title: Analysis of exogenous over-expression of 14-3-3σ/Stratifin in transgenic mouse skin carcinogenesis
Creators: name: McMenemy, Carol Marie
Publisher: name: University of Glasgow
Year: 2021
Subjects: subject: Stratifin, 14-3-3σ, 14-3-3, carcinogenesis, transgenic, skin, SCC, squamous cell carcinoma, mice, mouse model.

description: 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 &gt;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.