Tumours often become resistant to treatment, but how this occurs is poorly understood. An analysis of how the protein Rb affects tumour growth and the response to therapy might cast light on the problem

Rubin, Seth M

Sage, Julien

English

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UC Santa CruzUC Santa Cruz Previously Published WorksTitleManipulating the tumour-suppressor protein Rb in lung cancer reveals possible drug targetsPermalinkhttps://escholarship.org/uc/item/63s1z851JournalNature, 569(7756)ISSN0028-0836AuthorsRubin, Seth MSage, JulienPublication Date2019-05-01DOI10.1038/d41586-019-01319-y Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaCancer Print title: Manipulating a tumour suppressor protein  Online: Manipulating the tumor suppressor protein Rb in lung cancer reveals possible drug targets  Tumours often become resistant to treatment but how this occurs is poorly understood. An analysis of how the protein Rb affects tumour growth and therapy response has implications for cancer treatment and drug development. See Letter p.XXX  Seth M. Rubin & Julien Sage The development of cancer is invariably accompanied by cellular proteins performing unwanted activities or losing their function. Therapeutically targeting proteins that are more active than normal is conceptually straightforward — a drug needs to be found that, like a wrench thrown into a machine, specifically halts the malicious activity. By contrast, remedying the loss of a type of protein called a tumour suppressor, which normally opposes cancer growth, presents a conundrum. How can a protein be targeted by drugs if it is no longer present? One way to get around this problem and find new drug targets is to determine other consequences, such as molecular changes, that occur when a tumour suppressor is lost or does not function in a particular type of cancer. Writing in Nature, Walter et al.1 report their analysis of the role of the tumour suppressor protein retinoblastoma (Rb) using both an experimental system that allowed them to engineer the loss and re-activation of this protein in lung cancer in mice and human cancer cells grown in vitro[OK?].    Since the discovery of the tumour-suppressor activity of Rb from studies of children with a type of eye cancer and the subsequent identification of Rb loss or inactivation as a characteristic of many human tumours, this protein has served as a model for studying tumour suppressors in cancer2. Rb can act as a key brake that halts cell-cycle progression and it promotes cellular differentiation into specific cell types. Many cancers block Rb function by increasing the activity of enzymes called cyclin-dependent kinases (Cdks), which add phosphate groups to Rb to inactivate it3. There has been some success in the clinic with anticancer treatments that use inhibitor drugs that target Cdk4 and Cdk6 and prevent them from inactivating Rb. However, as would be expected, these inhibitors are not effective if Rb is not expressed, which might happen as a tumour evolves[OK? (or please revise to provide your preferred comment about Rb loss in tumours (for example, is Rb loss a common occurrence when treatment resistance occurs?)]4. A clear understanding of the consequences of Rb inactivation is lacking, which limits our ability to target tumours in which Rb is absent.    Walter and colleagues studied the consequences of Rb loss in a mouse model of a common form of lung cancer called lung adenocarcinoma. The authors genetically engineered the animals so they could control whether and when Rb was made in lung-cancer cells. This allowed them to make a key observation that the absence of Rb (a state termed an Rb knockout) promotes cancer development by causing an increase in both tumour growth and tumour spread to secondary sites in a process called metastasis. This provides some of the strongest in vivo evidence provided so far supporting a role for Rb loss not only in driving cancer initiation but also in promoting cancer progression.   The authors observed that the molecular changes that lead to cancer progression and metastasis in this lung cancer model are strikingly different depending on whether Rb was present or absent. If Rb was missing, compared to the situation when Rb was present but inactivated by Cdks, the cancer cells had an increased propensity to change their cellular identity to a less-differentiated state, which allows the disease to progress more rapidly. Cellular dedifferentiation in the absence of Rb has also been observed in prostate cancer5. Although the precise mechanism controlling cellular plasticity in this scenario is unknown, it might involve the activation of factors involved in cellular reprogramming, such as the transcription-factor protein Sox2, which is normally inhibited by Rb6.   Building on previous studies7,8 that investigated the consequences of Rb loss in mouse models of cancer, Walter and colleagues have deepened our understanding of Rb function by studying the effect of reinstating Rb expression in lung cancer months after the Rb-encoding gene was deleted by genetic engineering. This re-expression of Rb promoted the re-differentiation of undifferentiated cancer cells, thereby rendering the tumours less malignant. This observation is important because it suggests that strategies to re-activate Rb or to manipulate proteins that act downstream of Rb might have therapeutic benefits for the treatment of cancers that have lost Rb expression.  Another key observation made by Walter and colleagues is that tumours that lack Rb surprisingly do not upregulate the signalling pathway that contains the enzyme MAPK kinase, which is a pathway that is often activated in lung adenocarcinoma. This observation regarding the MAPK pathway had not been previously reported and is unexpected given that the upregulation of this pathway is a hallmark of this type of cancer. Their data strongly suggest that inactivation of Rb function is a major role of MAPK activation in lung cancer. MAPK signalling can cause Rb inactivation by several different mechanisms9. Walter et al. indicate that an increase of MAPK kinase signalling above normal in lung adenocarcinoma causes Rb inhibition because inactivation10 of the Cdk inhibitor protein called p27 causes the activation of Cdk2 — a Cdk family member that can3 phosphorylate and inactivate Rb.   The success of inhibitors of Cdk4 and Cdk6 in the treatment of some tumours indicates that the manipulation of Rb activity can stop cancer progression. However, the observation of therapy resistance is motivating the search for other drugs that could re-activate Rb and for the identification of additional targets that could be used in treatments for cancer cells that have lost Rb expression. Walter and colleagues’ success in identifying molecular and cellular consequences of Rb loss, such as cellular de-differentiation and an absence of up-regulation of MAPK signalling, will help with these efforts.   The authors’ observations regarding the interplay between MAPK and Cdk2 implicates Cdk2 as a potential drug target in lung cancers that have Rb but are nevertheless resistant to inhibitors of Cdk4 and Cdk6. In this context the combined inhibition of MAPK and Cdk2, or the combined inhibition of Cdk4, Cdk6 and Cdk2 might potently stop the growth of lung tumours by ensuring that Rb is not deactivated by phosphorylation (Fig. 1). However, more work will be needed to identify additional potential drug targets. Efforts should also be made to determine the sensitivity of a person’s cancer to inhibitors of Cdk4 and Cdk6 or Cdk2, including assessing a person’s genetic profile and monitoring alterations termed epigenetic changes that are modifications of DNA or DNA-binding proteins. It would be useful to try to identify targetable proteins or pathways that inhibit the cellular dedifferentiation that occurs upon Rb loss.   It will be essential to understand to what extent the principles of lung cancer development and progression are applicable to other types of cancer. The experimental system used by Walter et al. could help to address this challenge because their results might be relevant for understanding the effect of loss of tumour suppressor proteins in diverse contexts[OK?]. Studies of Rb have long provided a useful model system to investigate fundamental mechanisms of tumour suppression. Now they might also offer a way forward to uncover new anti-cancer strategies.  Seth M. Rubin is in the Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California 95064, USA. Julien Sage is in the Departments of Pediatrics and of Genetics, Stanford University, Stanford, California 94305, USA.  e-mails: srubin@ucsc.edu; julsage@stanford.edu 1. Walter, D. M. et al. Nature [ref will be completed by the subeditors when the PDF proof is prepared] (2019). 2.  Dyson, N. J. RB1: a prototype tumour suppressor and an enigma. Genes Dev 30, 1492-1502, doi:10.1101/gad.282145.116 (2016). 3. Rubin, S. M. Deciphering the retinoblastoma protein phosphorylation code. Trends Biochem Sci 38, 12-19, doi:10.1016/j.tibs.2012.10.007 (2013). 4. Klein, M. E., Kovatcheva, M., Davis, L. E., Tap, W. D. & Koff, A. CDK4/6 Inhibitors: The Mechanism of Action May Not Be as Simple as Once Thought. Cancer Cell 34, 9-20, doi:10.1016/j.ccell.2018.03.023 (2018). 5. Ku, S. Y. et al. Rb1 and Trp53 cooperate to suppress prostate cancer lineage plasticity, metastasis, and antiandrogen resistance. Science 355, 78-83, doi:10.1126/science.aah4199 (2017). 6. Kareta, M. S., Sage, J. & Wernig, M. Crosstalk between stem cell and cell cycle machineries. Curr Opin Cell Biol 37, 68-74, doi:10.1016/j.ceb.2015.10.001 (2015). 7. Ho, V. M., Schaffer, B. E., Karnezis, A. N., Park, K. S. & Sage, J. The retinoblastoma gene Rb and its family member p130 suppress lung adenocarcinoma induced by oncogenic K-Ras. Oncogene 28, 1393-1399, doi:onc2008491 [pii] 10.1038/onc.2008.491 (2009). 8. Rubin, B. P. et al. Evidence for an unanticipated relationship between undifferentiated pleomorphic sarcoma and embryonal rhabdomyosarcoma. Cancer Cell 19, 177-191, doi:10.1016/j.ccr.2010.12.023 (2011). 9. Meloche, S. & Pouyssegur, J. The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1- to S-phase transition. Oncogene 26, 3227-3239, doi:10.1038/sj.onc.1210414 (2007). 10. Donovan J.C , Milic, A. & Slingerland J. M. J. Biol. Chem. 276, :40888–40895 (2001).   Figure 1 | Rb protein in cancer cells. a, Rb is a type of protein known as tumour suppressor that can help to block cancer when it is active. A common feature of lung cancer is Rb inactivation by the addition of phosphate groups (P) to the protein. This can occur through a pathway that requires the action of the enzyme MAPK and the enzymes Cdk4 and Cdk6 or the enzyme Cdk2. b, Drugs currently in use in the clinic for lung cancer (green) block the action of MAPK or Cdk4 and Cdk6. Walter et al.1 studied a mouse model of lung cancer and report that Cdk2 has a major role in the inactivation of Rb. Therefore the development of drugs that target this protein might have promise as a new way to treat lung cancer. c, Rb expression is often lost in lung cancer. Walter and colleagues studied the consequence of Rb loss that they engineered in mouse models of lung cancer. This resulted in a loss of cellular differentiation and an increase in the spread of cancer cells to other sites in the body through a process called metastasis. Proteins implicated in such changes, like the proteins Sox2 or HMGA2, might offer targets to be investigated for future drug development.   |   RbProliferation (e.g. E2F)Metastasis  (e.g. HMGA2)Plasticity (e.g. Sox2)Cdk4/6Cdk2MAPKkinasesMAPK pathwayinhibitors Cdk2/4/6inhibitorsTherapeutic targets to activate Rb Targets to block tumor progressionupon loss of Rb

Manipulating the tumour-suppressor protein Rb in lung cancer reveals possible drug targets

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UC Santa CruzUC Santa Cruz Previously Published WorksTitleManipulating the tumour-suppressor protein Rb in lung cancer reveals possible drug targetsPermalinkhttps://escholarship.org/uc/item/0z07p3dmJournalNature, 569(7756)ISSN0028-0836AuthorsRubin, Seth MSage, JulienPublication Date2019-05-01DOI10.1038/d41586-019-01319-y Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaManipulating a tumour suppressorSETH M. RUBIN,Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California 95064, USA.JULIEN SAGEDepartments of Pediatrics and of Genetics, Stanford University, Stanford, California 94305, USA.AbstractTumours often become resistant to treatment, but how this occurs is poorly understood. An analysis of how the protein Rb affects tumour growth and the response to therapy might cast light on the problem.The development of cancer invariably involves cellular proteins that have unwanted effects or that become inactive. Therapeutically targeting proteins that are more active than normal is conceptually straightforward — a drug needs to be found that, like a wrench thrown into a machine, halts the malicious activity. By contrast, remedying the loss of a type of protein called a tumour suppressor, which normally opposes cancer growth, presents a conundrum. How can a protein be targeted by drugs if it is no longer present? One way of getting around this problem and finding new drug targets is to identify other effects, such as molecular changes, that occur when a tumour suppressor is lost or does not function. On page 424, Walter et al.1 report their analysis of the role of the tumour-suppressor protein retinoblastoma (Rb), using an experimental system that allowed them to engineer the loss and reactivation of this protein in lung cancer in mice.Since the discovery of Rb’s tumour-suppressor activity from studies of children with a type of eye cancer, and the subsequent identification of Rb loss or inactivation as a characteristic of many human tumours, this protein has served as a model for studying tumour suppressors in cancer2. Rb can act as a key brake on cell-cycle progression, and it promotes cellular differentiation into specific cell types, which slows the development of cancer. Many cancers block Rb function by increasing the activity of enzymes called cyclin-dependent kinases (Cdks), which inactivate Rb through phosphorylation3 (the addition of phosphate groups to it; Fig. 1a). Anticancer treatments involving inhibitor drugs that target Cdk4 and Cdk6 to prevent them from inactivating Rb have shown some success. However, as would be expected, these inhibitors are not effective if Rb is not expressed in the tumours4.Walter and colleagues studied the effects of Rb loss in a mouse model of a common form of lung cancer called lung adenocarcinoma. The authors used genetically engineered animals to control whether and when Rb was made in lung cancer cells. This showed that the absence srubin@ucsc.edu. HHS Public AccessAuthor manuscriptNature. Author manuscript; available in PMC 2020 May 04.Published in final edited form as:Nature. 2019 May ; 569(7756): 343–344. doi:10.1038/d41586-019-01319-y.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptof Rb (a state termed an Rb knockout) promotes cancer development by causing an increase in both tumour growth and its spread to secondary sites in a process called metastasis. The results provide some of the strongest in vivo evidence so far to support a role for Rb loss in driving not only cancer initiation, but also its progression.The authors observed that the molecular changes that lead to progression and metastasis in this lung cancer model are strikingly different, depending on whether Rb is present or absent. If Rb was missing, compared with the situation in which Rb was present but inactivated by Cdks, the cancer cells had an increased propensity to change their cellular identity to a less-differentiated state that allowed the disease to progress more rapidly. This state was accompanied by the expression of metastasis-promoting proteins such as HMGA2. Such cellular dedifferentiation in the absence of Rb has also been observed in prostate cancer5. Although the precise mechanism that controls cellular plasticity in this scenario is unknown, it might involve the activation of factors involved in cellular reprogramming, such as the transcription-factor protein SOX2, which is normally inhibited by Rb (ref. 6).Building on previous studies7,8 that investigated Rb loss in mouse models of cancer, Walter and colleagues studied the effect of reinstating Rb expression in mice with lung cancer months after the Rb-encoding gene had been deleted by genetic engineering. They found that re-expression of Rb promoted the redifferentiation of undifferentiated cancer cells, rendering the tumours less malignant. This observation is important, because it suggests that strategies to reactivate Rb or to manipulate proteins acting downstream of Rb might be therapeutically beneficial for treating cancers that have lost Rb expression.Another key observation made by Walter and colleagues is that tumours that lack Rb do not upregulate a signalling pathway containing the kinase MAPK, which is often activated in lung adenocarcinoma. This had not been reported previously and is unexpected, given that upregulation of the pathway is a hallmark of this type of cancer. The authors’ data strongly suggest that inactivating Rb is a major objective of MAPK activation in lung cancer that is no longer required in the absence of Rb.The clinical success of Cdk4 and Cdk6 inhibitors indicates that manipulation of Rb activity can stop cancer progression. However, the development of resistance to such therapies is motivating the search for other drugs that could mimic the reactivation of Rb function, and for new targets in combating cancer cells that have lost Rb expression. Walter and colleagues’ success in identifying molecular and cellular effects of Rb loss, such as cellular dedifferentiation and an absence of upregulation of MAPK signalling, will help with these efforts.MAPK signalling can inactivate Rb through several mechanisms9. Walter and colleagues’ results indicate that Rb inhibition results from increased MAPK signalling that inactivates10 a Cdk inhibitor protein called p27, thereby activating Cdk2 — a Cdk that can phosphorylate and thus inactivate Rb (ref. 3). The authors’ observations regarding the interplay between MAPK and Cdk2 implicate Cdk2 as a potential drug target in lung cancers that retain Rb but are resistant to Cdk4 and Cdk6 inhibitors. In this context, the combined inhibition of MAPK and Cdk2, or of Cdk4, Cdk6 and Cdk2, might potently stop the growth of lung tumours by RUBIN and SAGE Page 2Nature. Author manuscript; available in PMC 2020 May 04.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptensuring that Rb remains in its active state and is not associated with high levels of phosphorylation (Fig. 1b).However, more work is needed to identify further potential drug targets. Efforts should also be made to determine the sensitivity of a person’s cancer to inhibitors of Cdk4 and Cdk6 or Cdk2, including assessing the individual’s genetic profile and monitoring alterations termed epigenetic changes — modifications to DNA or DNA-binding proteins that can affect gene expression. Furthermore, it would be useful to identify targetable proteins or pathways that inhibit the cellular dedifferentiation that occurs after Rb loss (Fig. 1c).Finally, it will be essential to understand to what extent the principles of lung cancer development and progression are applicable to other types of cancer. The experimental system used by Walter et al. could help to address this challenge, because their results might elucidate the effect of loss of tumour-suppressor proteins in diverse contexts. Studies of Rb have long provided a useful model system in which to investigate fundamental mechanisms of tumour suppression. Now they might also offer a way forward in uncovering new anticancer strategies.References1. Walter DM et al. Nature 569,424–428 (2019).2. Dyson NJ Genes Dev. 30, 1492–1502 (2016). [PubMed: 27401552] 3. Rubin SM Trends Biochem. Sci 38, 12–19 (2013). [PubMed: 23218751] 4. Klein ME, Kovatcheva M, Davis LE, Tap WD & Koff A Cancer Cell 34, 9–20 (2018). [PubMed: 29731395] 5. Ku SY et al. Science 355, 78–83 (2017). [PubMed: 28059767] 6. Kareta MS, Sage J & Wernig M Curr. Opin. Cell Biol 37, 68–74(2015). [PubMed: 26520682] 7. Ho VM, Schaffer BE, Karnezis AN, Park KS & Sage J Oncogene 28, 1393–1399 (2009). [PubMed: 19151761] 8. Rubin BP et al. Cancer Cell 19,177–191 (2011). [PubMed: 21316601] 9. Meloche S & Pouysségur J Oncogene 26, 3227–3239 (2007). [PubMed: 17496918] 10. Donovan JCH, Milic A &Slingerland JM J. Biol. Chem 276, 40888–40895 (2001). [PubMed: 11527971] RUBIN and SAGE Page 3Nature. Author manuscript; available in PMC 2020 May 04.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptFigure 1|. The Rb protein in cancer cells.a, Rb is a type of protein known as a tumour suppressor, which can help to block cancer development. A common feature of lung cancer is Rb inactivation by the addition of phosphate groups (P) to the protein. This frequently occurs through a pathway that requires various enzymes: MAPK; Cdk4 and Cdk6; or Cdk2. b, Drugs that block the action of MAPK or Cdk4 and Cdk6 are in use in the clinic or under development for lung cancer treatment. Walter et al.1 studied a mouse model of lung cancer, and report that Cdk2 has a major role in inactivating Rb. Therefore, the development of drugs that target this protein might have promise in treating lung cancer, c, Rb expression is often lost in lung cancer cells (yellow). The authors studied the effect of Rb loss in mouse models of the cancer. The absence of Rb resulted in loss of cellular differentiation and an increase in the spread of cancer cells to other sites in the body through a process called metastasis. Proteins implicated in such changes, such as SOX2 and HMGA2, might offer targets for drug development.RUBIN and SAGE Page 4Nature. Author manuscript; available in PMC 2020 May 04.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscript

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Manipulating the tumour-suppressor protein Rb in lung cancer reveals possible drug targets.

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