Jesús M. Paramio

Functional roles of Akt signaling in mouse skin tumorigenesis

The mouse skin carcinogenesis protocol is a unique model for understanding the molecular events leading to oncogenic transformation. Mutations in the Ha-ras gene, and the presence of functional cyclin D1 and the EGF receptor, have proven to be important in this system. However, the signal transduction pathways connecting these elements during mouse skin carcinogenesis are poorly understood. This paper studies the relevance of the Akt and ERK pathways in the different stages of chemically induced mouse skin tumors. Akt activity increases throughout the entire process, and its early activation is detected prior to increased cyclin D1 expression. ERK activity rises only during the later stages of malignant conversion. The observed early increase in Akt activity appears to be due to raised PI-3K activity. Other factors acting on Akt such as ILK activation and decreased PTEN phosphatase activity appear to be involved at the conversion stage. To further confirm the involvement of Akt in this process, PB keratinocytes were transfected with Akt and subsequently injected into nude mice. The expression of Akt accelerates tumorigenesis and contributes to increased malignancy of these keratinocytes as demonstrated by the rate of appearance, the growth and the histological characteristics of the tumors. Collectively, these data provide evidence that Akt activation is one of the key elements during the different steps of mouse skin tumorigenesis.

Modulation of cell proliferation by cytokeratins K10 and K16

ABSTRACT The members of the large keratin family of cytoskeletal proteins are expressed in a carefully regulated tissue- and differentiation-specific manner. Although these proteins are thought to be involved in imparting mechanical integrity to epithelial cells, the functional significance of their complex differential expression is still unclear. Here we provide new data suggesting that the expression of particular keratins may influence cell proliferation. Specifically, we demonstrate that the ectopic expression of K10 inhibits the proliferation of human keratinocytes in culture, while K16 expression appears to promote the proliferation of these cells. Other keratins, such as K13 or K14, do not significantly alter this parameter. K10-induced inhibition is reversed by the coexpression of K16 but not that of K14. These results are coherent with the observed expression pattern of these proteins in the epidermis: basal, proliferative keratinocytes express K14; when they terminally differentiate, keratinocytes switch off K14 and start K10 expression, whereas in response to hyperproliferative stimuli, K16 replaces K10. The characteristics of this process indicate that K10 and K16 act on the retinoblastoma (Rb) pathway, as (i) K10-induced inhibition is hampered by cotransfection with viral oncoproteins which interfere with pRb but not with p53; (ii) K10-mediated cell growth arrest is rescued by the coexpression of specific cyclins, cyclin-dependent kinases (CDKs), or cyclin-CDK complexes; (iii) K10-induced inhibition does not take place in Rb-deficient cells but is restored in these cells by cotransfection with pRb or p107 but not p130; (iv) K16 efficiently rescues the cell growth arrest induced by pRb in HaCaT cells but not that induced by p107 or p130; and (v) pRb phosphorylation and cyclin D1 expression are reduced in K10-transfected cells and increased in K16-transfected cells. Finally, using K10 deletion mutants, we map this inhibitory function to the nonhelical terminal domains of K10, hypervariable regions in which keratin-specific functions are thought to reside, and demonstrate that the presence of one of these domains is sufficient to promote cell growth arrest.

Unique and overlapping functions of pRb and p107 in the control of proliferation and differentiation in epidermis.

The retinoblastoma gene product, pRb, plays a crucial role in cell cycle regulation, differentiation and inhibition of oncogenic transformation. pRb and its closely related family members p107 and p130 perform exclusive and overlapping functions during mouse development. The embryonic lethality of Rb-null animals restricts the phenotypic analysis of these mice to mid-gestation embryogenesis. We employed the Cre/loxP system to study the function of Rb in adult mouse stratified epithelium. RbF19/F19;K14cre mice displayed hyperplasia and hyperkeratosis in the epidermis with increased proliferation and aberrant expression of differentiation markers. In vitro, pRb is essential for the maintainance of the postmitotic state of terminally differentiated keratinocytes, preventing cell cycle re-entry. However, p107 compensates for the effects of Rb loss as the phenotypic abnormalities of RbF19/F19;K14cre keratinocytes in vivo and in vitro become more severe with the concurrent loss of p107 alleles. p107 alone appears to be dispensable for all these phenotypic changes, as the presence of a single Rb allele in a p107-null background rescues all these alterations. Luciferase reporter experiments indicate that these phenotypic alterations might be mediated by increased E2F activity. Our findings support a model in which pRb in conjunction with p107 plays a central role in regulating epidermal homeostasis.

Inhibition of Protein Kinase B (PKB) and PKCζ Mediates Keratin K10-Induced Cell Cycle Arrest

ABSTRACT The intermediate filament cytoskeleton is composed of keratins in all epithelial cells and imparts mechanical integrity to these cells. However, beyond this shared function, the functional significance of the carefully regulated tissue- and differentiation-specific expression of the large keratin family of cytoskeletal proteins remains unclear. We recently demonstrated that expression of keratin K10 or K16 may regulate the phosphorylation of the retinoblastoma protein (pRb), inhibiting (K10) or stimulating (K16) cell proliferation (J. M. Paramio, M. L. Casanova, C. Segrelles, S. Mittnacht, E. B. Lane, and J. L. Jorcano, Mol. Cell. Biol. 19:3086–3094, 1999). Here we show that keratin K10 function as a negative modulator of cell cycle progression involves changes in the phosphoinositide 3-kinase (PI-3K) signal transduction pathway. Physical interaction of K10 with Akt (protein kinase B [PKB]) and atypical PKCζ causes sequestration of these kinases within the cytoskeleton and inhibits their intracellular translocation. As a consequence, the expression of K10 impairs the activation of PKB and PKCζ. We also demonstrate that this inhibition impedes pRb phosphorylation and reduces the expression of cyclins D1 and E. Functional and biochemical data also demonstrate that the interaction between K10 and these kinases involves the non-α-helical amino domain of K10 (NTerm). Together, these results suggest new and essential roles for the keratins as modulators of specific signal transduction pathways.

PTEN tumour suppressor is linked to the cell cycle control through the retinoblastoma protein.

The tumour suppressor PTEN, also named MMAC1 or TEP1, is associated with a number of malignancies in human populations. This protein has a dual protein phosphatase activity, being also capable to dephosphorylate phosphatidylinositol 3,4,5 triphosphate. We have studied the mechanism of growth suppression attributable to PTEN. We observed that PTEN overexpression inhibits cell growth in a variety of normal and transformed, human and murine cells. Bromodeoxyuridine (BrdU) incorporation and TUNEL labelling experiments in transiently transfected cells demonstrate that this inhibition is due to a cell cycle arrest rather than induction of apoptosis. Given that PTEN is unable to cause cell growth arrest in retinoblastoma (Rb)-deficient cell lines, we have explored the possible requirement for pRb in the PTEN-induced inhibition of cell proliferation. We found that the co-expression of SV40 antigen, but not a mutant form (which binds exclusively to p53), and cyclin D1/cdk4 are able to overcome the PTEN-mediated growth suppression. In addition, the reintroduction of a functional pRb, but not its relatives p107 or p130, in Rb-deficient cells restores the sensitivity to PTEN-induced arrest. Finally, the hyperphosphorylation of transfected pRb is inhibited by PTEN co-expression and restored by PI-3K co-expression. Accordingly, PTEN gene is mostly expressed, in parallel to Akt, in mid-late G1 phase during cell cycle progression prior to pRb hyperphosphorylation. Finally, we have studied the signal transduction pathways modulated by PTEN expression. We found that PTEN-induced growth arrest can be rescued by the co-expression of active PI-3K and downstream effectors such as Akt or PDK1, and also certain small GTPases such as Rac1 and Cdc42, but not by active Ha-ras, raf or RhoA. Collectively, our data link the tumour suppressor activities of PTEN to the machinery controlling cell cycle through the modulation of signalling molecules whose final target is the functional inactivation of the retinoblastoma gene product.

Thyroid Hormone Receptor β1 Acts as a Potent Suppressor of Tumor Invasiveness and Metastasis

Loss of thyroid hormone receptors (TR) is a common feature in some tumors, although their role in tumor progression is currently unknown. We show here that expression of TRbeta1 in hepatocarcinoma and breast cancer cells reduces tumor growth, causes partial mesenchymal-to-epithelial cell transition, and has a striking inhibitory effect on invasiveness, extravasation, and metastasis formation in mice. In cultured cells, TRbeta1 abolishes anchorage-independent growth and migration, blocks responses to epidermal growth factor, insulin-like growth factor-I, and transforming growth factor beta, and regulates expression of genes that play a key role in tumorigenicity and metastatic growth. The receptor disrupts the mitogenic action of growth factors by suppressing activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling pathways that are crucial for cell proliferation and invasiveness. Furthermore, increased aggressiveness of skin tumors is found in genetically modified mice lacking TRs, further demonstrating the role of these receptors as inhibitors of tumor progression. These results define a novel role for the thyroid hormone receptor as a metastasis suppressor gene, providing a starting point for the development of novel therapeutic strategies for the treatment of human cancer.

Deficiency in p53 but not Retinoblastoma Induces the Transformation of Mesenchymal Stem Cells In vitro and Initiates Leiomyosarcoma In vivo

Sarcomas have been modeled in mice by the expression of specific fusion genes in mesenchymal stem cells (MSC), supporting the concept that MSCs might be the target initiating cell in sarcoma. In this study, we evaluated the potential oncogenic effects of p53 and/or retinoblastoma (Rb) deficiency in MSC transformation and sarcomagenesis. We derived wild-type, p53(-/-), Rb(-/-), and p53(-/-)Rb(-/-) MSC cultures and fully characterized their in vitro growth properties and in vivo tumorigenesis capabilities. In contrast with wild-type MSCs, Rb(-/-), p53(-/-), and p53(-/-)Rb(-/-) MSCs underwent in vitro transformation and showed severe alterations in culture homeostasis. More importantly, p53(-/-) and p53(-/-)Rb(-/-) MSCs, but not Rb(-/-) MSCs, were capable of tumor development in vivo after injection into immunodeficient mice. p53(-/-) or p53(-/-)Rb(-/-) MSCs originated leiomyosarcoma-like tumors, linking this type of smooth muscle sarcoma to p53 deficiency in fat tissue-derived MSCs. Sca1+ and Sca1 low/- cell populations isolated from ex vivo-established, transformed MSC lines from p53(-/-)Rb(-/-) tumors showed identical sarcomagenesis potential, with 100% tumor penetrance and identical latency, tumor weight, and histologic profile. Our findings define the differential roles of p53 and Rb in MSC transformation and offer proof-of-principle that MSCs could provide useful tools to dissect the sarcoma pathogenesis.

Molecular determinants of Akt-induced keratinocyte transformation

The PI3K/PTEN/Akt signaling pathway has emerged in recent years as a main player in human cancers, increasing proliferation and decreasing apoptosis of transformed cells, and thus becoming a potential target for therapeutic intervention. Our previous data have demonstrated that Akt-mediated signaling is of a key relevance in the mouse skin carcinogenesis system, one of the best-known models of experimental carcinogenesis. Here, we investigated the involvement of several pathways as mediators of Akt-induced increased proliferation and tumorigenesis in keratinocytes. Tumors produced by subcutaneous injection of Akt-transformed keratinocytes showed increased Foxo3a phosphorylation, but no major alterations in p21Cip1/WAF1, p27Kip1 or mdm2 expression and/or localization. In contrast, we found increased expression and nuclear localization of ΔNp63, β-catenin and Lef1. Concomitantly, we also found increased expression of c-myc and CycD1, targets of the β-catenin/Tcf pathway. Such increase is associated with increased phosphorylation and stabilization of c-myc protein as well as increased translation of c-myc and CycD1 due to mTOR activation. Using immunohistochemistry approaches in samples of oral dysplasias and human head and neck squamous cell carcinomas, we confirmed that increased Akt activation significantly correlates with increased ΔNp63 and CycD expression, c-myc phosphorylation and nuclear accumulation of β-catenin. Collectively, these results demonstrate that Akt is able to transform keratinocytes by specific mechanisms involving transcriptional and post-transcriptional processes.

Deregulated Activity of Akt in Epithelial Basal Cells Induces Spontaneous Tumors and Heightened Sensitivity to Skin Carcinogenesis

Aberrant activation of the phosphoinositide-3-kinase (PI3K)/PTEN/Akt pathway, leading to increased proliferation and decreased apoptosis, has been implicated in several human pathologies including cancer. Our previous data have shown that Akt-mediated signaling is an essential mediator in the mouse skin carcinogenesis system during both the tumor promotion and progression stages. In addition, overexpression of Akt is also able to transform keratinocytes through transcriptional and posttranscriptional processes. Here, we report the consequences of the increased expression of Akt1 (wtAkt) or constitutively active Akt1 (myrAkt) in the basal layer of stratified epithelia using the bovine keratin K5 promoter. These mice display alterations in epidermal proliferation and differentiation. In addition, transgenic mice with the highest levels of Akt expression developed spontaneous epithelial tumors in multiple organs with age. Furthermore, both wtAkt and myrAkt transgenic lines displayed heightened sensitivity to the epidermal proliferative effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and heightened sensitivity to two-stage skin carcinogenesis. Finally, enhanced susceptibility to two-stage carcinogenesis correlated with a more sustained proliferative response following treatment with TPA as well as sustained alterations in Akt downstream signaling pathways and elevations in cell cycle regulatory proteins. Collectively, the data provide direct support for an important role for Akt signaling in epithelial carcinogenesis in vivo, especially during the tumor promotion stage.

C/EBPalpha and beta couple interfollicular keratinocyte proliferation arrest to commitment and terminal differentiation

The transcriptional regulators that couple interfollicular basal keratinocyte proliferation arrest to commitment and differentiation are yet to be identified. Here we report that the basic region leucine zipper transcription factors C/EBPα and C/EBPβ are co-expressed in basal keratinocytes, and are coordinately upregulated as keratinocytes exit the basal layer and undergo terminal differentiation. Mice lacking both C/EBPα and β in the epidermis showed increased proliferation of basal keratinocytes and impaired commitment to differentiation. This led to ectopic expression of keratin 14 (K14) and ΔNp63 in suprabasal cells, decreased expression of spinous and granular layer proteins, parakeratosis and defective epidermal water barrier function. Knock-in mutagenesis revealed that C/EBP-E2F interaction was required for control of interfollicular epidermis (IFE) keratinocyte proliferation, but not for induction of spinous and granular layer markers, whereas C/EBP DNA binding was required for ΔNp63 downregulation and K1/K10 induction. Finally, loss of C/EBPα/β induced stem cell gene expression signatures in the epidermis. C/EBPs, therefore, couple basal keratinocyte cell cycle exit to commitment to differentiation through E2F repression and DNA binding, respectively, and may act to restrict the epidermal stem cell compartment.