Long non-coding RNAs in cutaneous squamous cell carcinoma

Long-term exposure to the sun UV-radiation is the leading cause for the development of skin cancer. Keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer and its incidence is increasing globally. Although the most frequent mutational targets in cSCC development have been characterized, a comprehensive understanding of the molecular events in cSCC pathogenesis remains incomplete. There is an obvious need for clinically useful prognostic biomarkers and therapeutic targets for recurrent and metastatic cSCCs. Long non-coding RNAs (lncRNAs) are a largely uncharacterized group of regulatory RNAs involved in various biological processes and their role in cancer progression is emerging. However, their role in cSCC is largely unknown. The main objective of this thesis was to investigate lncRNAs in order to identify new biomarkers for progression of cSCC and characterize novel therapeutic targets for recurrent and metastatic cSCC. In this study two tumorigenic lncRNAs in cSCC were identified, and based on their expression and function and with the permission of the HUGO Gene Nomenclature Committee they were named PICSAR and PRECSIT. They are specifically upregulated in cSCC cells in culture and in vivo and they contribute to cSCC progression by distinct mechanisms. PICSAR promotes cSCC cell growth by activating ERK1/2 via suppression of DUSP6 expression. Furthermore, PICSAR regulates adhesion and migration of cSCC cells by regulating integrin expression. PRECSIT expression in cSCC cells was shown to be regulated by the p53 pathway. Additionally, PRECSIT was found to regulate invasion of cSCC cells by regulating STAT3 signaling and expression of MMP-1, MMP-13, MMP-13, and MMP-10. In conclusion, lncRNAs PICSAR and PRECSIT may serve as novel biomarkers and putative therapeutic targets in cSCC.Jatkuva altistuminen auringon UV-säteilylle on suurin syy ihon levyepiteelisyövän (cutaneous squamous cell carcinoma, cSCC) kehittymiseen. Se on yleisin metastaattinen ihosyöpä maailmanlaajuisesti ja syöpätapausten määrä on kasvussa. Useita cSCC:n kehittymiseen liittyviä DNA-mutaatioita on tunnistettu, mutta kokonaisvaltainen käsitys syövän kehittymisen aikana tapahtuvista molekulaarisista muutoksista on yhä selvittämättä. Uusia merkkiaineita tarvitaan, jotta voitaisiin paremmin tunnistaa kehittyykö varhaisen vaiheen syöpä aggressiiviseksi metastasoivaksi syöväksi. Pitkät eikoodaavat RNA:t (long non-coding RNA, lncRNA) ovat varsin tuntematon ryhmä molekyylejä, joiden merkitystä syövässä on tutkittu laajalti, mutta niiden yhteyttä cSCC:hen ei juurikaan tunneta. Tämän väitöskirjatyön tavoitteena oli löytää ja karakterisoida lncRNA:ita, joita voitaisiin käyttää merkkiaineina nopeasti etenevän tai leviävän cSCC:n havaitsemisessa sekä uusien hoitomuotojen kehittämisessä. Tässä työssä havaittiin kaksi lncRNA:ta, jotka liittyvät cSCC:n kehittymiseen. Tutkimustulosten perusteella sekä ihmisen perimän kansainvälistä kartoitus- ja sekvensointihanketta johtavan organisaation nimeämiskomitean luvalla ne nimettiin PICSAR:iksi ja PRECSIT:iksi. Niiden ilmentyminen on koholla cSCC:ssä ja ne vaikuttavat cSCC:n kehittymiseen eri mekanismeilla. PICSAR edistää syövän kasvua aktivoimalla ERK1/2 kinaasia DUSP6:n kautta. Lisäksi se säätelee solujen kiinnittymistä ja liikkumista vaikuttamalla integriini-solureseptorien ilmentymiseen. PRECSIT:in ilmentymistä säädellään p53-signalointireitin kautta. PRECSIT säätelee cSCC-solujen invaasiota STAT3-signalointireitin välityksellä vaikuttamalla MMP-1, MMP-3, MMP-10 ja MMP-13 geenien ilmentymiseen. PICSAR ja PRECSIT voisivat toimia uusina merkkiaineina syövän diagnostiikassa ja uusien hoitomuotojen kehittämisessä cSCC:lle

Characterization of cancer-associated fibroblast heterogeneity in squamous cell carcinoma progression using single-cell RNA sequencing

Cutaneous squamous cell carcinoma (cSCC) is a keratinocyte cancer with a rapidly increasing incidence and one of the most common cancer types in the fair-skinned population. Chronic exposure to ultraviolet radiation (UVR) is its main risk factor and can lead to the development of a premalignant skin lesion, actinic keratosis (AK), which might further progress into cSCC. In addition, invasive cSCC can also develop from the in situ carcinoma Bowen’s disease (BD). During this progression, resident dermal fibroblasts are transformed into cancer-associated fibroblasts (CAFs) that are known to promote tumorigenesis. However, a detailed characterization of cSCC-related CAFs with respect to their fibroblast subpopulation-specific origin, heterogeneity, and tumor-promoting functions was still missing. Therefore, in this thesis, more than 115,000 single-cell transcriptomes from healthy human skin, BD and cSCC samples were analyzed. The results revealed two main CAF subpopulations with distinct functions and origins. Inflammatory CAFs (iCAFs) seemed to develop mainly from pro-inflammatory fibroblasts and presented immunoregulatory functions, including cellular interactions with immune cells in the tumor microenvironment (TME). On the other hand, myofibroblastic CAFs (myCAFs) were observed to originate mainly from healthy mesenchymal fibroblasts and were involved in extracellular matrix (ECM) remodeling processes. Furthermore, multiplexed RNA fluorescence in situ hybridization (FISH) assays not only confirmed both CAF subpopulations in human BD and cSCC, but also provided valuable information about the time window of CAF activation, as no CAFs could be observed in AK tissue sections. Interestingly, these findings could not be transferred to basal cell carcinoma (BCC), the second major keratinocyte cancer. Taken together, this thesis provides novel insights into CAF development, stratification, and functions during cSCC initiation and progression

MICRORNA FUNCTIONS IN UV-INDUCED CUTANEOUS SQUAMOUS CELL CARCINOMA

Cutaneous squamous cell carcinoma (cuSCC) is the second most common skin cancer, for which long term UV exposure and chronic wounding are the dominant risk factors. Despite these clinically established connections, little is understood about the early molecular response of human skin to UV exposure and its connection to acute wounding and cuSCC. Thus, our goal is to find common and specific signatures driven by UV-exposure and wounding as a means of developing new approaches for treating and preventing cuSCC. Here, we perform integrated analyses of RNA-seq and miR-seq on 3 datasets: (1) UV-unexposed and acute UV-exposed human skin, (2) public dataset on acute wound healing and (3) our previously published dataset on normal skin and cuSCC from humans. We find that biological signatures and processes regulated by acute UV exposure and wounding has profound similarity. Through RNA-seq and miR-seq on matched normal skin and cuSCC tumors from humans and a UV-driven mouse model, as well as acute UV-exposed human skin, we were able to identify a group of miRs that change both in cuSCC development and following UV exposure. We previously reported that miR-21-5p and miR-31-5p overexpression correlates with the development of UV-induced cuSCC in human. This is also true for our analysis where we find that these miRs as well as miR-21-3p are upregulated by more than 6-fold in cuSCC (compared to normal skin) and more than 2.5-fold in UV-exposed skin (compared to unexposed skin). In addition, we identify that miR-340-5p and let-7i-5p are novel candidates that have not been previously linked to either cuSCC development or the UV response of human skin. This suggests that these changes in miRNA-RNA are important early events that regulated by both UV-exposure and wounding which eventually can promote cuSCC initiation. Thus, our findings suggest that UV-exposed skin, wound and cuSCC share various common signatures, which can be potentially validated as chemopreventive targets for cuSCC

The LKB1-SIK pathway: dysregulation in melanomagenesis and regulated use in skin cancer prevention

The presence of dark melanin (eumelanin) within human epidermis represents one of the strongest predictors of skin cancer risk. Topical rescue of eumelanin synthesis, previously achieved in “redhaired” Mc1r-deficient mice, demonstrated significant protection against UV damage and skin carcinogenesis. However, application of a topical strategy for human skin pigmentation has not been achieved, largely due to the greater barrier function of human epidermis. Salt Inducible Kinase (SIK) has been demonstrated to regulate MITF, the master regulator of pigment gene expression, through its effects on CREB regulated transcription coactivator (CRTC) and CRE binding protein (CREB) activity. Here, we describe the development of small molecule SIK inhibitors that were optimized for human skin penetration, resulting in MITF upregulation and induction of melanogenesis. When topically applied, pigment production was induced in Mc1r-deficient mice and normal human skin. These findings are the first to demonstrate a realistic pathway towards UV-independent topical modulation of human skin pigmentation, potentially impacting UV protection and skin cancer risk. Although MITF normally functions as a regulator of pigmentation, if amplified MITF can serve as a melanoma oncogene shown to cooperate with BRAF (V600E) to induce tumorigenic transformation of melanocytes . Only 10% of melanomas carry an MITF amplification emphasizing the need to identify pathways that modulate MITF expression . Liver kinase B1 (LKB1) regulates many cancer-relevant cell phenotypes and is a known SIK inducer . However, the interaction of the LKB1-SIK pathway and MITF in melanoma formation is not fully understood. Overall 49% of human melanomas in The Cancer Genome Atlas contain aberrations in LKB1, SIK1, SIK2, SIK3, CRTC1, CRTC2, CRTC3, or MITF . Here, we report that the LKB1-SIK axis can negatively regulate MITF expression, and our data suggests this is through increasing CRTC2 cytoplasmic localization. Rescue of LKB1 in LKB1-null G361 melanoma cells suppresses cell proliferation. MAP kinase pathway activation suppresses MITF, and knockdown of all three SIK isoforms rescues MITF expression in NRAS (Q61R) expressing melanocytes. Overall, our findings establish SIK and LKB1 as regulators of the CRTC-CREB-MITF pathway and through this regulation, potentially play a critical role as tumor suppressors in melanoma oncogenesis

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

Tumor Angiogenesis

Tumor angiogenesis is the main process responsible for the formation of new blood vessels that promote tumor growth and metastasis. This process is driven by potent pro-angiogenic factors that are predominant in the tumor environment and are produced by both malignant cells and the host cells recruited to the tumor site. Tumor environment is characterized by the imbalance between pro-angiogenic and anti-angiogenic factors, which drives the construction of numerous but structurally defective vessels. These poorly perfused and abnormal vessels significantly contribute to the tumor pathology not only by supporting the expansion of the tumor mass but also by promoting chronic inflammation, enhancing thrombosis, impeding drug delivery, and disseminating tumor cells. These problems associated with tumor vasculature continue to attract great attention of scientists and clinicians interested in advancing the understanding of tumor biology and development of new drugs. This book complies a series of reviews that cover a broad spectrum of current topics related to the pathology of tumor blood vessels including mechanisms inducing new vessels, identification of new targets for inhibition of tumor angiogenesis, and potential clinical use of known and novel anti-angiogenic therapies. The book provides an update on tumor angiogenesis that could be useful for oncologists, cancer researchers and biologists with interests in vascular and endothelial cell behavior in the context of cancer

Roles of poly(ADP-ribose) polymerase-1 in the ultraviolet radiation-induced skin carcinogenesis

L'exposition aux rayons ultraviolets (UV) est essentielle à la vie et bénéfique pour la santé humaine. Cependant, la surexposition aux UV solaires, en particulier aux UVB, rayons les plus énergétiques atteignant la surface terrestre, peut entrainer des cancers de la peau chez l'être-humain comme les cancers de la peau de type non-mélanome (NMSC). La capacité des UVB à initier des NMSC provient principalement de leurs habilités à causer des dommages directs à l'ADN, tels que les dimères cyclobutyliques de pyrimidine (CPD) et les produits pyrimidine-pyrimidone (6-4PP), qui sont pris en charge par le mécanisme de réparation par excision de nucléotide (NER). L'incidence croissante de NMSC chez les patients déficients pour l'une des protéines de la NER souligne l'importance d'un processus fonctionnel. Par conséquent, une meilleure compréhension des mécanismes moléculaires de la NER permettrait de mettre en évidence de nouvelles cibles thérapeutiques pour la prévention ou le traitement des cancers de la peau. L'une des premières réponses cellulaires aux dommages CPD/6-4PP induits par UVB dans la peau des mammifères est l'activation de l'enzyme nucléaire poly(ADP-ribose) polymérase-1 (PARP1) qui catalyse la formation de polymères d'ADP-ribose. Les précédents travaux de notre laboratoire et d'autres équipes ont démontré que PARP1 et son activité enzymatique facilitent la NER en collaboration avec la protéine UV-damaged DNA binding protein 2 (DDB2), qui va aussi s'accumuler rapidement aux sites CPD/6-4PP pendant la phase de reconnaissance des dommages à l'ADN de la NER. Cependant, plusieurs aspects des interactions de PARP1 avec DDB2 et avec les dommages directs à l'ADN sont inconnus. Ainsi, le premier objectif de mon projet de doctorat a été de caractériser précisément la nature de la liaison de PARP1 aux dommages CPD/6-4PP induits par UV vis-à-vis la protéine DDB2. Mes recherches ont mis en évidence l'empreinte asymétrique formée par PARP1 de -12 à +9 nucléotides de chaque côté des dommages CPD/6-4PP en présence ou en absence de DDB2. Nous avons également démontré que PARP1 augmente l'affinité de DDB2 pour les dommages CPD/6-4PP. De plus, les résultats de notre étude indiquent un rôle de PARP1 indépendant de DDB2 pendant la phase de reconnaissance des dommages à l'ADN. Cibler PARP1 et son rôle dans les voies de réparation des dommages à l'ADN est l'une des stratégies les plus efficaces développées ces dernières années pour le traitement des cancers des ovaires et du sein. L'application translationnelle de mon projet de doctorat a alors été de comprendre le rôle de PARP1 dans la NER dans le contexte des NMSC. À cet égard, nous avons développé un modèle PARP1-KO dans la lignée de souris SKH-1, qui est un modèle largement adopté pour étudier les NMSC induits par UVB. Puisque les souris SKH-1 développent principalement des carcinomes spinocellulaires (CSC) cutanés après une exposition chronique aux UVB, notre étude rapporte le rôle de PARP1 dans le développement des CSC. En utilisant les souris nouvellement créées SKH-1 PARP1-KO et les souris SKH-1 PARP1-WT avec ou sans application topique d'inhibiteurs de PARP, nous avons mis en évidence que l'absence de PARP1 ou de son activité dans la peau des souris SKH-1 mâles et femelles réduit significativement le fardeau tumoral des CSC et prolonge la période de latence du développement tumoral. L'étude hebdomadaire de l'apparition et de la croissance de tumeurs tout au long du protocole révèlent aussi que cibler PARP1 est très efficace pour ralentir, à l'étape pré-maligne, le développement de CSC. Nos résultats sont surprenants à la lumière des propriétés onco-suppressives rapportées de PARP1 et de son activité catalytique dans des cas de cancérogenèse induits par des dommages à l'ADN causés par des agents alkylants, ainsi que de la susceptibilité croissante des souris knock-out pour d'autres protéines de la NER à développer des CSC induits par UVB. Le rôle de PARP1 dans les mécanismes cellulaires induits par UVB autres que la NER, comme la mort cellulaire et les modulations immunes, pourrait expliquer nos observations. Alors que d'autres analyses sont nécessaires pour comprendre le rôle de PARP1 dans ces mécanismes, notre étude met en avant l'utilisation potentielle d'inhibiteurs de PARP comme nouvel agent chimiopréventif contre les CSC induits par UVB.The exposure to solar ultraviolet radiation (UV) is essential to life and beneficial to human health. However, an overexposure to terrestrial solar UV, especially its most energetic component UVB, can cause skin cancers including the non-melanoma skin cancers (NMSC) in humans. The NMSC initiating properties of UVB arise predominantly from their ability to cause direct DNA damage such as cyclobutane pyrimidine dimers (CPD) and 6-4photoproducts (6-4PP), which are repaired via nucleotide excision repair (NER) pathway. The increased incidence of NMSC in patients with hereditary defects in NER pathway proteins underscores the importance of efficient NER in humans. Therefore, detailed understanding of the molecular operation of NER pathway can provide novel therapeutic targets for the prevention or treatment of skin cancers. One of the earliest responses of the mammalian skin cells to UVB-induced CPD or 6-4PP is the activation of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP1), which catalyzes the formation of polymers of ADP-ribose (PAR). The previous work from other teams and our laboratory have shown that PARP1 and its enzymatic activity facilitate NER in collaboration with UV-damaged DNA binding protein 2 (DDB2), which also rapidly accumulates at the CPD/6-4PP site during the DNA damage recognition stage of NER. However, many aspects of interaction of PARP1 with DDB2 and direct DNA damage are not understood. Therefore, the first aim of my doctoral project was to characterize the precise nature of binding of PARP1 vis-à-vis DDB2 at UV-induced CPD/6-4PP. My doctoral research demonstrates that PARP1 casts asymmetric footprint from −12 to +9 nucleotides on either side of the CPD/6-4PP in presence or absence of DDB2. We also demonstrated that PARP1 facilitates the binding of DDB2 to CPD/6-4PP. Moreover, our study reports DDB2-independent role of PARP1 during the DNA damage recognition phase in NER. Targeting the role of PARP1 in DNA strand break repair pathways has emerged as one of the successful strategies for the treatment of ovarian and breast cancers in last decade. Consequently, the ultimate translational goal of my doctoral project was to understand the implication of NER facilitating role of PARP1 in NMSC. In this regard, we first developed a PARP1-KO model in the albino hairless SKH-1 mouse strain, which is a widely adopted mouse model to study UVB-induced NMSC. Since SKH-1 mice mainly develop cutaneous squamous cell carcinoma (SCC) upon chronic UVB-exposure, our present study reports the role of PARP1 in development of SCC. Using the newly developed PARP1-KO and PARP1-WT SKH-1 mice with or without topical application of PARP inhibitor, we report that the absence of PARP1 or its activity in skin of both male and female SKH-1 mice significantly reduces the SCC tumor burden and prolongs the tumor latency period. The analyses of appearance and growth of individual tumors on a weekly basis during this protocol also revealed that targeting of PARP1 was most effective in suppressing the premalignant stage of the SCC development. Our results are surprising in light of the reported onco-suppressive property of PARP1 and its catalytic activity in alkylating DNA damage-induced tumorigenesis and the increased susceptibility of other NER protein knock-out mice to UVB-induced SCC. We reason that the roles of PARP1 in UVB-induced cellular processes other than NER, such as cell death and immune modulations, can account for our observation. While further studies are required to understand these roles of PARP1 in UVB-induced cellular processes, our study underscores the potential for use of PARP inhibitors as a novel chemopreventive agents against UVB-induced SCC

Personalized Medicine in the Field of Inflammatory Skin Disorders

Skin inflammation is associated with a wide range of conditions which represent major health issues worldwide. Skin and mucosal surfaces represent the primary interface between the human body and the environment, susceptible to numerous factors whose action results in diseases produced by chemical substances, mechanical trauma, microbial agents, radiation, etc. Inflammation, a complex network of interactions between soluble molecules and cells, represents the main modality of the skin’s response to injuries. Numerous studies have revealed close links between chronic inflammation, oxidative stress, and carcinogenesis. Chronic inflammation induces the activation of various cell types and an increase in the production of reactive oxygen species, promoting the initiation of a malignant process. Identifying specific biomarkers is essential for understanding molecular mechanisms and developing therapies appropriate to the patient’s characteristics.Personalized medicine is an emerging field of medicine that has the potential to predict which therapy will be safe and efficacious for specific patients using an individual’s genetic profile to guide decisions regarding the diagnosis, treatment, as well as prevention of disease. This book gathers articles that present recent advancements in research involving the mechanisms that underlie the development of inflammatory skin disorders, skin and mucosal inflammation in general

Regulation of circadian clock genes by UVB radiation and vitamin D : a pilot study in human epidermal keratinocytes during different stages of skin photocarcinogenicity.

Several cellular processes are constantly under regulation of a timekeeping system, known as the circadian clock (CC), with the main regulatory genes involved referred to as the circadian clock genes (CCGs). In spite of a master pacemaker existing in the suprachiasmatic nucleus (SCN) of the hypothalamus, several clock systems have been shown to operate independently in peripheral tissues, including the skin. CCGs interact with cellular processes by both regulating them and being regulated by them, a prime example of this being the bilateral relationship between CC and ultraviolet B radiation (UV-B): On one hand, UV-radiation regulates expression of CCGs in many cell types, and on the other, it has recently been shown that expression of CCGs modulates susceptibility for UV-B-induced cellular damage, including the formation of pyrimidine dimers and other DNA-lesions that are a hallmark of photocarcinogenesis. It was the aim of this study to gain further insights into the CCGs’ putative role for UVB-induced skin photocarcinogenesis. In particular, we aimed to investigate UV-effects on CC and whether these may at least in part be mediated by 1,25(OH)2D3 (D3), the active form of vitamin D, that both depends on UV-B for its synthesis and is known to protect the skin from UV-B’s damaging properties. We treated cells with D3, UV-B and a combination of the two and measured expression of two core clock genes, brain and muscle ARNT-like 1 (BMAL1) and Period-2 (Per2), both over several time points and in cells representing: normal (Normal Human Epidermal Keratinocytes – NHEK; p53 wild type), precancerous (HaCaT keratinocytes; mutated p53 status) and cancerous keratinocytes (Squamous Cell Carcinoma SCL-1; p53 null status). We also assessed the role of vitamin D receptor (VDR) and aryl hydrocarbon receptor (AhR) pathways by measuring UVB-induced damage, repair and cellular toxicity after treatment with D3 and UVB and under chemical antagonization of either/both VDR and/or AhR. Untreated HaCaT cells showed circadian rhythmicity (length of individual periods approximately 24 hours) for expression of BMAL1 and Per2, that were induced by UVB (p<0,001), which also resulted in significant increase in cellular toxicity 24h after treatment (p<0,05). In contrast, treatment with 1,25-dihydroxyvitamin D had only marginal effects (no visual effect on expression of BMAL1, trend for a marginal reduction of expression of Per2). While UV-B induced expression of Per2 in NHEK, HaCaT and SCL-1 cells, UVB-induced upregulation of BMAL1 was only detected in NHEKs and HaCaT, but not in SCL-1 cells. In regards to direct effects and interaction of UVB and D3 and the roles of VDR and AhR on DNA damage and repair, no safe conclusions can be drawn from this study. HaCaT keratinocytes express BMAL1 and Per2 with circadian rhythmicity. Our findings do not support the hypothesis that the UVB-induced upregulation of these CCGs may be mediated via UVB-induced synthesis of vitamin D. Comparing epidermal keratinocytes representing different stages of skin photocarcinogenesis, we provide further evidence for an independently operating timekeeping system in human skin cells, that is physiologically regulated by UVB. The destruction of this mechanism in malignant keratinocytes points to a contribution of CCGs for skin photocarcinogenesis