Carmen Segrelles

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.

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.

Differential expression and functionally co-operative roles for the retinoblastoma family of proteins in epidermal differentiation.

Terminal differentiation requires cell cycle withdrawal, suggesting the involvement of negative cell cycle controllers in the process. We have analysed the involvement of the retinoblastoma family of proteins (pRb, p107 and p130) in epidermal proliferation and differentiation. These proteins play key roles as inhibitors of cell cycle progression and are involved in muscle and neuron differentiation. We found that during in vitro differentiation of human HaCaT keratinocytes, pRb, p107 and p130 are sequentially expressed, in contrast to the co-expression observed during cell cycle progression in the same cells. Immunofluorescence studies on skin sections revealed the presence of pRb and p107 in basal and suprabasal cell layers, whilst p130 is restricted to cells already committed to differentiation in the suprabasal compartments. To explore the functional significance of the differential expression of these proteins, transfection experiments were performed in HaCaT keratinocytes. We observed that the forced over-expression of pRb, p107 or p130 individually did not induce differentiation of the transfected cells. However, the co-transfection of pRb and p107 induced the expression of early differentiation markers (keratin k10) and triple transfectants pRb+p107+p130 expressed markers representative of later stages of epidermal differentiation (involucrin). Finally, we observed that these three proteins repress keratinocyte proliferation, although to a different extent (p107>pRb⩾p130). These results indicate that the members of the pRb family play specific, yet co-ordinated roles during epidermal differentiation, and that the ordered progression along the different stages of this process results from the effects of different combinations of these proteins.

Expression, localization, and activity of glycogen synthase kinase 3β during mouse skin tumorigenesis

Glycogen synthase kinase 3 (GSK‐3) is a protein kinase that plays essential roles in the control of several developmental, metabolic, and apoptotic processes. Owing to its negative actions on several oncogenic insults, it has been considered a putative functional tumor suppressor. We studied the expression, activity, and localization of GSK‐3β during the process of chemically induced two‐stage mouse skin carcinogenesis and also in the tumors generated upon subcutaneous injection of Akt‐transformed keratinocytes. We found that GSK‐3 activity was downregulated at the later stages of promotion by tyrosine 216 dephosphorylation and serine 9 phosphorylation. The data obtained with Akt‐transformed keratinocytes clearly suggested the involvement of Akt in serine 9 phosphorylation of GSK‐3β. Finally, besides functional inactivation, significant basal activity of GSK‐3β was detected in all cases, indicating that this enzyme provides essential functions to malignant keratinocytes.

Abnormal epidermal differentiation and impaired epithelial-mesenchymal tissue interactions in mice lacking the retinoblastoma relatives p107 and p130

The functions of p107 and p130, members of the retinoblastoma family, include the control of cell cycle progression and differentiation in several tissues. Our previous studies suggested a role for p107 and p130 in keratinocyte differentiation in vitro. We now extend these data using knockout animal models. We found impaired terminal differentiation in the interfollicular keratinocytes of p107/p130-double-null mice epidermis. In addition, we observed a decreased number of hair follicles and a clear developmental delay in hair, whiskers and tooth germs. Skin grafts of p107/p130-deficient epidermis onto NOD/scid mice showed altered differentiation and hyperproliferation of the interfollicular keratinocytes, thus demonstrating that the absence of p107 and p130 results in the deficient control of differentiation in keratinocytes in a cell-autonomous manner. Besides normal hair formation, follicular cysts, misoriented and dysplastic follicles, together with aberrant hair cycling, were also observed in the p107/p130 skin transplants. Finally, the hair abnormalities in p107/p130-null skin were associated with altered Bmp4-dependent signaling including decreasedΔ Np63 expression. These results indicate an essential role for p107 and p130 in the epithelial-mesenchimal interactions.

EMT and induction of miR-21 mediate metastasis development in Trp53-deficient tumours

Missense mutations in TP53 gene promote metastasis in human tumours. However, little is known about the complete loss of function of p53 in tumour metastasis. Here we show that squamous cell carcinomas generated by the specific ablation of Trp53 gene in mouse epidermis are highly metastatic. Biochemical and genome-wide mRNA and miRNA analyses demonstrated that metastases are associated with the early induction of epithelial-mesenchymal transition (EMT) and deregulated miRNA expression in primary tumours. Increased expression of miR-21 was observed in undifferentiated, prometastatic mouse tumours and in human tumours characterized by p53 mutations and distant metastasis. The augmented expression of miR-21, mediated by active mTOR and Stat3 signalling, conferred increased invasive properties to mouse keratinocytes in vitro and in vivo, whereas blockade of miR-21 in a metastatic spindle cell line inhibits metastasis development. Collectively these data identify novel molecular mechanisms leading to metastasis in vivo originated by p53 loss in epithelia.