[Cell Cycle 3:5, 621-624; May 2004]; ©2004 Landes Bioscience
Pathways Sufficient to Induce Epidermal Carcinogenesis
California USA
*Correspondence to: Paul A. Khavari; Program in Epithelial Biology; 269 Campus Drive, Room 2145; Stanford, California 94305 USA; Tel.: 650.725.5266; Fax: 650.723.8762; Email:
[email protected] Received 03/12/04; Accepted 03/15/04
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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=860
Abnormal epidermal proliferation is characteristic of a number of disorders, including the two most common cancers in the United States, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Both cancers display a disruption in the normal homeostatic balance between cell division and programmed cell death. While abnormal activation of the sonic hedgehog/patched pathway has been established as sufficient to induce hallmark features of BCC in both human and murine epidermis,1-4 pathways sufficient to convert normal epidermis into SCC have been less well defined. Building on findings that indicate a potent role for Ras and NF-κB in normal epidermal growth regulation,5-9 recent work indicates that activation of Ras signaling in concert with inhibition of NF-κB function is entirely sufficient to transform normal human epidermis into tumor tissue with all the cardinal features of SCC.
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Palo Alto Healthcare System; Palo Alto, California USA
2Program in Epithelial Biology; Stanford University School of Medicine; Stanford,
KEY WORDS
Using a limited number of defined genetic elements to drive primary human cells to a malignant phenotype that mimics spontaneously occurring cancer offers a number of attractive features. First, it has the potential to advance mechanistic understanding of the interplay between aberrantly regulated cellular signaling pathways in a fashion that is not readily accomplished with highly passaged cell lines. This advantage results from having direct experimental control over each of the multiple steps required to drive human tumorigenesis, a control that is absent in cell lines that have already commonly incorporated multiple adaptive changes that support proliferation. Additionally, while the bulk of the work on mammalian cellular growth regulation and neoplasia has been performed using murine cells, it is desirable to perform mechanistic studies in the more biomedically relevant human tissue context. The importance of human tissue models has been further underscored the observation that murine and human oncoproteins can engage different downstream signaling effector pathways.10 Furthermore, it is well known that human tissues often differ significantly from their murine counterparts in terms of architecture, metabolism and DNA repair. For example, human skin displays a significantly thicker epidermis than mouse. Moreover, primary human cells are generally much more resistant to malignant transformation. Consistent with this, photocarcinogenesis experiments of human skin grafts on immune deficient mice demonstrate a far greater ease of induction of epidermal neoplasia in surrounding murine skin compared to centrally placed human skin graft tissue.11 These observations have highlighted the need for human tissue-based studies. To study cancer development in a more medically relevant setting, recent studies have focused on human cells. Using such tissues as kidney and breast, investigators have introduced a number of functional alterations to transform cells into neoplasms,12-14 including:
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JNK IκKs
squamous cell carcinoma basal cell carcinoma phosphotidylinositol-3 kinases Ral guanine nucleotide exchange factors c-jun N-terminal kinase IkB kinases
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ABBREVIATIONS
INDUCTION OF HUMAN TISSUE CARCINOGENESIS FROM PRIMARY CELLS
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skin, cancer, squamous cell carcinoma, Ras, NF-κB
SCC BCC PI3Ks RalGEFs
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ABSTRACT
Todd W. Ridky1 Paul A. Khavari2,* 1VA
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Spotlight on Human Cell Transformation
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Ras activation Rb inactivation p53 inactivation telomerase activation and protein phosphatase 2A inhibition
Interestingly, cells from different tissues responded differently in these experiments. Up until recently, the tissue responsible for the two most common cancers in the United States, cutaneous epidermis, had not been studied. Recent studies in skin have shown that simultaneous induction of Ras, via expression of oncogenic Ras, along with blockade of NF-κB, via expression of IκBα, is sufficient to convert normal human epidermal tissue into an invasive neoplasia indistinguishable from SCC. This tumorigenic phenotype is therefore produced with only two nonviral genetic elements that alter activity of two well known www.landesbioscience.com
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Ras proteins are membrane-bound GTPases that transmit signals from a number of cell surface growth factor and extracellular matrix receptors to the nucleus.15 They were initially identified in rat sarcoma viruses and were found to have cellular proto-oncogene homologs, H-Ras, N-Ras and K-Ras. Mammalian K-Ras exists in two splice variants, K-Ras4A and K-Ras4B. Ras proteins demonstrate functional redundancy from yeast to mammals15 and signal through at least three major downstream effector arms including Raf kinases, phosphotidylinositol-3 kinases (PI3Ks), and Ral guanine nucleotide exchange factors (RalGEFs). The relative contributions of these individual pathways and their functional consequences vary with cell and tissue type,16,17 with the most thoroughly studied pathway being the Raf/Mek/MAP kinase cascade. The contributions of these major Ras effector pathways are under study17 and their relative roles in epidermal homeostasis are unknown. In epidermal cells, controversy has existed regarding the effects of Ras and its Raf/MEK/ERK effectors. In part, this has been due to an inability thus far to delete epidermal Ras function genetically. In skin, there is no overt phenotype in HRAS-/-,18 NRAS-/-,19 HRAS-//NRAS-/- mice20 and KRAS-/-/NRAS-/- chimeras.21 Mice with disruption of all 3 RAS isoform genes have not been successfully produced due to embryonic lethality seen in KRAS-/- animals.21 The fact that both RAS isoform genes must be deleted in yeast in order to see a phenotype suggests that truly null Ras tissue in mammals may only be obtained by removing at least HRAS, NRAS and KRAS, and possibly additional RAS-related genes as well.15 Consistent with Ras redundancy, there is a compensatory increase in other Ras isoforms in H-Rasdeficient skin.18 In spite of the difficulties in obtaining genetic deletion of epidermal Ras function, recent efforts have further defined the effects of Ras loss-of-function in epidermis. Expression of dominantnegative Ras in the epidermis of transgenic mice induces terminal differentiation and drastically decreases basal cell proliferation, leading to a complete loss of epidermal self-renewal that results in widespread erosions and death.5 Although definitive proof still awaits genetic deletion or knockdown studies, these dominant-negative overexpression data suggest that Ras normally promotes epidermal proliferation and opposes differentiation. Consistent with a capacity for Ras in regulating epidermal homeostasis, studies using inducible gain-of-function Ras mutants in primary human keratinocyte in vitro and transgenic epidermis in vivo demonstrate that conditional activation of Ras signaling in epidermis stimulates growth and suppresses differentiation.5,7 Similar cell and tissue effects were seen with induction of the Ras effector, Raf, indicating that this downstream pathway is sufficient to mediate most of the observed Ras effects.7 While Ras induced marked tissue hyperplasia in post-natal adult epidermal tissues, it fails to induce invasive epidermal cancer, with all observed changes entirely reversible upon cessation of Ras activation.7 This stands in contrast to earlier work expressing oncogenic Ras in epidermis through embryogenesis, which led to changes consistent with SCC,22 however, these former studies may reflect impacts operative within specific windows of development as reported in the case of Myc in visceral tissues.23 This collection of complementary studies collectively indicate that Ras and Raf function may act in adult epidermis to support proliferative capacity and oppose differentiation, two effects of central importance in carcinogenesis.
NF-κB/Rel proteins comprise a conserved family of transcription factors implicated in regulating inflammation, morphogenesis, apoptosis, differentiation and cell growth, with overall effects highly dependent on cell context. In mammals there are 5 NF-κB subunits, RelA (p65), p50, p52, RelB and c-Rel.24 NF-κB gene regulatory proteins function as hetero- and homodimers, with RelA/p50 heterodimers the most abundantly detected in skin and other tissues.25,26 NF-κB signaling has long been known be directly controlled at several layers of upstream regulators, including IκBα proteins, which bind directly to NF-κB and inhibit signaling through cytoplasmic sequestration as well as the IκB kinases (IKKs), which negatively regulate IκB, thereby activating NF-κB. In some settings, NF-κB can serve as a growth promoting oncogene, while playing a growth inhibitory role in others. Overexpressing NF-κB subunits in epidermis leads to growth inhibition, while NF-κB blockade via IκBα overexpression or pharmacologic inhibitors leads to hyperplasia in both murine and human epidermis.27,28 These data suggest that the physiologic role of NF-κB in skin may be to act as an important anti-proliferative safeguard. It was therefore surprising that mice deficient in the 4 out of 5 NF-κB subunits initially examined lacked epidermis-intrinsic growth abnormalities.29-33 In addition, NF-κB appears to be uninvolved in IKK1-induced epidermal differentiation,27,34 suggesting that IKK effects are attributable to NF-κB-independent targets.35 However, NF-κB DNA binding activity remains inducible in primary keratinocytes isolated from both Ikk1-/- and Ikk2-/- mice in response to stimuli such as TNFα.36-38 Additionally, IKK-independent NF-κB target genes have been identified in cells deficient in IKKγ, IKK1 and IKK2.39 Therefore, the phenotypic changes in epidermis that result from the loss of individual upstream IKK subunits do not reflect complete impairment of downstream NF-κB function. Moreover, NF-κB proteins display considerable genetic redundancy24,26 and all 5 subunits are expressed in epidermis.8 Notably, homeostasis in adult epidermis deficient for the RelA/p65 NF-κB subunit had not been studied due to the embryonic lethality from hepatic apoptosis that is seen in RelA-/- mice,40 leaving open a potential role for RelA in this setting. Recently, RelA-/- epidermis has been shown to display cell autonomous hyperproliferation independent of altered differentiation or inflammation.9 This increased cell division is accompanied by induction of epidermal c-jun N-terminal kinase (JNK) signaling and requires TNFR1 because Tnfr1 deletion in this setting restored both proliferation and JNK levels to normal. The epidermal hyperplasia seen with NF-κB inhibition is blocked by pharmacologic and genetic interference with JNK activation indicating that RelA plays a nonredundant role in epidermal growth inhibition by opposing proliferative signals dependent on intact TNFR1 and JNK function.9 Epidermal hyperplasia due to inhibited NF-κB function respects normal tissue boundaries but, unlike the situation seen with Ras activation, it maintains typical differentiation patterns, indicating that significant additional changes are required for conversion to malignancy.
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RAS FUNCTION IN EPIDERMIS
NF-κB FUNCTION IN EPIDERMIS
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and ubiquitous cell signaling pathways. Recent work has shed light on the effects of these pathways in epidermal tissue and provided insight into their oncogenic potency in this setting.
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RAS, NF-κB AND EPIDERMAL TUMORIGENESIS Normal epidermis is a self-renewing tissue that maintains homeostasis via precise control of cellular proliferation. Epidermal progenitor cells possess enormous proliferative potential, which is engaged at a slow basal rate throughout an individual’s lifespan, with upregulated cell division kinetics occurring during wound healing and some inflammatory conditions. Benign and malignant neoplasias represent
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How does NF-κB blockade cooperate with Ras to induce epidermal neoplasia? While Ras can contribute to G1 progression, strong constitutive signaling from oncogenic Ras by itself causes irreversible growth inhibition. This cell cycle arrest is elicited by oncogenic Ras in many primary cell types and is believed to serve as a safeguard against Ras-driven neoplasia. In an effort to define the mechanism for this keratinocyte growth arrest, Ras effects on G1/S transition regulators were examined. In this setting, Ras induced previously identified targets, including cyclin D1 as well as the cyclin dependent kinase inhibitors p21CIP1, p16INK4A, and p15INK4B.46 Ras also augmented levels of hypo-phosphorylated Rb, which inhibits G1 progression. Expression of other proteins implicated in Ras cell cycle regulation in different settings, such as p14ARF, p27KIP1, and p53 were unchanged in epidermal cells. Unexpectedly, Ras decreased CDK4 protein levels without altering the other G1 kinases, CDK2 and CDK6. That CDK4 downregulation is itself the major component arresting cell cycle progression in the face of high levels of oncogenic Ras was demonstrated by CDK4 expression with oncogenic Ras, an intervention that bypassed Ras cell cycle arrest.46 Interestingly, IκBα expression blocked CDK4 suppression and permitted continued replication. CDK4 expression alone was found to support tumorigenesis in concert with oncogenic Ras, underscoring the importance of this effect of NF-κB blockade. Therefore, activation of the Ras pathway and inhibition of the NF-κB pathway are sufficient to achieve direct induction of epidermal SCC-like tumors in human tissue. NF-κB blockade in this setting maintains CDK4 protein levels in the face of signal transmission by oncogenic Ras that would normally suppress it and trigger G1 arrest. Of interest, Ras activates NF-κB in primary epidermal cells and NF-κB activation itself down-regulates CDK4.44 This finding suggests that oncogenic Ras activates a growth inhibitory safeguard mechanism involving NF-κB suppression of CDK4 protein, although definitive evidence linking these proteins in a direct sequential pathway requires further studies. A physiologically relevant model of human SCC has thus now been developed employing only two genetic elements from well-known cell signaling pathways involved in cell growth regulation. This model provides the opportunity to characterize genes of potential importance in SCC pathogenesis and to begin to test new approaches to molecular therapy for epidermal cancer.
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a disruption of the normal proliferative balance and likely engage many of the same pathways involved in physiologic proliferative responses. In light of data indicating potent effects for both NF-κB and Ras on epidermal growth and for Ras on differentiation, a logical question concerns whether these dominant regulators may play a role in facilitating epidermal cancer. Epidermal SCC is the second most common cancer in the United States, after epidermal BCC, with an annual incidence estimated 250,000.41 Activating mutations in RAS genes can occur in human42 and in experimental SCC induction in mice.43 However, it has been shown recently that the majority of spontaneous human SCCs display increased levels of active Ras and MAPK induction without mutations in H, N or K-RAS genes themselves.44 This suggests an analogous situation to that seen in breast cancer where RAS mutations are rarely seen but where Ras is activated due to a number of factors, including overexpression of upstream receptor tyrosine kinases. Consistent with a role for Ras in spontaneously occurring SCC, cDNA gene expression profiling demonstrates strong induction of known Ras target genes in human SCC patient tissue versus normal site-matched skin.45 These data point to Ras, or its downstream effectors, as a potentially important element in epidermal neoplasia. A potential role for NF-κB in epidermal neoplasia is less well studied. To date, there have been no systematic studies published searching for mutations in the NF-κB pathway in human SCC. There is, however, some evidence to indicate that NF-κB function is inhibited in spontaneous human epidermal SCC. First, the p65/ RelA NF-κB subunit was found to be retained in the cytoplasm, where it is inactive, in patient SCCs.44 Second, IκBα protein levels were found to be elevated in a majority of SCC specimens and levels of phosphorylated IκBα were decreased, consistent with decreased activation of NF-κB pathway function in SCC.44 Consistent with these human observations, sustained NF-κB inhibition in murine skin leads to epidermal tumors with features of SCC.28 Taken together, these observations are consistent with a role for both NF-κB inhibition and Ras activation in SCC, however, they do not establish that altered function of either gene plays a causative role in human SCC. Moreover, these data do not establish that Ras induction and NF-κB blockade in epidermis are oncogenic in their own right. Systematic analyses with larger human SCC sample sizes as well as further functional studies are needed to probe a potential role for Ras and NF-κB in epidermal carcinogenesis. Recent work has addressed the potential neoplasia-inducing impacts of activating Ras and blocking NF-κB in epidermis. Constitutive expression of oncogenic Ras in human epidermal cells leads to permanent cell cycle arrest. Concomitant expression of IκBα bypasses this arrest. Ras and IκBα coexpressing cells not only proliferate, they also form lethal tumors when administered subcutaneously, intravenously or by surface grafting to immune deficient mice.44 These tumors are histologically indistinguishable from common human SCC. Ras and IκBα-induced tumors also display protein expression changes characteristic of SCC, including decreased E-cadherin as well as increased VEGF and MMPs.44 Remarkably, human epidermal tumors triggered by Ras and IκBα coexpression occur rapidly, are polyclonal and lack evidence of genetic instability or genomic abnormalities. Unlike prior human transformation studies, these cells were not subjected to prolonged periods of potentially mutagenic drug selection in long-term cell culture but were used as primary cells directly after gene transfer. All of these observations argue for a direct transforming effect of these proteins in epidermis.
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