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Arf Tumor Suppression: Stem Cells and Cancer
Summary: Charles Sherr studies tumor-suppressor–dependent signaling networks that prevent progression through the mammalian cell division cycle in response to activated oncogenes and that are inactivated in virtually all forms of cancer. A tumor-suppressor network dominated by the retinoblastoma protein (Rb), p53, and the Ink4-Arf locus may have evolved to regulate stem cell self-renewal and tissue homeostasis. This concept explains why three intimately linked and coregulated tumor-suppressor genes (Ink4a, Ink4b, and Arf) are conserved in mammals, despite the risk of their frequent codeletion in cancers.
Cancers arise due to mutations that impinge upon two general families of molecular regulators that govern cell division. Gain-of-function mutations that activate cellular oncogenes result in the production of deregulated proteins that stimulate uncontrolled cell proliferation. A second class of tumor-suppressor genes opposes the actions of oncogenes, and their loss of function predisposes to cancer. The realization that oncogenes and tumor-suppressor genes encode proteins that govern processes such as gene expression, cell differentiation, tissue development, and responses to environmental stress, and that mutations affecting their functions constitutively deregulate signaling networks important for cell proliferation have provided key mechanistic insights into tumor formation.
A Hallmark of Cancer: Inactivation of the Rb and p53 Network
Transformation of normal cells to cancer cells commonly involves the disruption of signaling networks regulated by two canonical tumor-suppressor genes encoding the retinoblastoma protein (Rb) and the p53 transcription factor. Although the functions of Rb and p53 are not normally required for cell division, both proteins govern protective "checkpoint" responses that are triggered by DNA damage or by oncogene stimulation. Activation of Rb and p53 induces global changes in gene expression, which either arrest cell division or induce suicide (apoptosis) of irreversibly damaged cells. Disruption of Rb and p53 compromises these protective programs, allowing incipient cancer cells to continue to divide, to consolidate mutational damage, and, ultimately, to form tumors.
Lesions affecting other genes governing the Rb and p53 signaling network can mimic Rb and p53 loss of function. Among these are mutations that disrupt the Ink4-Arf gene cluster, a compact genetic locus that specifies three additional tumor-suppressor proteins: p16Ink4a, p15Ink4b, and p19Arf (p14ARF in humans). Expression of the p16Ink4a and p15Ink4b proteins in response to oncogenic stress leads to Rb activation, whereas synthesis of p19Arf induces p53. Therefore, chromosomal deletions that encompass the Ink4-Arf locus simultaneously compromise the activities of both Rb and p53 and represent one of the most frequent genetic events in cancer.
Arf Tumor Suppression in Acute Lymphoblastic Leukemia
The Arf gene selectively responds to abnormally sustained and elevated thresholds of proliferative signals generated by oncogene activation. Inappropriate growth-promoting signals trigger the Arf-p53 circuit, resulting in the proliferative arrest or elimination of cells that have sustained oncogene mutations. However, cells that suffer Arf deletions, much like those lacking functional p53, fail to undergo cell cycle arrest or apoptosis in response to oncogenic stimulation and are more highly tumorigenic.
Most hematological malignancies are initiated by chromosomal translocations or mutations that inappropriately activate cellular oncogenes, and investigations of leukemias and lymphomas have provided fertile ground for gaining insights into when Arf is induced in the course of disease. The Philadelphia chromosome (Ph), the first tumor-specific cytogenetic anomaly identified, results from a reciprocal translocation that adjoins the human ABL oncogene on chromosome 9 to a breakpoint cluster region (BCR) on chromosome 22 to produce the BCR-ABL fusion protein, a constitutively active protein tyrosine kinase. This translocation is the founding lesion of two distinct hematological malignancies—chronic myelogenous leukemia (CML), which initially presents as a relatively benign myeloproliferative disorder, and Ph+ acute lymphoblastic leukemia (ALL), a more aggressive B cell tumor.
CML affects immature hematopoietic progenitor cells in which the human INK4-ARF locus is normally silenced and INK4-ARF is seldom deleted. In contrast, in more mature lymphoblastic cells that arise in Ph+ ALL, INK4-ARF is induced and plays a protective role in the early stages of disease. However, rare cells that sustain INK4-ARF deletions emerge as highly aggressive malignant clones that have acquired a capacity for continuous self-renewal. A survey of patients with newly diagnosed Ph+ ALL revealed that almost two-thirds of them had already sustained biallelic INK4-ARF deletions in their leukemic blasts before therapy could be initiated. This suggests that inactivation of the locus contributes to the more aggressive features of Ph+ ALL versus CML and to much reduced therapeutic responsiveness.
Despite the dramatic clinical efficacy of BCR-ABL kinase inhibitors (imatinib, dasatinib, and nilotinib) in treating CML patients in chronic phase, these drugs fail to provide durable therapeutic benefit in those with Ph+ ALL or in CML patients in lymphoid blast crisis. Our investigations of BCR-ABL–driven B cell ALL in mouse model systems indicated that Arf inactivation enhances the biological "fitness" of Ph+ ALL cells in the hematopoietic microenvironment through a tumor cell–nonautonomous mechanism. This process diminishes the efficiency with which targeted therapy can eradicate residual disease and facilitates the subsequent emergence of drug-resistant clones that have acquired BCR-ABL kinase mutations. Hence, deletion of INK4-ARF and mutations in the BCR-ABL kinase collaborate through separate mechanisms to hamper the potentially beneficial effects of targeted therapy in Ph+ ALL.
ALL also arises in T lymphocytes bearing mutations in NOTCH receptors, and virtually all such T-ALLs sustain INK4-ARF deletions. Modeling of mutant Notch1-induced T-ALL in the mouse provided further evidence for a developmental stage-specific role of Arf in tumor suppression. Specifically, retention of the Arf locus had relatively modest activity in suppressing the formation of T-ALLs arising from bone marrow–derived progenitors in which the Ink4-Arf locus is epigenetically silenced and p19Arf is not expressed. In striking contrast, retention of Arf in thymocyte-derived donor cells delayed disease onset and penetrance, tumor-suppressive effects that were reversed by Arf deletion. Thus, the prevalence of INK4-ARF deletions in human NOTCH-induced T-ALL argues that this malignancy arises from committed T cells in which the INK4-ARF locus can be engaged, and not from their less mature bone marrow progenitors in which the locus is silenced.
p53-Independent Functions of Arf
Although the prevailing paradigm is that the tumor-suppressive functions of Arf are mediated through p53, mice lacking both Arf and p53 develop a broader spectrum of cancers than animals lacking either gene alone, implying that the Arf protein also has p53-independent functions. In this regard, Arf can induce the sumoylation of many cellular proteins by inhibiting the activity of Senp3, a desumoylating protease. Given that the modification of target proteins by sumoylation influences many cellular processes, including protein transport, ribosomal biogenesis, DNA replication and repair, and mitosis, Arf-induced sumoylation is likely to underlie its p53-independent tumor-suppressor activity. Pinpointing the most relevant targets is a major challenge.
Visualizing Arf Expression in Living Mice
By replacing p19Arf-coding sequences in the mouse genome with a DNA cassette specifying green fluorescent protein (GFP), we were able to visualize Arf promoter activity in homozygous ArfGFP/GFP animals. Because these mice lack functional p19Arf activity, they are highly prone to tumor development. Cancers that arise spontaneously in ArfGFP/GFP mice, and more rapidly appearing tumors in animals engineered to coexpress activated oncogenes, exhibit vivid green fluorescence. By contrast, green fluorescent cells were not detected in most normal tissues of these animals. These results highlight a key feature of Arf regulation—namely, the Arf promoter is insulated from responding to physiologic signals conveyed by proteins that drive normal cell proliferation; yet, Arf is expressed when abnormally elevated signaling thresholds are triggered by mutationally activated oncogenes.
Nonetheless, the fact that inactivation of Arf leads to spontaneous tumor formation implies that the protein must be at least transiently expressed in normal tissues in order to eliminate rare cells that have sustained oncogenic mutations. With this in mind, we generated a new reporter mouse in which the cDNA encoding Cre recombinase was introduced in place of GFP downstream of the Arf promoter. We reasoned that interbreeding Arf-Cre mice to indicator strains engineered to synthesize Cre-induced β-galactosidase (β-Gal) or yellow fluorescent protein (YFP) would not only allow identification of cells in which the Arf promoter had been transiently activated but would also define their cellular progeny by generating marked "clones." These lineage-tracing experiments indicate that Arf is transiently expressed in cell populations that arise from certain tissue stem cells, consistent with the idea that Arf acts as part of a molecular switch that limits stem cell self-renewal. Arf silencing may therefore be required for immortalization of both normal stem cells and abnormally self-renewing cancer cells.
As of May 30, 2012
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