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. Author manuscript; available in PMC: 2011 Jun 1.
Published in final edited form as: Cell Cycle. 2010 Jun 1;9(11):2118–2123. doi: 10.4161/cc.9.11.11726

Racing to block tumorigenesis after pRb loss: an innocuous point mutation wins with synthetic lethality

Frederick Bauzon 1, Liang Zhu 1
PMCID: PMC3044209  NIHMSID: NIHMS248937  PMID: 20505340

Abstract

A major goal of tumor suppressor research is to neutralize the tumorigenic effects of their loss. Since loss of pRb does not induce tumorigenesis in many types of cells, natural mechanisms may neutralize the tumorigenic effects of pRb loss in these cells. For susceptible cells, neutralizing the tumorigenic effects of pRb loss could logically be achieved by correcting the deregulated activities of pRb targets to render pRb-deficient cells less abnormal. This line of research has unexpectedly revealed that knocking out the pRb target Skp2 did not render Rb1 deficient cells less abnormal but, rather, induced apoptosis in them, thereby completely blocking tumorigenesis in Rb1+/- mice and after targeted deletion of Rb1 in pituitary intermediate lobe (IL). Skp2 is a substrate-recruiting component of the SCFSkp2 E3 biquitin ligase; one of its substrates is Thr187-phosphorylated p27Kip1. A p27T187A knockin (KI) mutation phenocopied Skp2 knockout (KO) in inducing apoptosis following Rb1 loss. Thus, Skp2 KO or p27T187A KI are synthetic lethal with pRb inactivation. Since homozygous p27T187A KI mutations show no adverse effects in mice, inhibiting p27T187 phosphorylation or p27T187p ubiquitination could be a highly therapeutic and minimally toxic intervention strategy for pRb deficiency-induced tumorigenesis.

Keywords: pRb, E2F, Skp2, p27, synthetic lethal

Introduction

The retinoblastoma protein, pRb, is a prototype tumor suppressor 1. Children who inherited an inactivated RB1 allele develop retinoblastoma with full-penetrance and tumor cells invariably lose the remaining functional allele of RB1. Alfred Knudson in 1971 proposed a two-hit hypothesis for pRb deficiency-induced tumorigenesis 2. The first-hit is the inherited inactivating mutation in one allele of the RB1 gene; the second hit is the spontaneous somatic loss of the other wild type allele of RB1. Since spontaneous somatic loss of a gene is a low-frequency event, the full-penetrance development of retinoblastomas in these children suggests an indispensable tumor suppressor role of pRb in the retina, i.e. once a cell in the retina becomes RB1-/-, it develops into a retinoblastoma with certainty. Furthermore, retinoblastomas in these children are often multi-focal and bilateral, which provide further evidence for the essential tumor suppression role of the RB1 gene.

Engineered Rb1+/- mice spontaneously develop tumors with full-penetrance, and tumor cells are invariably Rb1-/- 3. These mice provide experimental evidence for the two-hit hypothesis for pRb loss-induced tumorigenesis and faithful animal models for studying pRb and treating pRb-deficient tumors. Here, we review how far these studies have advanced.

Neutralize the tumorigenic effects of pRb loss by naturally built-in mechanisms

RB1 heterozygous patients and Rb1+/- mice develop only a small spectrum of tumors following two-hit loss of pRb. Within this spectrum of tumors, only retinoblastomas in people and pituitary IL tumors in mice are fully penetrant. Assuming that spontaneous gene loss is occurring with similar frequencies in various types of cells, the above findings indicate that pRb loss does not induce tumorigenesis in many types of cells.

The cell type specific susceptibility to pRb loss-induced tumorigenesis is definitively demonstrated when both alleles of Rb1 were artificially deleted in specific types of cells in mice with targeted expression of recombinases. As expected, artificial Rb1 deletion greatly accelerated tumorigenesis in tissues that are susceptible to tumorigenesis in Rb1+/- mice such as the pituitary IL 4. In tissues that do not spontaneously develop tumorigenesis in Rb1+/- mice, artificial deletion of Rb1 in them still failed to induce tumorigenesis. The list of this type of resistant cells is growing; some examples are the lung 5, bone 6, 7, and the prostate 8.

Another technique to artificially generate Rb1-/- cells in various tissues is to generate Rb1-/- ES cells and inject them into Rb1+/+ blastocysts to create chimeic mice of Rb1+/+ and Rb1-/- cells. Although Rb1-/- cells were found in all organs and tissues, these chimeric mice still predominantly develop pituitary IL tumors. The kinetics of IL tumor development is increased since spontaneous loss of the wild type allele of Rb1 is already artificially accomplished 9, 10.

The restriction of tumorigenesis to a few susceptible tissues indicate that certain naturally built-in mechanisms must exist to safe-guard against Rb1-deficiency induced tumorigenesis in resistant cells, as discussed below.

Functional compensation by pRb family members

Perhaps the simplest mechanism to prevent tumorigenesis following Rb1 loss is compensation by other members of the pRb family p107 and p130. These two family members share structure and functional similarities with pRb to very high degrees. In cell proliferation assays, ectopic expression of any of these three proteins could induce G1 cell cycle arrest to similar degrees. Of course, the most important difference among these three proteins is that mutations of pRb are found in human tumors and Rb1+/- mice develop spontaneous tumorigenesis with full-penetrance, while mutations in p107 and p130 have not been found in human tumors and p107 KO and p130 KO mice do not spontaneously develop tumors.

While these facts indicate that p107 and p130 are not tumor suppressors, mouse genetics studies have demonstrated that p107 and p130 could play tumor suppressor roles when pRb is lost. The best-studied tissue in this respect is the retina. Rb1+/- mice do not develop retinoblastomas, and recombinase-mediated artificial deletions of Rb1 are also unable to induce retinblastomas. However, combined deletion of Rb1 and p107 or Rb1 and p130 induced retinoblastoma 11-14. These findings clearly demonstrate that one reason for the lack of retinoblastoma in Rb1+/- mice is that p107 and p130 can compensate pRb loss in this tissue, and suggest that the existence of three pRb family members likely provides a simple mechanism to neutralize the tumorigenic effects of pRb loss in certain types of cells and tissues.

Activation of safe-guard mechanisms

A relatively recent advance in cancer research is that oncogenic signals also activate proliferation arrest, apoptosis, senescence, or various combinations of these to neutralize their tumorigenic effects. Thus, tumorigenesis will only be successful when these safe-guard mechanisms are inactivated 15.

The best-understood oncogenic signal generated by pRb loss is the activation of E2F transcription factors, which inappropriately activate expression of genes whose protein products promote proliferation. This oncogenic mechanism is used in parallel to activate a number of safe-guard mechanisms, as discussed below.

“The pRb-E2F1-apoptotic genes” safe-guard mechanism

While E2F transcription factors can promote cell proliferation, they, in particular the E2F1 member, can also induce apoptosis by activating expression of apoptotic genes 16, 17. Since the same proteins stimulate cell proliferation and cell death, this safe-guard mechanism seems to provide the “safest” defense against pRb loss-induced tumorigenesis.

“The pRb-E2F-ARF-MDM2-p53” safe-guard mechanism

E2F can indirectly induce apoptosis through activating expression of ARF, whose promoter can be activated by E2F 18. ARF binds and inhibits MDM2 to activate p53, which has both cell cycle arrest and apoptotic functions. The importance of “The pRb-E2F-ARF-MDM2-p53” safe-guard mechanism is supported by findings that deletion of p53 is required for tumorigenesis following pRb loss in a number of tissues such as the lung 5, bone 6, 7, and the prostate 8 in mice. The counter mechanisms to cripple this safe-guard mechanism are inactivation of ARF, overexpression of MDM2, or inactivation of p53, all of which are frequent in various tumors.

“The pRb-E2F-ARF-MDM2-p53” safe-guard mechanism may provide an explanation for the extreme susceptibility of retina to pRb loss-induced tumorigenesis. A recent study identified the cone cells to be the cell of origin for retinobloastomas and, strikingly, normal cone cells already contain high levels of MDM2 due to cone-specific expression of RXRγ, which binds to the MDM2 promoter to promote its high level expression 19. In mice, MDM4 may play similar roles in retinoblastomas induced by inactivation of Rb1 and its family members 20.

“The pRb-E2F-prenylation genes-Ras” safe-guard mechanism

Safe-guard mechanism also works in tissues that are susceptible to pRb loss-induced tumorigenesis. C cell adenocarcinomas of the thyroid gland occur with 50-75% penetrance in Rb1+/- mice after spontaneous loss of the wild type allele of Rb1. Yet, C cell tumorigenesis in Rb1+/- mice is still greatly accelerated by deletion of Nras, revealing that Nras functions to suppress pRb loss-induced tumorigenesis in C cells 21. A new study showed that this effect of Nras is also activated by E2F as a result of pRb loss 22. Nras exists in inactive GDP-bound state and active GTP-bound state. One first step to activate Nras-GDP is its prenylation to allow its association with the inner side of the cellular membrane, where it is converted to Nras-GTP by guanine nucleotide exchange factors (GEFs). One prenylation enzyme called farnesyl diphosphate synthase is an E2F regulated gene. E2Fs, particularly E2F1 and E2F3, also regulate expression of SREBP (sterol regulatory element-binding protein), which in turn regulates expression of certain prenyltransferases. Thus, loss of pRb induces higher levels of prenylation enzymes leading to more Nras prenylation and subsequent activation. Normal activated Nras can induce senescence in the absence of pRb but in a manner dependent on pRb family member p130 in C cells.

Neutralize the tumorigenic effects of pRb loss by experimentally correcting the effects of pRb loss on its targets

In cells that are susceptible to pRb loss-induced tumorigenesis, the effects of pRb loss on its targets should be the driving force of tumorigenesis. Identifying pRb targets and correcting the effects of pRb loss on these targets is a logical approach to neutralizing the tumorigenic effects of pRb loss. The literature has reported more than 100 pRb targets identified mostly based on their physical and functional interactions with pRb in vitro. The physiological significance of a number of pRb targets has been determined in mouse models of pRb deficiency-induced tumorigenesis. For meaningful pRb targets, one would expect that if the effects of pRb loss upon these targets could be experimentally corrected, the loss of pRb would become inconsequent and, hence, pRb loss-induced tumorigenesis would not occur. Below, the most studied pRb targets are discussed in this light.

pRb and E2F

The role of various E2Fs in pRb loss-induced tumorigenesis has been determined by generating Rb1+/- mice with combined deletion of a particular E2F. E2F1 was the first E2F tested and the results demonstrate that pituitary tumorigenesis in Rb1+/-E2F1-/- mice was significantly reduced in frequency and delayed in progression 23. Thus, although E2F1 can induce apoptosis as well as proliferation, it actually plays an oncogenic role in pRb deficiency-induced tumorigenesis.

Inactivation of E2F3 also delayed pituitary tumorigenesis in Rb1+/- mice, confirming that deregulated E2F3 activity contributes to tumorigenesis following pRb loss 24. However, in the same Rb1+/- mice, inactivation of E2F3 increased the aggressiveness and metastasis of thyroid tumors and induced tumorigenesis in a number of other types of tissues that were not susceptible to tumorigenesis in Rb1+/-E2F3+/+ mice. The simplest interpretation of this finding is that E2F3 has a tumor suppressor role that is complementary with pRb in these tissues.

Since E2F4 exhibited transcription repression activity in vitro, Rb1+/-E2F4-/- mice were expected to have enhanced tumor phenotypes than Rb1+/-E2F4+/+ mice. To the contrary, these mice showed significantly delayed pituitary tumorigenesis than Rb1+/-E2F4+/+ mice, providing genetic evidence that E2F4 is in fact promoting tumorigenesis 25. How could releasing repression of E2F inhibit tumorigenesis? Biochemical studies with Rb1-/-E2F4-/- MEFs revealed that the lack of E2F4 led p107 and p130 to bind more E2Fs 1, 2, and 3. Thus, in vivo studies indicate that the general nature of the pRb-E2F relationship is that between a tumor suppressor and its antagonists.

pRb and Id2

Proliferation and differentiation are generally mutually exclusive processes and a hallmark of tumorigenesis is increased proliferation accompanied with compromised differentiation. Specific differentiation is often activated by basic-helix-loop-helix type transcription factors that bind to gene promoters as dimmers. The Id proteins contain helix-loop-helix motif but not the basic DNA binding motif. Thus, Id proteins can dimmerize with a basic-helix-loop-helix transcription factor to form hetero-dimmers that cannot bind DNA. The Id2 member of the Id protein family physically interact with pRb and these two proteins functionally antagonize each other.

Pituitary tumorigenesis is significantly delayed in Rb1+/-Id2-/- mice, indicating that Id2 plays important roles in tumorigenesis following pRb loss 26. It was shown that in the absence of Id2, pituitary melanocyte differentiation was initiated prematurely, which could explain the inhibition of tumorigenesis. Proliferation of pituitary tumor cells and tumor angiogenesis were also inhibited, revealing that Id2 plays tumorigenic roles at multiple levels.

pRb and Ras

Identification of Ras proteins as targets of pRb was originally made with genetic studies in that Rb1-/- MEFs contained elevated levels of activated Nras and Kras 27. Since Kras KO is embryonic lethal, Rb1+/-Kras+/- mice were generated to determine its role in tumorigenesis following pRb loss 28. The results showed that deletion of one allele of Kras did not reduce the incidence of pituitary tumorigenesis, but pituitary tumors in Rb1+/-Kras+/- mice were generally smaller than those in Rb1+/-Kras+/+ mice and Rb1+/-Kras+/- mice survived longer than Rb1+/-Kras+/+ mice. Tumor cells in Rb1+/-Kras+/- mice showed decreased proliferation and increased differentiation, suggesting that Kras plays important roles in both proliferation and differentiation downstream of pRb. Interestingly, Rb1+/-Kras+/+ mice and Rb1+/-Kras+/- mice showed similar degrees of thyroid C cell tumorigenesis, suggesting that loss of one allele of Kras did not affect tumorigenesis in this cell type.

Nras-/- mice are normal and the role of Nras in tumorigenesis following pRb loss was therefore examined in Rb1+/-Nras-/- mice 21. Compared with Rb1+/-Nras+/+ mice, Rb1+/-Nras-/- mice developed fewer and smaller pituitary tumors and survived longer. Pituitary tumors developed in Rb1+/-Nras-/- mice showed higher degrees of differentiation but similar degrees of proliferation compared with tumors developed in Rb1+/-Nras+/+ mice. These findings suggest that activation of Nras following Rb1 deletion promotes tumorigenesis mainly through inhibiting differentiation.

Surprisingly, thyroid C cell tumorigenesis was greatly enhanced in Rb1+/-Nras-/- mice; the tumors were more proliferative, poorly differentiated, invasive, and highly metastatic to distant sites such as the lung, kidney, and liver. As discussed above, the mechanism underlying this tumor-promoting effect is the loss of a pRb-E2F-prenylation enzyme-Nras safe-guard mechanism in the thyroid C cells following loss of pRb. This effect is highly specific to thyroid C cells since Rb1+/-Nras-/- mice developed fewer pituitary tumors and do not develop additional type of tumors.

Limitations and opportunities of this approach

Deletion of a pRb target to determine the role of the activation of this target in pRb loss-induced tumorigenesis also eliminates the normal function of this target. This concern is less serious when the normal function of this target has been shown to be dispensable for a given cell type under study.

Since currently reported studies only delete one pRb target at a time, one wonders whether deleting more pRb targets together would more effectively neutralize the tumorigenic effects of pRb loss, or whether one particular pRb target whose deletion does not affect pRb loss-induced tumorigenesis would in fact play an essential role that could be compensated by another pRb target. However, although deleting multiple pRb targets appears attractive for this purpose, it is often not feasible. This is perhaps best demonstrated by the finding that deleting three E2F family members, E2F1, 2, and 3 (3a and 3b), caused embryonic lethality 29. This finding may also imply that therapeutically inactivating multiple E2Fs would have intolerable side effects.

Correcting the effects of pRb loss on only one pRb target may create a situation that is incompatible with cell homeostasis. In the simplest form, pRb loss may deregulate one target to induce more proliferation and another target to provide enhanced survival support. When only the survival-promoting target is inactivated, the outcome of deregulated proliferation would be cell death rather than transformation. Normal cells (with functional pRb) may not need the enhanced survival support since they are not aberrantly proliferating. This scenario forms the basis for a phenomenon well-known in classic genetics called synthetic lethality, which may in fact be the most effective way to neutralize the tumorigenic effects of pRb loss, as discussed below.

Synthetic lethal mutations to neutralize the tumorigenic effects of pRb loss

Conventional cancer therapeutics aim to kill proliferating cells while sparing non-proliferating cells. This approach frequently poses intolerable side effects since the body contains normally highly proliferating cells such as the hematopoietic cells and the mucosa epithelial cells of the intestine. More modern cancer therapeutics inhibits the activities of specific molecules that are altered in cancer cells (the so-called targeted therapies). When the targeted molecules are also present and play important roles in normal cells, targeted therapies either still have significant side effects or have a very narrow and less-than-robust therapeutic index. Also, certain oncogenic mutations may not be “druggable”.

More recent efforts have focused on identifying alterations that are synthetic lethal with the cancer-causing mutations. In this scenario, one mutation is the oncogenic event and present only in cancer cells, the other mutation is the alteration affected by the therapeutics. Since these two alterations are synthetic lethal, only cancer cells are killed and side effects should be non-existent. In reality, an important issue is whether the therapeutically inflicted alteration is truly harmless in cells without the oncogenic alteration, since it is not unreasonable to expect that most functions present in normal cells have roles of various degrees of importance.

How to identify mutations that are synthetic lethal to a specific oncogenic mutation? Synthetic lethal mutations have been identified based on “educated guesses”. An example is the identification of PARP [poly(ADP-ribose) polymerase] inhibition as synthetic lethal with BRCA1 or BRCA2 mutations, based on the prediction that inhibition of PARP would leave the DNA lesions caused by BRCA1/2 mutations unrepaired to cause apoptosis 30, 31. Most of the time however, synthetic lethal relationships are not easy to predict based on existing knowledge. Unbiased screens using chemical libraries or gene knockdown approaches therefore are often necessary. For one example, three gene knockdown screens have been reported that identified synthetic lethal mutations for the Kras oncogene 32-34. Three different groups of synthetic lethal mutations were identified in these three studies. One resides in the mitotic kinase PLK1, the anaphase-promoting complex/cyclosome, and the proteasome affecting cell cycle progression through mitosis. The other is the serine/threonine kinase STK33 regulating mitochondria-mediated apoptosis. The third one is the non-canonical IkappaB kinase TBK1, which could activate NF-kappaB anti-apoptotic signals involving c-Rel and BCL-XL. The therapeutic effectiveness of these synthetic lethal mutations and their potential side effects await testing with animal models.

Educated-guess approaches to Identifying alterations that are synthetic lethal with pRb loss

Since deregulated E2F1 can stimulate both proliferation and apoptosis, it is logically thought that alterations that favor E2F1’s apoptotic activity would preferentially induce pRb-deficient tumor cells. The first such attempt originated from the finding that cyclin A/Cdk2 could bind, phosphorylate, and inhibit E2F1. Expression of cyclin A and Cdk2 are upregulated by deregulated E2F after pRb loss and their inhibitory effects on E2F1 may ensure survival of pRb-deficient tumor cells. Disruption of cyclin A-E2F1 binding may therefore induce apoptosis in pRb-deficient cells (since they contain deregulated E2F1) but not in normal cells (since their E2F1 is not deregulated). After the cyclin A binding sequences in E2F1 were identified, a peptide mimicry was used to block cyclin A-E2F1 interaction. This blocking peptide induced apoptosis in cultured pRb –deficient tumor cells but not in non-transformed cells 35.

The recent discovery that PI3K signaling can inhibit E2F1’s ability to activate expression of apoptotic genes but not its ability to activate expression of proliferation genes should provide another avenue to identify alterations that are synthetic lethal to pRb loss 36, 37. Chemical inhibitors of PI3K are already in various stages of development.

Deleting the pRb target Skp2 is synthetic lethal to pRb loss

As discussed above, experimentally correcting the effects of pRb on one of its targets has the potential to kill pRb-deficient cells with a synthetic lethal mutation. Inactivating the pRb target Skp2 in Rb1+/- mice provided the first experimental evidence for this prediction 38. Skp2 was identified as a pRb target when pRb was inducibly re-expressed in pRb-deficient osteosarcoma cell Saos-2 in a time course 39. It was found that p27 protein accumulation took place before the reductions in protein levels of many E2F target genes. Skp2 functions as a substrate-recruiting subunit for the SCFSkp2 E3 ubiquitin ligase whose best-studied target is p27 after p27T187 is phosphorylated. pRb binds to Skp2 to prevent it from binding to T187-phosphorylated p27, thereby leading to p27 accumulation following pRb re-expression.

Skp2-/- mice are smaller and contain enlarged nuclei in a number of organs such as the liver and kidney with accompanying p27 protein accumulation 40. The pituitary and thyroid glands appear indistinguishable from their wild type counterparts. To test the in vivo role of Skp2 in pRb loss-induced tumorigenesis, Rb1+/-Skp2-/- mice were generated. These mice were completely tumor free, identifying Skp2 as the first pRb target that is essential for tumorigenesis following loss of pRb 38.

To study how Skp2 deletion blocked tumorigenesis in Rb1+/- mice, targeted deletion of Rb1 was carried out in malenocytes of the pituitary IL using POMC-Cre and Rb1lox/lox 38. While spontaneous loss of the wild type allele of Rb1 in Rb+/- mice would generate a few Rb1-/- melanotrophs, pRb is deleted in all melanotrophs in the pituitary of POMC-Cre;Rb1lox/loxSkp2+/+ and POMC-Cre;Rb1lox/loxSkp2-/- mice as demonstrated by a reporter strain. In this setting, deletion of Rb1 induced tumorigenesis across the entire IL in POMC-Cre;Rb1lox/loxSkp2+/+ mice but ablation of the IL in POMC-Cre;Rb1lox/loxSkp2-/- mice due to massive apoptosis. Deregulated melanotroph proliferation was not inhibited in the absence of Skp2. These findings dramatically reveal that deletion of Skp2 is synthetic lethal with deletion of Rb1 in mouse melanotrophs.

Blocking phosphorylation of p27T187 is also synthetic lethal to pRb loss

Identification of Skp2 deletion as synthetic lethal with Rb1 deletion raises inevitable new questions. First, Skp2 is a multi-function protein; it would therefore be important to determine which function of Skp2 is relevant for the synthetic lethal relationship with pRb loss.

In its capacity as the substrate recruiting subunit of the SCFSkp2 E3 ubiquitin ligase, Skp2 targets a growing list of substrates including p27, p21, p57 41. The best-established Skp2 substrate is p27 phosphorylated on T187 by cyclin-dependent kinases. Importantly however, the in vivo role of this well-established biochemical reaction has remained unclear due to divergent phenotypes of Skp2 KO mice and p27T187A KI mice 42. Skp2 KO mice are smaller than normal and show p27 protein accumulation in many tissues, while p27T187A KI mice are slightly larger than normal and do not have p27 protein accumulation. These genetic evidence indicate that Skp2 plays an important role in down-regulating p27 protein levels but Skp2 plays this role through mechanisms other than its involvement in SCFSkp2 E3 ubiquitin ligase targeting p27T187p; how Skp2 KO induces p27 protein accumulation therefore remains unknown.

Lack of abnormalities in p27T187A KI mice indicates that the SCFSkp2-mediated ubiquitination of p27T187p is dispensable for p27 protein degradation in vivo and for normal mouse development and physiology. Another study further demonstrated that p27T187A KI mice and WT mice were similarly susceptible to tumorigenesis induced by activated Kras or by chemical carcinogen ENU 43.

Independent of its role in SCFSkp2 E3 ubiquitin ligase, Skp2 can directly bind to cyclin A to protect it from inhibition by p27 family inhibitors 44, 45 and can bind to p300 to prevent p53 acetylation and inactivation 46. These two mechanisms have been shown to provide survival support in a number of cancer cell lines.

Second, although Skp2 KO does not induce apoptosis in various tissues during mouse development, Skp2 KO mice have a number of abnormalities such as small body sizes and enlarged cell nuclei in a number of tissues 40. This suggests that inhibiting Skp2 might have significant side effects. If a specific function of Skp2 is responsible for synthetic lethality with pRb loss and inhibiting this function does not produce these abnormalities of Skp2 KO, it would provide a better therapeutic target.

The finding that pRb binds Skp2 to prevent it from interacting with T187-phosphorylated p27 39 provided a basis for hypothesizing that Skp2 function in the SCFSkp2-mediated ubiquitination of p27T187p is responsible for synthetic lethality with pRb loss, and this hypothesis was best tested with the p27T187A KI mice.

POMC-Cre;Rb1lox/loxp27T187A/T187A mice were generated to test the above hypothesis. The results show that pituitary tumorigenesis was blocked by melanotroph apoptosis in these mice to similar, albeit not identical, degrees as seen in POMC-Cre;Rb1lox/loxSkp2-/- mice. In addition to identifying a specific function of Skp2 that is largely responsible for its synthetic lethal relationship with pRb loss, this finding for the first time revealed an in vivo role of SCFSkp2-mediated ubiquitination of p27T187p. The latter point suggests that therapeutically inhibiting p27T187 phosphorylation or SCFSkp2-mediated ubiquitination of p27T187p should have no or minimal side effects.

Summary and future studies

In retrospect, the approach to correcting the effects of pRb loss on only one of its targets indeed has the potential to identify synthetic lethal mutations for pRb loss (Fig. 1). The revelation that deleting Skp2, or inhibiting p27 ubiquitination by the SCFSkp2 E3 ubiquitin ligase, did not inhibit aberrant proliferation but induced apoptosis in pRb deficient cells was a surprise since the best-known function of p27 is to inhibit proliferation. The next challenge is to understand how a slight increase in p27 protein levels following pRb loss triggers apoptosis. Two major inducers of apoptosis following pRb loss are E2F1 and p53, and it will be important to determine which or whether both are playing a role in apoptosis in this setting. If either one, or both, does play a role, there is still a molecular gap to be filled between p27 and E2F1 or p53. p27 may also induce apoptosis independent of E2F1 or p53.

Figure 1.

Figure 1

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Consequences of pRb loss. a. pRb functions to suppress tumorigenesis via its targets. Only E2Fs and Skp2 are listed among the more than 100 pRb targets. One function of Skp2 is to target p27T187p for ubiquitination, and this function is inhibited by pRb. b. Loss of pRb leads to deregulation of its targets (indicated by red color). Deregulation of Skp2 leads to ubiquitination and degradation of p27 (indicated by grey color). The outcome of pRb loss is tumorigenesis. c. When Skp2 is also deleted, p27 is prevented from ubiquitination and degradation. The outcome of pRb loss in this scenario becomes apoptosis. d. When p27T187 is mutated to p27T187A, p27 is also prevented from ubiquitination and degradation, and the outcome of pRb loss also becomes apoptosis.

Another direction for future studies is to determine whether Skp2 deletion or p27T187A mutation is synthetic lethal with pRb loss in tumorigenesis of various other types of tissues. This line of research will identify candidate tumor types for clinical application of this synthetic lethal therapeutic strategy. Inactivation of p53 is often necessary for tumorigenesis following pRb loss in various types of tissues such the lung, bone, and prostate. If this synthetic lethal relationship depends on p53 to induce apoptosis, it might prove to be effective for a very limited spectrum of tumors. As is often the case, the identification of synthetic lethal mutations for pRb loss has provided the proof-of-principle for a new paradigm for understanding and treating pRb loss-induced tumorigenesis, many critical aspects of this paradigm are now in need to be addressed.

Acknowledgments

We thank members of our laboratory for helpful discussions. This work was supported by grant RO1CA131421 from the NIH-NCI. L.Z. is a recipient of the Irma T. Hirschl Career Scientist Award.

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