) and tarsiers (Pollock and Mullin, 1987), both have lost gulonolactone oxidase (GLO) activity, the final enzyme in ascorbate synthesis. Why?
The de novo synthesis of ascorbate, particularly in the presence of intracellular ROS, represents a significant sink for G6P in animals with GLO activity (Figure 1). Just as the catabolism of G6P to glucose and P i via G6PC activity had to be disabled by conversion from the canonical GCAG to the GAAT sequence motif in the G6PC promoter of anthropoid primates to enable accumulation of G6P, so too, other sources of G6P loss had to be rendered inactive. Loss of GLO activity in Haplorrhine primates removed a pathway that would have consumed large amounts of G6P and would thereby have severely inhibited kill switch function. Thus, the accumulation of G6P required for the irreversible uncompetitive inhibition kinetics of DHEA toward G6PD, essential for the full expression of the anthropoid primate-specific kill switch tumor 5 suppression system, appears to require both the GAAT sequence motif which disabled catabolism of G6P by G6PC, and the loss of GLO activity. Anthropoid primates must therefore obtain ascorbate in their diets, which appears to have been an equitable tradeoff to enjoy optimization of the kill switch tumor suppression mechanism. Figure 1. Synthesis of ascorbic acid acts as a sink for G6P in the presence of intracellular ROS. GLO activity is disabled in Haplorrhine primates, enabling accumulation of G6P in the presence of intracellular ROS. Both the GAAT sequence motif that disables DHEA-mediated induction of G6PC activity, and inactivation of GLO were required for optimization of the anthropoid primate-specific kill switch tumor suppression system. SS, Steroid Sulfatase; DHEAS (black type), extracellular DHEAS; DHEAS (red type), intracellular DHEAS.

S2.4 Step-wise evolution of the kill switch tumor suppression system enabled increased body size and lifespan of anthropoid primates
It is possible that GLO became superfluous in basal Haplorrhine primates and tarsiers because, like modern tarsiers, they were insectivorous and ingested sufficient vitamin C in their insect diet, insects representing a good source of vitamin C and most other nutrients (Finke, 2015). But as noted above, other possibilities also exist. We maintain cancer risk at the requisite 4% (Supplemental Section 1) in the PAHcontaminated landscape they inhabited. The anthropoid primates, however, had to increase their body size substantially to exploit new niches within the PAHcontaminated PETM. In order to increase their body size, improvements to the elementary form of the kill switch in basal primates and tarsiers were necessary. As noted above, one such major improvement was the conversion from the canonical GCAG sequence motif to GAAT, which disabled the catabolism of G6P by G6PC, enabling higher levels of intracellular G6P accumulation and thereby enhancing kill switch function. Another major improvement was a dramatic increase in the concentration of circulating DHEAS, to levels as much as forty-fold higher than in Strepsirrhine primates (Figure 3 in Nyce, 2018). These dramatic increases in circulating DHEAS trace the evolution toward greater body size and lifespan in Haplorrhine compared to Strepsirrhine primates. (The DHEAS levels in tarsiers remain unknown, as 7 they are a heavily protected endangered species. However, based upon their canonical GCAG G6PC promoter motif, we suspect that they will be in the range of Strepsirrhine, not Haplorrhine primates.) In any case, anthropoid primates diverged from tarsiers, increasing body size in ways that tarsiers (and Strepsirrhine primates) never did. The anthropoid primate-specific kill switch tumor suppressor system, characterized by extraordinarily high circulating DHEAS, the GAAT G6PC promoter sequence motif, and the inactivation of GLO, enabled this dramatic increase in body size and lifespan.
The picture that emerges is one in which circulating DHEAS, which opposes cortisol and thereby moderates the fight-or-flight response, may have originally appeared in primates to enable the formation of social groups much larger than would otherwise have been possible. Existing as a member of a large community would have significant survival value, with respect both to detecting and defending against predators, and providing more variability in the selection of mates. Subsequently, the Haplorrhine branch of the primate tree emerged with the deletion of GLO activity, activating a rudimentary form of the kill switch that may have enabled exploitation of PAHcontaminated resources within the fire-ravaged landscapes of the PETM. Then, in order to expand niche exploitation by increasing body size and lifespan, anthropoid primates emerged with their GAAT sequence motif in the G6PC promoter, improving kill switch function by optimizing G6P accumulation above that enabled by GLO deletion. Finally, selection for increased levels of circulating DHEAS occurred, culminating in humans, the only anthropoid primate to harness fire ( Figure 2). The harnessing of fire may not have been possible without the kill switch tumor suppression mechanism already being present at an early time in hominin evolution. The species-specific PAH exposure that 8 would have occurred by consuming heat processed food, and by smoke inhalation that was unavoidable in the unventilated habitats of early Homo species, would likely have selected for higher and higher levels of circulating DHEAS, accounting for the fact that Homo sapiens have by far the highest levels of any anthropoid species.

S2.5 Conclusion
It cannot be coincidental that three major, primate-specific evolutionary eventscirculating DHEAS, inactivation of GLO resulting in inability to synthesize vitamin C, and the GAAT sequence motif of G6PC-have a common intersection in the enabling of irreversible uncompetitive inhibition of G6PD which requires high concentrations of substrate and inhibitor; DHEAS by acting as a safe, circulating form of the uncompetitive inhibitor, DHEA; and GLO inactivation and the GAAT G6PC sequence motif combining to enable accumulation of G6P substrate. Determining if reconstitution of circulating DHEAS will restore the kill switch tumor suppression system, and if such restoration will normalize human lifetime cancer risk to the 4% of other long-lived vertebrate species (Abegglen et al., 2015), is an experiment that can only be performed in humans. But unlike DHEA, DHEAS should be a safe pharmacological test substance.
In view of this fact, and the dramatic increases in worldwide cancer cases predicted for the near future, we recommend that consideration be given to including the cost of such clinical trials in the National Cancer Institute's 2020 budget, which has just been opened for consideration.