Analysis of exogenous over-expression of 14-3-3σ/Stratifin in transgenic mouse skin carcinogenesis

Cutaneous squamous cell carcinoma is the second most common skin cancer and the most commonly diagnosed cancer capable of metastasis diagnosed in Caucasians. While many are surgically curable, survival rates for those which metastasise to regional lymph nodes and beyond are dismal in comparison, with 5-year-survival estimates averaging around 30%. There is a clear need, therefore, to establish better prognostic markers and systemic therapeutics to avoid or treat metastatic disease. While chemical carcinogenesis of mouse skin has been the mainstay of cSCC research for decades, this process introduces a raft of mutations which are not integral to cancer development and the papillomas generated have a low rate of conversion; therefore, transgenic models mimicking the initiation and promotion steps have been developed to study specific pathways. Using a modified human Keratin 1 (HK1) promoter, oncogenes H-Ras and Fos have been targeted to the epidermis to study their involvement in initiation and promotion and, together, generated aggressive yet benign papillomas. Further work found that induced ablation of the Pten tumour suppressor, which results in deregulation of the PI3K/Akt pathway, reliably caused conversion to well-differentiated SCC in this HK1.ras/fos-Δ5Pten model, marked by loss of the major TSG, p53, due to upregulating of its main inhibitor, Mdm2. Thus, the first area of study investigated the p53-Mdm2 interaction, which identified the chaperone protein 14-3-3σ, known as Stratifin, as a positive regulator of p53. Stratifin was found to persist after loss of p53 in HK1.ras/fos-Δ5Pten wdSCCs, alongside cell cycle regulator p21, but was lost as tumours converted to poorly-differentiated SCC. This apparent tumour suppressor role fit with much of the literature which described its functions in differentiation and cell cycle arrest, as well as its role in protecting p53. In light of this, a transgenic mouse model was obtained which overexpressed Stratifin in the skin and hair follicles using a Keratin 14 promoter (K14.stratifin), with the intention of suppressing tumour conversion in the HK1.ras/fos-Δ5Pten model of carcinogenesis, in part via p53 protection. The first step introduced the K14.stratifin transgene into this multistage model and, unexpectedly, initial HK1.fos/K14.stratifin mice developed rapidly growing keratotic tumours on the ears which were found to convert to malignancy at an early stage. Detailed analysis found that these tumours seem to recapitulate an under-recognised subtype of cutaneous SCC arising from the hair follicle (HF) cells; fSCC. Further investigating discovered that mice harbouring the K14.stratifin transgene aberrantly express murine K1 in the HF, indicating that these fSCCs were possible due to spurious HK1.fos expression in these cells alongside elevated Stratifin, becoming the first model to directly link Stratifin and Fos deregulation in malignancy. Moreover, despite strong expression of p53 in these tumours, ablation of the p53 gene did not have any observable effect on this model indicating a p53-independent Stratifin pathway is responsible for the fSCC tumour aetiology. This model may be the first to implicate Stratifin and Fos in the aetiology of fSCC and, once validated in humans, may provide greater information on this poorly understood SCC variant. Further to this, K14.stratifin was then co-expressed with HK1.ras, which alone produces benign wound-dependent papillomas. Here, two very distinct tumour types were observed: Type 1 tumours were wound-dependent and slow growing, though eventually developed areas of carcinoma and invasion, while Type 2 tumours did not require ongoing wound-promotion and grew rapidly, converting to SCC in under 2 weeks from inception. Analysis of p53 again showed strong expression even in clearly malignant tumours of both types, while its downstream effector, p21, appeared to be strongly active in the nuclei of Type 1 tumours, yet confined to the cytoplasm in Type 2s. Ablation of p53 reconfirmed an odd “p53 paradox” previously reported in HK1.ras mice, in which tumours fail to form if p53 is ablated prior to growth of the papilloma. Here, however, K14.ras.p53flx/flx.stratifin mice developed inflamed hyperplasia with clear signs of localised invasion, while K14.ras.p53flx/flx controls appeared grossly and histologically normal, indicating the cancer-promotion function of Stratifin overexpression despite no overt tumour development. To observe the apparent oncogenic effects of K14.stratifin expression on a p53-null background in the absence of other known oncogenic activation, K14.p53flx/flx.stratifin mice were generated. Wound-promoted skin appeared grossly similar to control mice lacking Stratifin overexpression; however, histological analysis at >4 months found hyperchromatic nuclei and areas of invasion suggestive of early malignancy, again, without precursor benign tumour development. Similarly, when K14.stratifin was subsequently expressed on a Pten-null background, its inclusion clearly exacerbated the Cowden Syndrome-like K14.Ptenflx/flx phenotype, with much more pronounced inflamed hyperplasia, hyperkeratosis, and hair defects present. Moreover, wound-promoted ear skin in these K14.Ptenflx/flx.stratifin mice showed clear evidence of carcinogenesis and cell invasion on histological examination. Again, this did not involve a benign tumour intermediary, further indicating that Stratifin acts as an oncogenic agent which is involved in producing aggressive, invasive carcinoma directly, not requiring development of an overt tumour prior to malignant conversion; hence the poor prognosis reported in internal tumours which overexpress Stratifin. On a dual p53-null/Pten-null background, Stratifin overexpression caused development of small wound-independent tumours requiring biopsy at an early stage, unlike the p53- and Pten-null backgrounds alone. These were found to have varied histology, though all appeared to involve HF aberration and possible involvement of HF stem cells in tumour generation, which requires confirmation. Wound-dependent tumours did not form at the ear tag of these mice in the relatively short time prior to sacrifice. Thus, the wound-independent tumours may have acquired further genetic lesions or have been linked to the cycling of the HFs, which occurs differently in ear skin. Finally, keratinocytes were isolated from transgenic pups expressing HK1.fos and K14.stratifin, alone and together, to examine the phenotypes of primary cell cultures and assess their malignant potential. Here, it was found that primary HK1.fos, K14.stratifin, and HK1.fos/K14.stratifin cultures did not grow from clonal density and were susceptible to calcium-induced differentiation. Cell lines derived from these initial cultures were subjected to the same tests and while HK1.fos and K14.stratifin lines still responded to increased Ca2+ concentrations, several HK1.fos/K14.stratifin lines exhibited a much-reduced differentiation response. One such line, FS2, was also able to grow from clonal density in both low and high Ca2+ medium, though the other, FS3m, which exhibited an unusual morphology somewhat akin to spinous layer cells in low Ca2+, was unable to form colonies at this low density even in low Ca2+ conditions. Western blot analysis found that these lines expressed approx. 50- and 12-fold more Stratifin than normal, respectively, thus, their different phenotypes and behaviours may be directly linked to the level of Stratifin present, as its functions are highly context-dependent. Stratifin overexpression was also found to be linked to increased migratory potential, with monogenic K14.stratifin cells closing the gap in a scratch assay experiment almost twice as fast as the next line, FS2, while HK1.fos and normal ICR cells took far longer. These data reflect the observations in vivo and so the lines generated may be useful in helping to elucidate protein interactions and pathways underlying the fSCC phenotype. In summary, this model not only clearly demonstrates the oncogenic activity of Stratifin overexpression—as opposed to a tumour suppressive role—but also appears to be the first to show direct co-operation between Fos or Ras activation and Stratifin overexpression in cutaneous carcinogenesis. In the case of Fos and Stratifin, their co-operation appears to specifically mimic the aetiology of rare human fSCC. Given the plethora of mutations observable in SCC aetiology, it may be that Stratifin overexpression in combination with other parameters could become a useful biomarker of tumour prognosis and final tumour outcome also applicable to diverse carcinoma types, not just cutaneous SCC

14-3-3σ/Stratifin and p21 limit AKT-related malignant progression in skin carcinogenesis following MDM2-associated p53 loss

To study mechanisms driving/inhibiting skin carcinogenesis, stage-specific expression of 14-3-3σ (Stratifin) was analyzed in skin carcinogenesis driven by activated rasHa/fos expression (HK1.ras/fos) and ablation of PTEN-mediated AKT regulation (K14.creP/Δ5PTENflx/flx). Consistent with 14-3-3σ roles in epidermal differentiation, HK1.ras hyperplasia and papillomas displayed elevated 14-3-3σ expression in supra-basal keratinocytes, paralleled by supra-basal p-MDM2166 activation and sporadic p-AKT473 expression. In bi-genic HK1.fos/Δ5PTENflx/flx hyperplasia, basal-layer 14-3-3σ expression appeared, and alongside p53/p21, was associated with keratinocyte differentiation and keratoacanthoma etiology. Tri-genic HK1.ras/fos-Δ5PTENflx/flx hyperplasia/papillomas initially displayed increased basal-layer 14-3-3σ, suggesting attempts to maintain supra-basal p-MDM2166 and protect basal-layer p53. However, HK1.ras/fos-Δ5PTENflx/flx papillomas exhibited increasing basal-layer p-MDM2166 activation that reduced p53, which coincided with malignant conversion. Despite p53 loss, 14-3-3σ expression persisted in well-differentiated squamous cell carcinomas (wdSCCs) and alongside elevated p21, limited malignant progression via inhibiting p-AKT1473 expression; until 14-3-3σ/p21 loss facilitated progression to aggressive SCC exhibiting uniform p-AKT1473. Analysis of TPA-promoted HK1.ras-Δ5PTENflx/flx mouse skin, demonstrated early loss of 14-3-3σ/p53/p21 in hyperplasia and papillomas, with increased p-MDM2166/p-AKT1473 that resulted in rapid malignant conversion and progression to poorly differentiated SCC. In 2D/3D cultures, membranous 14-3-3σ expression observed in normal HaCaT and SP1ras61 papilloma keratinocytes was unexpectedly detected in malignant T52ras61/v-fos SCC cells cultured in monolayers, but not invasive 3D-cells. Collectively, these data suggest 14-3-3σ/Stratifin exerts suppressive roles in papillomatogenesis via MDM2/p53-dependent mechanisms; while persistent p53-independent expression in early wdSCC may involve p21-mediated AKT1 inhibition to limit malignant progression

Discovery of potent and selective MRCK inhibitors with therapeutic effect on skin cancer

The myotonic dystrophy-related Cdc42-binding kinases MRCKα and MRCKβ contribute to the regulation of actin-myosin cytoskeleton organization and dynamics, acting in concert with the Rho-associated coiled-coil kinases ROCK1 and ROCK2. The absence of highly potent and selective MRCK inhibitors has resulted in relatively little knowledge of the potential roles of these kinases in cancer. Here we report the discovery of the azaindole compounds BDP8900 and BDP9066 as potent and selective MRCK inhibitors that reduce substrate phosphorylation, leading to morphological changes in cancer cells along with inhibition of their motility and invasive character. In over 750 human cancer cell lines tested, BDP8900 and BDP9066 displayed consistent anti-proliferative effects with greatest activity in hematological cancer cells. Mass spectrometry identified MRCKα S1003 as an autophosphorylation site, enabling development of a phosphorylation-sensitive antibody tool to report on MRCKα status in tumor specimens. In a two-stage chemical carcinogenesis model of murine squamous cell carcinoma, topical treatments reduced MRCKα S1003 autophosphorylation and skin papilloma outgrowth. In parallel work, we validated a phospho-selective antibody with the capability to monitor drug pharmacodynamics. Taken together, our findings establish an important oncogenic role for MRCK in cancer, and they offer an initial preclinical proof of concept for MRCK inhibition as a valid therapeutic strategy

Transgenic epidermal 14-3-3σ/stratifin overexpression cooperates with Pten ablation to give a limited carcinoma in situ phenotype

PTEN is a major tumour suppressor gene that negatively regulates the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway. The phenotypes associated with PTEN mutation are dose dependent, i.e. mutant allele heterozygosity (PTEN+/–) results in a less severe phenotype than full ablation (PTEN–/–). In the skin, loss of PTEN results in Cowden disease, which has been recapitulated in mice by ablation of exon 5, which encodes the core catalytic motif responsible for inhibition of AKT activation. This was achieved using an inducible Cre-loxP system driven by a K14 promoter, generating a Ptenflx/flx genotype, which resulted in mild hyperplasia, dysplasia and hyperkeratosis. These mice also develop a noticeably ‘woolly’ appearance to the fur, likely due to impaired follicle development. However, alone, the K14.cre/Ptenflx/flx genotype did not result in overt neoplasia for over 12 months. Another important molecule in the epidermis is 14-3-3σ/stratifin, an adapter protein named for its importance in the development of stratified epithelia. Among the many functions of the 14-3-3 superfamily, stratifin is associated with keratinocyte spatial awareness; roles in epidermal differentiation and proliferation; and is well known to be induced by elevated p53 levels following DNA damage, thus inhibiting Mdm2 in a positive feedback loop. 14-3-3σ/stratifin has been shown to possess tumour-suppressive functions such as direct inhibition of oncogenic AKT activities, hence the study with PTEN. Conversely, studies of human carcinomas have found elevated 14-3-3σ/stratifin in malignant squamous cell carcinoma relative to normal adjacent tissues, thereby suggesting possible oncogenic activity. Supporting this idea previously, in conjunction with overexpression of oncogenes Fos and H-Ras, overexpression of stratifin via a K14 promoter (K14.stratifin) resulted in novel carcinomas with several distinctive features – some suggesting a follicular origin. Given this latter observation, here K14.stratifin was expressed concurrently with partial (Ptenwt/flx) and full (Ptenflx/flx) Pten ablation. Preliminary results showed that the gross and histological phenotypes of the K14.stratifin/Ptenflx/flx mice were exacerbated by elevated stratifin expression. While the severity of the induced phenotype differed slightly between mice, histological analysis suggests that carcinoma in situ frequently developed, with evidence of local invasion. These initial results suggest that, as in previously demonstrated models, overexpression of epidermal stratifin results in oncogenic activity rather than rescue of the Cowden phenotype

Inducible 14-3-3 Sigma/Stratifin Ablation Accelerates Malignant Progression in HK1.ras/fos-Δ5PTENflx Transgenic Mouse Skin Carcinogenesis

In human carcinogenesis, given the plethora of pathways associated with 14-3-3σ and their complex interactions, the causality of 14-3-3σ deregulation remains elusive, with both tumour suppressive and oncogenic roles. To study mechanisms driving/inhibiting skin carcinogenesis, stage-specific expression of endogenous 14-3-3σ (stratifin) was analysed in skin carcinogenesis driven by activated rasHa/fos expression (HK1.ras/fos) and ablation of phosphatase and tensin homolog (PTEN)-mediated Akt regulation (K14.creP/Δ5PTENflx) and via conditional RU486-inducible 14-3-3σ knockout (K14.creP-Δ14-3-3). Consistent with 14-3-3σ roles in the commitment of keratinocytes to differentiate, bigenic HK1.fos/Δ5PTENflx hyperplasia expressed 14-3-3σ in basal layers and paralleled keratin K1 expression which appeared alongside elevated p53/p21 to increase keratinocyte differentiation leading to a keratoacanthoma (KA) aetiology. Trigenic HK1.ras/fos-Δ5PTENflx hyperplasia/papillomas also displayed increased basal-layer 14-3-3σ, suggesting attempts to protect basal layer p53 tumour suppressor gene function, given that 14-3-3σ acts as a chaperone protein to remove/relocate MDM2 for degradation, thus maintaining p53 levels. With time, HK1.ras/fos-Δ5PTENflx papillomas exhibited reduced p53 and increased p-MDM2166 activity in basal layers, which coincided with malignant conversion. Surprisingly, despite this p53 loss, 14-3-3σ expression persisted in well-differentiated squamous cell carcinomas (wdSCC) and, alongside elevated p21, appeared to limit further malignant progression via inhibiting p-Akt1473 expression. To further assess causality, 14-3-3σ functions were ablated in this model via an RU486-inducible cre/loxP system expressed from a keratin 14 (K14) promoter (K14.creP-Δ14-3-3σ). In bigenic HK1.fos-Δ14-3-3σ, preliminary data suggest loss of 14-3-3σ functions resulted in increased hyperplasia and keratosis similar to that observed in bigenic HK1.fos/Δ5PTENflx hyperplasia. Functional 14-3-3σ ablation in HK1.ras.fos/Δ5PTENflx/Δ14-3-3σflx genotypes apparently failed to accelerate papillomatogenesis, suggesting elements of redundancy in terms of tumour promotion; however, loss of 14-3-3σ facilitated increased p-MDM2166 and p53 loss, resulting in malignant conversion with progression to aggressive SCC. Collectively, these data suggest that 14-3-3σ/stratifin exerts suppressive roles in papillomatogenesis via MDM2/p53-dependent mechanisms, while persistent expression in wdSCCs suggests p53-independent scenarios that may involve p21-mediated Akt1 inhibition. However, if ablated, this limit of early-stage malignant progression is lost and 14-3-3σ loss leads to rapid progression to aggressive, p21-negative SCCs exhibiting uniform p-Akt1473activation

E-cadherin loss cooperates with rasHa activation to drivemalignant progression in transgenic mouse skincarcinogenesis

To investigate the stage at which E-cadherin loss may drive carcinogenesis, E-cadherin expression has been investigated in mouse skin carcinogenesis driven by ras and fos activation (HK1.ras; HK1.fos), and conditional (cre/lox) PTEN knockout exclusively in the epidermis. Analysis of endogenous E-cadherin expression in stage-specific HK1.ras/fos/Δ5PTEN tumours found increased levels in papilloma basal layer keratinocytes, but following p53 loss and subsequent malignant conversion, E-cadherin expression became reduced at the invasive front of resultant, well-differentiated, squamous cell carcinomas (wdSCCs). To validate the causality of this E-cadherin loss observed in malignant progression, RU486-inducible, conditional E-cadherin knockout was introduced into regression-prone papillomas exhibited by HK1.ras mice, employing the cre/LoxP system (K14cre.ΔE-cadflx/flx). Initial observations in RU486-treated K14cre.ΔE-cadflx/flx mice found that E-cadherin ablation induced a mild hyperplasia, with some altered differentiation in basal layer and follicular keratinocytes but no tumours. In contrast, while E-cadherin loss in HK1.ras.K14cre.ΔE-cadflx/flx mice did not appear to alter HK1.ras-mediated papillomatogenesis, malignant conversion was now observed and, moreover, subsequent wdSCCs showed a rapid progression to aggressive SCC. Further analysis found that malignant conversion was associated with p53 loss, while malignant progression also implicated β-catenin activation; increased nuclear expression appeared in the invasive basal layer keratinocytes. The SCC aetiology also suggested the mechanism of tumour invasion may involve an early collective mode and later, in cooperation with β-catenin activation, this progressed to a more aggressive mode of individual cell invasion. Collectively, these data show that conditional ablation of E-cadherin cooperates with rasHa activation and p53 loss at later stages following malignant conversion of papillomas, to induce a rapid tumour progression due to cell–cell adhesion failures and β-catenin signalling

Inducible 14-3-3 Sigma/Stratifin Ablation Cooperates with rasHa Activation to Accelerate Papillomatogenesis and Induce Malignant Conversion in Transgenic Mouse Skin Carcinogenesis

In carcinogenesis, the 14-3-3σ isoform (stratifin) is implicated by being downregulated in a mode of classic tumour suppression gene (TSG) loss and also via elevated oncogenic roles; with both scenarios appearing to be hallmarks of 14-3-3σ deregulation, depending on tumour stage or context. Mouse skin models highlight tumour-suppressive roles, as the Er/Er+/– repeated epilation stain possesses germline mutations that result in hyperplasia, failed follicular differentiation and susceptibility to squamous cell carcinoma (SCC). In transgenic models, 14-3-3σ knockout supports early roles, resulting in rapid papilloma formation in classic two-stage DMBA/TPA chemical carcinogenesis; however, our previous studies in this model demonstrated targeted overexpression of 14-3-3σ led to SCC of a follicular origin in collaboration with Fos and well-differentiated SCC in collaboration with rasHa. Therefore, to study mechanisms driving/inhibiting skin carcinogenesis, stage-specific expression of endogenous 14-3-3σ (stratifin) was analysed in early skin carcinogenesis driven by activated rasHa expression (HK1.ras) and subsequently via conditional RU486-inducible 14-3-3σ knockout (K14.creP-Δ14-3-3σ). Consistent with 14-3-3σ roles previously observed in epidermal differentiation, HK1.ras hyperplasia and early papillomas displayed elevated 14-3-3σ expression in suprabasal keratinocytes and in occasional K1-positive basal keratinocytes as they committed to differentiate. Older HK1.ras mice exhibited increasing 14-3-3σ positivity in papilloma basal layers, suggesting attempts to protect basal-layer p53 TSG function, given that 14-3-3σ acts as a chaperone protein to remove/relocate MDM2 for degradation, thus maintaining p53 levels. Indeed, such papillomas expressed activated p-MDM2166 confined to suprabasal layers, resulting in strong basal layer p53 expression and thus HK1.ras papillomas lacked malignant conversion. In mice where 14-3-3σ functions were ablated via an RU486-inducible Cre/loxP system expressed from a keratin 14 (K14) promoter (K14.creP-Δ14-3-3σ), treated HK1.ras-K14.Δ14-3-3σ mice exhibited a rapid papillomatogenesis and produced larger papillomas. These papillomas also exhibited rapid conversion to malignancy, associated with increased activated p-MDM2166 expression in basal layers and resultant p53 loss. These observations suggest that 14-3-3σ plays tumour suppressive roles involving p53 protection, where failure to regulate MDM2 activity led to p53 loss and susceptibility to malignant conversion

Inducible E-cadherin Ablation Drives Malignant Progression in HK1.ras.fos.PTENflx/flx Carcinogenesis and Induces Conversion of HK1.fos.PTENflx/flx Keratoacanthoma

Inducible ablation of E-cadherin expression has been investigated in mouse skin carcinogenesis driven by Ras and Fos activation (HK1.ras; HK1.fos), and conditional (cre/lox) PTEN knockout exclusively in the epidermis. Previously, analysis of endogenous E-cadherin expression in stage-specific HK1.ras/fos/Δ5PTEN tumours showed E-cadherin expression became reduced at the invasive front of well-differentiated squamous cell carcinomas (wdSCCs) following p53 loss, and where retention of p21 minimized Akt activation and further progression. To validate stage-specific causality of E-cadherin loss, RU486-inducible conditional E-cadherin knockout was introduced into HK1.ras/fos/Δ5PTENflx/flx genotypes employing the cre/LoxP system (K14cre-ΔE-cadflx/flx). Initial observations in RU486-treated compound HK1.ras/fos/ΔPTENflx/flx-ΔE-cadflx/flx mice found that E-cadherin ablation appeared to accelerate malignant conversion, again associated with p53 loss; however, the main effect was on malignant progression and invasion, as wdSCCs displayed rapid progression to aggressive SCCs. Malignant progression also implicated β-catenin activation, finding increased nuclear expression, alongside loss of membranous expression, in the invasive basal layer keratinocytes, together with loss of p21 and increased p-Akt1 activity. In addition, preliminary data for E-cadherin loss in HK1.fos/Δ5PTEN keratoacanthoma aetiology now showed malignant conversion to invasive wdSCC, again associated with increased β-catenin activation. These wdSCCs may have retained a degree of correct adhesion signalling, as basal layer HK1.fos/Δ5PTEN-ΔE-cadflx/flx keratinocytes exhibited both membranous and nuclear p-β-catenin expression; hence, in the absence of Ras, progression stalled at wdSCC. Collectively, these data show that, in some contexts (hyperplasia/papillomas), E-cadherin loss can be tolerated via increased differentiation, while in carcinogenesis, following overt tumour appearance, conditional ablation of E-cadherin drives invasion and malignant progression, consistent with roles in cell–cell adhesion and deregulated signalling to β-catenin

14-3-3σ/Stratifin Expression in HK1.Ras/Fos-Δ5ptenflx Transgenic Mouse Skin Carcinogenesis Reveals an Early, Inhibitory Role; Lost During MDM2 Activated/P53 Inactivated Malignant Progression

The mechanisms driving or inhibiting skin carcinogenesis depend upon the contexts created by temporal acquisition of each mutation when pitted against the sentinel systems that have evolved to resist progression at each stage. To model this process, transgenic mice have been established that express activated rasHa and fos oncogenes together with RU486-inducible [K14.creP/lox-P] PTEN [5PTENflx] mutation exclusively in the epidermis. HK1.ras/fos/Δ5PTEN mice exhibit papillomas that convert to well-differentiated SCC following p53 loss and progress to SCCs following subsequent p21 loss/p-AKT1 activation. In addition, recently a suprabasal-to-basal increase in MDM2 activation [p-MDM2166] was observed associated with this p53 loss. Thus, given that 14-3-3σ/stratifin is a chaperone protein which removes/relocates MDM2 for degradation, 14-3-3σ/stratifin expression status in papillomatogenesis and progression to carcinoma was investigated. Already 14-3-3σ/Stratifin is known to play pivotal roles in keratinocyte growth, differentiation, and carcinogenesis but its exact protective [or potentially oncogenic] functions in differing tumour contexts remain unclear, as some studies show that 14-3-3σ/stratifin acts as a tumour suppressor and loss drives cancer; whilst others show increased expression associates with tumour invasion. Initially, compared to normal skin, RU486-treated tri-genic ras/fos/PTENflx epidermis expressed elevated 14-3-3σ/stratifin in resultant hyperplasia, and 14-3-3σ/stratifin remained strongly expressed in both basal and suprabasal layers of p53+ve /p21+ve papillomas. However, 14-3-3σ/stratifin expression diminished following malignant conversion, concomitant with p53 loss but persistent p21 expression. New HK1.ras/fos/5PTEN data demonstrate that this 14-3-3σ reduction/loss in basal layer keratinocytes was also associated with the suprabasal-to-basal expression of activated p-MDM2166; consistent with a mechanism of reduced basal layer p53 expression driving the susceptibility of late-stage papillomas to malignant conversion. Further, in 3D living skin assays, reduction/loss in 14-3-3σ expression was associated with an increased invasive potential. Collectively these data suggest that, in this context, 14-3-3σ plays important tumour suppressive roles in papillomas which inhibit MDM2 function and help maintain p53. However, diminished 14-3-3σ expression may facilitate MDM2-mediated loss of p53 which aids malignant conversion