Macrophages Mediate a Switch between Canonical and Non-Canonical Wnt Pathways in Canine Mammary Tumors

Objective

According to the current hypothesis, tumor-associated macrophages (TAMs) are “corrupted” by cancer cells and subsequently facilitate, rather than inhibit, tumor metastasis. Because the molecular mechanisms of cancer cell–TAM interactions are complicated and controversial we aimed to better define this phenomenon.

Methods and Results

Using microRNA microarrays, Real-time qPCR and Western blot we showed that co-culture of canine mammary tumor cells with TAMs or treatment with macrophage-conditioned medium inhibited the canonical Wnt pathway and activated the non-canonical Wnt pathway in tumor cells. We also showed that co-culture of TAMs with tumor cells increased expression of canonical Wnt inhibitors in TAMs. Subsequently, we demonstrated macrophage-induced invasive growth patterns and epithelial–mesenchymal transition of tumor cells. Validation of these results in canine mammary carcinoma tissues (n = 50) and xenograft tumors indicated the activation of non-canonical and canonical Wnt pathways in metastatic tumors and non-metastatic malignancies, respectively. Activation of non-canonical Wnt pathway correlated with number of TAMs.

Conclusions

We demonstrated that TAMs mediate a “switch” between canonical and non-canonical Wnt signaling pathways in canine mammary tumors, leading to increased tumor invasion and metastasis.Interestingly, similar changes in neoplastic cells were observed in the presence of macrophage-conditioned medium or live macrophages. These observations indicate that rather than being “corrupted” by cancer cells, TAMs constitutively secrete canonical Wnt inhibitors that decrease tumor proliferation and development, but as a side effect, they induce the non-canonical Wnt pathway, which leads to tumor metastasis.These data challenge the conventional understanding of TAM–cancer cell interactions.     

Wogonin Induced Calreticulin/Annexin A1 Exposure Dictates the Immunogenicity of Cancer Cells in a PERK/AKT Dependent Manner

In response to ionizing irradiation and certain chemotherapeutic agents, dying tumor cells elicit a potent anticancer immune response. However, the potential effect of wogonin (5,7-dihydroxy-8-methoxyflavone) on cancer immunogenicity has not been studied. Here we demonstrated for the first time that wogonin elicits a potent antitumor immunity effect by inducing the translocation of calreticulin (CRT) and Annexin A1 to cell plasma membrane as well as the release of high-mobility group protein 1 (HMGB1) and ATP. Signal pathways involved in this process were studied. We found that wogonin-induced reactive oxygen species (ROS) production causes an endoplasmic reticulum (ER) stress response, including the phosphorylation of PERK (PKR-like endoplasmic reticulum kinase)/PKR (protein kinase R) and eIF2α (eukaryotic initiation factor 2α), which served as upstream signal for the activation of phosphoinositide 3-kinase (PI3K)/AKT, inducing calreticulin (CRT)/Annexin A1 cell membrane translocation. P22/CHP, a Ca²⁺-binding protein, was associated with CRT and was required for CRT translocation to cell membrane. The releases of HMGB1 and ATP from wogonin treated MFC cells, alone or together with other possible factors, activated dendritic cells and induced cytokine releases. In vivo study confirmed that immunization with wogonin-pretreated tumor cells vaccination significantly inhibited homoplastic grafted gastric tumor growth in mice and a possible inflammatory response was involved. In conclusion, the activation of PI3K pathway elicited by ER stress induced CRT/Annexin A1 translocation (“eat me” signal) and HMGB1 release, mediating wogonin-induced immunity of tumor cell vaccine. This indicated that wogonin is a novel effective candidate of immunotherapy against gastric tumor.     

A Conditional Mouse Mutant in the Tumor Suppressor SdhD Gene Unveils a Link between p21WAF1/Cip1 Induction and Mitochondrial Dysfunction

Mutations in mitochondrial complex II (MCII; succinate dehydrogenase, Sdh) genes cause familiar pheochromocytoma/paraganglioma tumors. Several mechanisms have been proposed to account for Sdh-mutation-induced tumorigenesis, the most accepted of which is based on the constitutive expression of the hypoxia-inducible factor 1α (Hif1α) at normal oxygen tension, a theory referred to as “pseudo-hypoxic drive”. Other molecular processes, such as oxidative stress, apoptosis, or chromatin remodeling have been also proposed to play a causative role. Nevertheless, the actual contribution of each of these mechanisms has not been definitively established. Moreover, the biological factors that determine the tissue-specificity of these tumors have not been identified. In this work, we made use of the inducible SDHD-ESR mouse, a conditional mutant in the SdhD gene, which encodes the small subunit of MCII, and that acts as a tumor suppressor gene in humans. The analysis of the Hif1α pathway in SDHD-ESR tissues and in two newly derived cell lines after complete SdhD loss -a requirement for hereditary paraganglioma type-1 tumor formation in humans- partially recapitulated the “pseudo-hypoxic” response and rendered inconsistent results. Therefore, we performed microarray analysis of adrenal medulla and kidney in order to identify other early gene expression changes elicited by SdhD deletion. Our results revealed that each mutant tissue displayed different variations in their gene expression profiles affecting to different biological processes. However, we found that the Cdkn1a gene was up-regulated in both tissues. This gene encodes the cyclin-dependent kinase inhibitor p21^WAF1/Cip1, a factor implicated in cell cycle, senescence, and cancer. The two SDHD-ESR cell lines also showed accumulation of this protein. This new and unprecedented evidence for a link between SdhD dysfunction and p21^WAF1/Cip1 will open new avenues for the study of the mechanisms that cause tumors in Sdh mutants. Finally, we discuss the actual role of Hif1α in tumorigenesis.     

Strategies of oncogenic microbes to deal with WW domain-containing oxidoreductase

WW domain-containing oxidoreductase (WWOX) is a well-documented tumor suppressor protein that controls growth, survival, and metastasis of malignant cells. To counteract WWOX’s suppressive effects, cancer cells have developed many strategies either to downregulate WWOX expression or to functionally inactivate WWOX. Relatively unknown is, in the context of those cancers associated with certain viruses or bacteria, how the oncogenic pathogens deal with WWOX. Here we review recent studies showing different strategies utilized by three cancer-associated pathogens. Helicobactor pylori reduces WWOX expression through promoter hypermethylation, an epigenetic mechanism also occurring in many other cancer cells. WWOX has a potential to block canonical NF-κB activation and tumorigenesis induced by Tax, an oncoprotein of human T-cell leukemia virus. Tax successfully overcomes the blockage by inhibiting WWOX expression through activation of the non-canonical NF-κB pathway. On the other hand, latent membrane protein 2A of Epstein–Barr virus physically interacts with WWOX and redirects its function to trigger a signaling pathway that upregulates matrix metalloproteinase 9 and cancer cell invasion. These reports may be just “the tip of the iceberg” regarding multiple interactions between WWOX and oncogenic microbes. Further studies in this direction should expand our understanding of infection-driven oncogenesis.     

BaxΔ2 Is a Novel Bax Isoform Unique to Microsatellite Unstable Tumors

The pro-death Bcl-2 family protein and tumor suppressor Bax is frequently mutated in tumors with microsatellite instability (MSI). The mutation often results in a “Bax negative” phenotype and therefore is generally thought to be beneficial to the development of the tumor. Here, we report the identification of a novel Bax isoform, BaxΔ2, which is unique to microsatellite unstable tumors. BaxΔ2 is generated by a unique combination of a microsatellite deletion in Bax exon 3 and alternative splicing of Bax exon 2. Consistently, BaxΔ2 is only detected in MSI cell lines and primary tumors. BaxΔ2 is a potent cell death inducer but does not directly target mitochondria. In addition, BaxΔ2 sensitizes certain MSI tumor cells to a subset of chemotherapeutic agents, such as adriamycin. Thus, our data provide evidence that mutation and alternative splicing of tumor suppressors such as Bax are not always beneficial to tumor development but can be detrimental instead.     

“Velcro” Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein α (SIRPα) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis

CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the “don''t-eat-me” signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined “Velcro” engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that “Velcro” engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy.     

Testing Models of the APC Tumor Suppressor/β-Catenin Interaction Reshapes Our View of the Destruction Complex in Wnt Signaling

The Wnt pathway is a conserved signal transduction pathway that contributes to normal development and adult homeostasis, but is also misregulated in human diseases such as cancer. The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling inactivated in >80% of colorectal cancers. APC participates in a multiprotein “destruction complex” that targets the proto-oncogene β-catenin for ubiquitin-mediated proteolysis; however, the mechanistic role of APC in the destruction complex remains unknown. Several models of APC function have recently been proposed, many of which have emphasized the importance of phosphorylation of high-affinity β-catenin-binding sites [20-amino-acid repeats (20Rs)] on APC. Here we test these models by generating a Drosophila APC2 mutant lacking all β-catenin-binding 20Rs and performing functional studies in human colon cancer cell lines and Drosophila embryos. Our results are inconsistent with current models, as we find that β-catenin binding to the 20Rs of APC is not required for destruction complex activity. In addition, we generate an APC2 mutant lacking all β-catenin-binding sites (including the 15Rs) and find that a direct β-catenin/APC interaction is also not essential for β-catenin destruction, although it increases destruction complex efficiency in certain developmental contexts. Overall, our findings support a model whereby β-catenin-binding sites on APC do not provide a critical mechanistic function per se, but rather dock β-catenin in the destruction complex to increase the efficiency of β-catenin destruction. Furthermore, in Drosophila embryos expressing some APC2 mutant transgenes we observe a separation of β-catenin destruction and Wg/Wnt signaling outputs and suggest that cytoplasmic retention of β-catenin likely accounts for this difference.     

WWOX: A fragile tumor suppressor

WWOX, the WW domain-containing oxidoreductase gene at chromosome region 16q23.3–q24.1, spanning chromosomal fragile site FRA16D, encodes the 46 kDa Wwox protein, a tumor suppressor that is lost or reduced in expression in a wide variety of cancers, including breast, prostate, ovarian, and lung. The function of Wwox as a tumor suppressor implies that it serves a function in the prevention of carcinogenesis. Indeed, in vitro studies show that Wwox protein interacts with many binding partners to regulate cellular apoptosis, proliferation, and/or maturation. It has been reported that newborn Wwox knockout mice exhibit nascent osteosarcomas while Wwox^+/− mice exhibit increased incidence of spontaneous and induced tumors. Furthermore, absence or reduction of Wwox expression in mouse xenograft models results in increased tumorigenesis, which can be rescued by Wwox re-expression, though there is not universal agreement among investigators regarding the role of Wwox loss in these experimental models. Despite this proposed tumor suppressor function, the overlap of the human WWOX locus with FRA16D sensitizes the gene to protein-inactivating deletions caused by replication stress. The high frequency of deletions within the WWOX locus in cancers of various types, without the hallmark protein inactivation-associated mutations of “classical” tumor suppressors, has led to the proposal that WWOX deletions in cancers are passenger events that occur in early cancer progenitor cells due to fragility of the genetic locus, rather than driver events which provide the cancer cell a selective advantage. Recently, a proposed epigenetic cause of chromosomal fragility has suggested a novel mechanism for early fragile site instability and has implications regarding the involvement of tumor suppressor genes at chromosomal fragile sites in cancer. In this review, we provide an overview of the evidence for WWOX as a tumor suppressor gene and put this into the context of fragility associated with the FRA16D locus.     

The two-hit theory hits 50

Few ideas in cancer genetics have been as influential as the “two-hit” theory of tumor suppressors. This idea was introduced in 1971 by Al Knudson in a paper in the Proceedings of the National Academy of Science and forms the basis for our current understanding of the role of mutations in cancer. In this theoretical discussion proposing a genetic basis for retinoblastoma, a childhood cancer of the retina, Knudson posited that these tumors arise from two inactivating mutations, targeting both alleles of a putative tumor suppressor gene. While this work built on earlier proposals that cancers are the result of mutations in more than one gene, it was the first to propose a plausible mechanism by which single genes that are affected by germ-line mutations in heritable cancers could also cause spontaneous, nonheritable tumors when mutated in somatic tissues. Remarkably, Knudson described the existence and properties of a retinoblastoma tumor suppressor gene a full 15 years before the gene was cloned.

Let’s put ourselves, if we may, into the mindset of cancer researchers a half century ago. By the early 1970s, we have come to accept the idea that cancer is a genetic disease resulting from mutations in particular genes. We also know that chromosomal aberrations occur in many cancers, including loss or gain of genetic material, but the identity, and even the existence, of cancer driver genes and how they might operate is entirely unknown to us. Recently reported somatic cell fusion experiments, in which normal human cells have been induced to fuse with malignant rodent cells, suggest that normal cells possess dominant tumor-suppressive properties and that these properties are associated with particular chromosomes (Harris et al., 1969), but the genes that confer such suppression, and how they might do so, are obscure. In addition, there is considerable evidence that that most cancers involve more than a single mutational event (Nordling, 1953; Ashley, 1969). On the other hand, some of our fellow cancer researchers have also shown that acutely transforming retroviruses can rapidly induce cancers in their hosts, suggesting that, at least in avian and mammalian species, single “oncogenes” can transform cells and cause tumors (Huebner and Todaro, 1969). Finally, we know that certain cancer predispositions can be passed down from parent to child, even if the genetic basis for this phenomenon remains uncharacterized.Among the many questions that puzzled our midcentury scientist were: how can these various findings—some of which suggest a multigene cause for cancer and others that suggest a single event—be reconciled? What is the relationship between dominant oncogenes and recessive “anti-oncogenes?” Also, are the genetic mechanisms underlying relatively rare inherited forms of cancer related to the more common forms of this disease, which seem to occur spontaneously?The pediatric cancer retinoblastoma represented a particularly compelling model in which to address this last question. This type of cancer affects retinoblast cells in the developing eye and typically presents in childhood. Curiously, some affected patients were known to develop an early, aggressive, often bilateral form of the disease, and, if they survived, could pass susceptibility to retinoblastoma to their children. Other children developed such tumors later in childhood, never presented with bilateral disease, and did not impart additional risk to their offspring. This cancer drew the attention of Alfred Knudson, at that time a 49-year-old physician studying heritable metabolic disorders. Following a study of patient charts that dated back decades, he suggested in his seminal 1971 National Academy of Science paper that, in both familial and nonfamilial cases of retinoblastoma, the number of mutations required to initiate this tumor was two, that they must occur before retinal cells differentiate, and that the gene or genes affected likely act in a recessive manner (Knudson, 1971). The ideas presented in this paper continue to guide our thinking about cancer genetics to the present day.While the impact and implications of the 1971 paper were profound, the paper itself was profoundly simple. It was four pages long. It had a single author. It neither listed nor required any grant support. It showed no blots (Southern published his eponymous technique in 1975, PCR was more than a decade away, and restriction enzymes were not yet discovered), no sequences (DNA sequencing methods were introduced in 1975), and but one simple figure, showing a straight line and a hyperbolic line on a log scale (Figure 1). In a way, its analytic methods represented a style of science that, while not too uncommon at that time, later fell into relative disfavor as experimental molecular techniques allowed for genes to be isolated, sequenced, mutated, and introduced into cells and animals. By the mid-1970s, gene jockeys were in, and theoretical biologists were out.Open in a separate windowFIGURE 1:The plot from which Knudson proposed the two-hit hypothesis (Knudson, 1971, with the permission of the National Academy of Sciences, USA).Interestingly, Knudson’s statistical approach anticipated much of today’s cancer research literature; that is to say, the work consisted entirely of dataset analysis and mathematical modeling. The numbers being crunched were rather simple by today’s standards: the age of onset of retinoblastoma in pediatric cases, whether these children developed unilateral or bilateral disease, how many tumors were present, and whether these tumors were hereditary or not. The key facts were that the familial cases tended to present at a younger age, were often bilateral, and, in a related point, could arise as multiple independent tumors. He also noted that not everyone who inherited the mutation(s) actually developed tumors; some retinoblastomas skipped a generation. This feature, plus a knowledge of how many cells comprise the retina, suggested that the affected gene(s) was recessive and allowed Knudson to infer a mutational frequency rate per cell division that closely matched previous predictions and was consistent with the observed tumor burden in familial cases.From these data, and unassisted by any form of computer, Knudson used curve-fitting and Poisson statistics to derive an important conclusion: the incidence curve for heritable cases fit a model in which the development of retinoblastoma required not one but two mutational events, or two “hits.” Whether these events were disabling mutations in each of the two alleles of a hypothetical retinoblastoma gene (as indeed proved to be the case) or instead were activating mutations in one allele each of two separate genes, could not be ascertained at that time, though the observation that retinoblastoma cells sometimes lost part of chromosome 13 favored the first interpretation. A decade later, the case for the two-hit theory received crucial experimental support when Cavenee and colleagues applied restriction site polymorphism analysis to retinoblastomas (Cavenee et al., 1983). These studies showed that retinoblastomas commonly display loss of polymorphic restriction sites, consistent with the idea that these tumors involve damage to one allele of an RB gene and subsequent loss of the second copy. The two-hit theory provided an appealing genetic model that could be used to explain both heritable and spontaneous cases of retinoblastoma: the former had one hit in a tumor suppressor gene in the germline and only required one more hit in a somatic retinal cell, whereas the latter required that the first and second mutation to occur in a somatic cell. This model explained why spontaneous cases of retinoblastoma occurred later in life and were never bilateral, as the number of stem cells, the mutation rate, and the amount of time for retinoblasts to terminally differentiate was insufficient for more than one tumor to initiate. The result of these analyses led to a clear prediction regarding the existence and properties of tumor suppressor genes, predictions that have largely withstood the test of time.It would be more than a decade before the first “two-hit” gene, RB, was mapped, isolated, and sequenced (Friend et al., 1986; Lee et al., 1987) and even longer before its biochemical role in regulating cell proliferation was understood in any detail. However, in the meanwhile, dozens of other tumor suppressor genes were characterized, most governed by the rules laid out by Knudson in his 1971 paper.Looking back from a space of 50 years, the 1971 work profoundly reoriented our thinking about cancer genetics in a way that few other single works have done. Importantly, it led to testable predictions that were later—in some cases, much later—proved true. That is not to say, however, that the two-hit theory itself has not evolved. For example, Knudson himself was the one of the first to recognize the possibility that haploinsufficiency (i.e., a one-hit scenario) could alter cellular behavior in ways that contributed to tumorigenesis even in the absence of a second hit. In fact, he spent the last decade of his career studying such effects in cells derived from cancer-prone families (Berger et al., 2011; Peri et al., 2017). Haploinsufficiency was first experimentally verified in mouse models of the Cdkn2a (p27^kip1) tumor suppressor. Mice lacking one allele of Cdkn1b were larger than their littermates, but smaller than those lacking both alleles. Crucially, the heterozygous mice were more prone to tumorigenesis when treated with various mutagens or when bred to oncogene expressing mice (Fero et al., 1998). Many other examples of haploinsufficiency were subsequently described. In this respect, Knudson’s initial two-hit theory was perhaps too parsimonious in its division of tumor suppressor genes into recessive and dominant categories. Most of the proteins encoded by tumor suppressor genes might more aptly be considered as rheostats than as on/off switches: gene dosage matters, and rigid threshold effects are not always seen. To add to the complexity, over the past decades it has become clear that mutations in tumor suppressor genes can also result in dominant-negative or even neomorphic functions, in which the mutant protein carries out functions that are different than those performed the wild-type form (Takiar et al., 2017). To extend our light-switch analogy, the key feature of neomorphic tumor suppressor proteins isn’t whether they act as rheostats or on/off switches, but whether they turn on the stereo instead of the lights. To make matters even more interesting, certain tumor suppressors are suppressors only in particular contexts; that is, depending, as it were, on the time of day and the particulars of the room they’re in, they can act either as on or as off switches. For example, Notch, a central mediator of cell-to-cell signaling, is endowed with both tumor suppressor and tumor-promoting activities that are highly cell and context dependent (Dotto, 2008). Several other tumor suppressor genes display a similar duality (Datta et al., 2020).Another key modification of the two-hit theory is that, despite the simplicity and enduring appeal of the number “two” in its title, the theory applies best to tumor initiation, not necessarily to tumor growth and development. In fact, even in retinoblastoma, it quickly became apparent that two hits are not enough to cause full-blown cancer, and additional “third” hits are required. That is to say, RB1 inactivation is necessary for retinoblastoma tumor initiation but not sufficient for full malignant transformation (Wang et al., 1994; Sellers and Kaelin, 1997).The mapping, cloning, and characterization of additional tumor suppressor genes enabled Kinzler and Vogelstein to propose that these genes fell into at least two general classes: gatekeepers and caretakers (Kinzler and Vogelstein, 1997). The former represented most of the classical tumor suppressor genes, including APC, NF1, NF2, RB1, TSC1/2, VHL, and WT1. These gatekeepers regulate cell division and/or survival through their interaction with elements of signal transduction pathways, and their loss directly initiates growth of the incipient tumor. In contrast, the caretakers, such as ATM, BRCA1 and BRCA2, and FANCA, are involved in maintaining genome integrity through their actions in various aspects of DNA unwinding and repair. In this model, mutational inactivation of such caretaker genes leads to genetic instabilities, increasing the number of mutations of all genes, inactivating gatekeepers and activating oncogenes.In the intervening half century since the initial Knudson paper appeared, the range and variety of mechanisms for tumor suppressor gene inactivation has been more completely defined, incorporating epigenetic as well as genetic events. RB1 itself provides a good example, as silencing of expression of this gene by methylation of CpG islands in its promoter has been noted in sporadic cases (Sakai et al., 1991; Ohtani-Fujita et al., 1993; Greger et al., 1994). In these cases, an epigenetic mechanism of gene inactivation was supported by the lack of mutations in the RB gene sequence. A similar phenomenon has been reported for the VHL gene in spontaneous clear-cell renal carcinoma (Herman et al., 1994) as well as other tumor suppressor genes.Interestingly, at about the same time these ideas were being formulated, Knudson’s colleague at the Institute for Cancer Research (now the Fox Chase Cancer Center), Beatrice Mintz, was busy demonstrating that the cells comprising the tumor cell microenvironment exerted a suppressive effect on cancer cells (Mintz and Illmensee, 1975). In this scenario, loss of a single allele of a tumor suppressor gene in a fibroblast or an immune cell might well impact the growth of an adjacent cancer cell with single or biallelic loss of the same tumor suppressor. A good example of this phenomenon can be seen in one of Knudson’s enduring interests, neurofibromatosis (NF) type 1 syndrome, associated with the tumor suppressor NF1. Here, malignant Schwann cells show biallelic loss of the NF1 gene, just as predicted by proper “Knudsonian” two-hit mechanics, and the surrounding immune cells are hemizygous for NF1 (i.e., have one hit) due to germline mutation. Importantly, these microenvironment cells have to be hemizygous for tumors to develop, as demonstrated by transplantation studies in conditional mouse models (Yang et al., 2008). Such stromal effects have led to the idea that there is a third category of tumor suppressor genes—the landscapers—that predispose to cancer by contributing to a more tumor-conducive stroma (Kinzler and Vogelstein, 1998).Knudson lived to see many of the proteins encoded by tumor suppressor genes functionally linked by virtue of their effects on common signaling pathways that regulate the cell cycle, apoptosis, and protein synthesis. He was particularly interested in determining whether some or all of the tumor suppressor genes that are mutated in phakomatoses—heritable neurocutaneous cancer syndromes that include Cowden’s disease (PTEN), Gorlin’s disease (PTH), juvenile polyposis (SMAD4), Peutz-Jeghers (LKB1), neurofibromatosis type 1 (NF1) and -2 (NF2), tuberous sclerosis (TSC1 and -2), and Von Hippel Lindau (VHL)—could somehow be shown to act in a single pathway, as in fact we now know many of them do. He used to refer to this idea as his “grand unification theory” for tumor suppressor genes.Regarding therapeutics, I think Knudson, whom I knew well as a friend, colleague, and mentor at Fox Chase, would have been disappointed at our relative lack of progress in devising effective treatments for many types of cancers driven by tumor suppressor gene mutations. For example, despite the fact that TP53 is the single most commonly mutated gene in cancer, knowledge of TP53 status has not readily translated into targeted therapies. Part of the reason for this relative lack of progress is obvious: it is much easier to disrupt the action of an oncoprotein than to fix a broken tumor suppressor protein. Direct targeting is not possible if a protein isn’t expressed, and that is the scenario in many tumor cells driven by tumor suppressor gene mutations. Instead, the dominant strategy in this situation has been to target downstream signaling elements, for example, by impeding mitogen-activated protein kinase signaling in NF1-mutant tumors or mTORC1 in TSC-mutant tumors. On the other hand, we have been able to exploit vulnerabilities of cancers with certain caretaker gene mutations, such as PALB2, BRCA1, and BRCA2, as these cells become solely dependent on PARP for DNA repair, rendering them susceptible to small molecule inhibitors of this enzyme. Other “synthetic lethal” strategies have been proposed for various additional tumor suppressor genes (Nijman and Friend, 2013). Finally, given recent advances in gene editing and gene replacement methodologies, it is not unreasonable to think that long before the next 50 years have passed, we will be able to repair damaged tumor suppressor genes in tumor cells and/or replace them with undamaged alleles. If so, we will have come full circle, using a genetic cure for a genetic disease.