Abstract 5734: The effect of PRAME on retinoid response and cell proliferation in cutaneous and head and neck squamous cell carcinoma

Abstract As cells undergo terminal differentiation, they adopt their tissue-specific functions and permanently exit the cell cycle. Inducing differentiation of premalignant cells and malignant cells are proposed strategies for cancer prevention and treatment, respectively. Retinoids, compounds related to retinol, drive terminal differentiation of numerous cell types. These compounds exhibit efficacy for the prevention of cutaneous squamous cell carcinomas (cSCC) and head and neck squamous cell carcinomas (HNSCCs), and their incorporation into treatment plans for these cancers is supported by laboratory and clinical studies. Preferentially Expressed Antigen in Melanoma (PRAME) is a cancer-testis antigen that represses retinoid signaling, and is associated with adverse outcomes in a plethora of malignancies. Although PRAME is known to be expressed in subsets of cutaneous SCC (cSCC) and head and neck SCC (HNSCC) tumors, its functions, prognostic and therapeutic significance have never been investigated in these cancers. We hypothesize that PRAME expression in SCC cells confers resistance to the anti-neoplastic effects of retinoids and supports cell proliferation. PRAME expression was evaluated in human cSCC tumors, and in cSCC and HNSCC cell lines by immunoblotting and qRT-PCR. PRAME-overexpressing immortalized keratinocyte, cSCC and HNSCC cell lines were generated. shRNA-mediated knockdown of PRAME was performed in a cSCC and a HNSCC cell line. Cells were treated with all-trans retinoic acid (ATRA) for 24, 48 or 72 hours. Expression of differentiation markers was assessed by immunoblotting and qRT-PCR of markers of differentiation. Cell counting assays, immunoblot analysis of cell cycle genes and Ki67 immunofluorescence staining were used to assess proliferation. PRAME expression is detected in subsets of cSCC tumors and in select SCC cell lines. Overexpression of PRAME in HNSCC cells enhanced cell proliferation compared to control cells. Treatment with ATRA did not promote differentiation of PRAME-expressing cells. Furthermore, PRAME overexpression attenuated the anti-proliferative effect of ATRA in HNSCC cells. We conclude that PRAME enhances proliferation of malignant keratinocytes in vitro and may confer resistance to retinoid-induced differentiation and proliferation arrest. Investigations to assess the prognostic and therapeutic significance of PRAME expression in SCCs are warranted. Citation Format: Brandon Liam Ramchatesingh, Ivan Litvinov. The effect of PRAME on retinoid response and cell proliferation in cutaneous and head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5734.</jats:p

Abstract 2039: The role of HORMAD1 in DNA damage repair in squamous cell carcinomas

Abstract Genomic instability is a prominent hallmark of cancer, however the dynamic mechanisms that drive and sustain genomic instability remain elusive. Research demonstrates that numerous cancers with increased levels of genomic instability express meiosis genes and undergo a process called meiomitosis. Cancer meiomitosis is the orchestrated activation of both mitotic and meiotic machineries in neoplastic cells that confer a selective advantage. Specifically, meiCT (meiosis-specific cancer/testis) genes that specialize in the reductional division of germ cells in meiosis I are ectopically expressed in several cancers, particularly in squamous cell carcinomas. The meiCT gene, HORMAD1 is a meiosis specific protein that functions to ensure that a sufficient number of DSBs are formed for appropriate meiotic progression and to maintain genome integrity throughout homologous recombination. Studies show that HORMAD1 is significantly upregulated in several cancers and is regarded as a potentially important oncogene that plays a role in sustaining increased genomic instability. With the use of shRNA mediated knockdown of HORMAD1, we evaluated the effects ofHORMAD1 on genomic instability and survival in squamous cell carcinomas (SCCs). Our results demonstrate that HORMAD1 knockdown in SCC cancer cell lines leads to an increase in genomic instability denoted by an increase in double strand breaks, micronuclei formation and ploidy. Furthermore, shHORMAD1 results in increased sensitivity to etoposide treatment leading to increased centrosome formation and reduced cell survival. We also demonstrate thatHORMAD1 modulates a functional and balanced level of genomic instability by eliciting an DNA damage response through homologous recombination mechanisms that allows SCCs to thrive following DNA damage. Citation Format: Jennifer Gantchev, Amelia Martinez Villarreal, Brandon Ramchatesingh, Ivan V. Litvinov. The role of HORMAD1 in DNA damage repair in squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2039.</jats:p

Abstract 98: The role of HORMAD1 in transformation of keratinocytes following vemurafenib treatment

Abstract BRAF inhibitor (BRAFi) therapies are highly effective treatments for metastatic melanoma. However, BRAFi monotherapies are associated with a significant risk of squamous cell carcinoma (SCC) development in melanoma patients. Consequently, BRAF inhibition results in an unanticipated activation of the MAPK pathway in surrounding cells that harbour wildtype BRAF. Fortunately, a combination approach with both a BRAFi and the selective MAPK inhibitor (MEKi), trametinib, attenuates BRAFi-induced risk of SCCs. Despite the fact that this combination therapy is widely used in clinical practice, the mechanisms associated with SCC induction and why this effect is attenuated with a MEK inhibitor remain poorly understood. HORMAD1, a meiosis specific gene, has been shown to have an impact on genomic instability and proliferation in the development of SCCs. Moreover, recent work in our lab has demonstrated that the MAPK pathway regulates HORMAD1 expression in SCC cell lines. Therefore, we sought to investigate a potential role of HORMAD1 in the risk and development of SCCs as a mechanism of BRAFi that is attenuated with the co-administration of the MEKi, trametinib. Using the BRAFi, vemurafenib, we inhibited the kinase activity of activated BRAF (V600E) and the MEKi, trametinib to evaluate the expression of HORMAD1 in melanoma and normal keratinocyte cell lines. To determine the effects of these two inhibitors, a cell proliferation assay was conducted followed by the evaluation of various components of cell cycle regulation and genomic instability. We found that HORMAD1 protein expression levels increased following treatment with the BRAFi, vemurafenib, in the normal keratinocyte cell line, HaCaT, but not in the melanoma cell line, A375. In contrast, HORMAD1 expression decreased following treatment with the MEKi, trametinib in both HaCaT and A375. Cell proliferation assays depict a vulnerability to both vemurafenib and trametinib in both normal and melanoma cell lines, however, HaCaT cells appear to be more susceptible to increased levels of genomic instability following treatment with vemurafenib when compared to A375. Our results suggest that HORMAD1 likely plays a key role in the development of SCCs by increasing genomic instability and proliferation in normal keratinocytes following BRAFi monotreatment. The increased risk of SCC formation is attenuated by dampening HORMAD1 levels with the co-administration of the MEKi, trametinib. Citation Format: Jennifer Gantchev, Amelia Martinez Villarreal, Brandon Ramchatesingh, Ivan V. Litvinov. The role of HORMAD1 in transformation of keratinocytes following vemurafenib treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 98.</jats:p

Pharmacological Agents Used in the Prevention and Treatment of Actinic Keratosis: A Review

Actinic keratosis (AK) is among the most commonly diagnosed skin diseases with potentially life-threatening repercussions if left untreated. Usage of pharmacologic agents represents one of many therapeutic strategies that can be used to help manage these lesions. Ongoing research into these compounds continues to change our clinical understanding as to which agents most benefit particular patient populations. Indeed, factors such as past personal medical history, lesion location and tolerability of therapy only represent a few considerations that clinicians must account for when prescribing appropriate treatment. This review focuses on specific drugs used in either the prevention or treatment of AKs. Nicotinamide, acitretin and topical 5-fluorouracil (5-FU) continue to be used with fidelity in the chemoprevention of actinic keratosis, although some uncertainty persists in regard to which agents should be used in immunocompetent vs. immunodeficient/immunosuppressed patients. Topical 5-FU, including combination formulations with either calcipotriol or salicylic acid, as well as imiquimod, diclofenac and photodynamic light therapy are all accepted treatment strategies employed to target and eliminate AKs. Five percent of 5-FU is regarded as the most effective therapy in the condition, although the literature has conflictingly shown that lower concentrations of the drug might also be as effective. Topical diclofenac (3%) appears to be less efficacious than 5% 5-FU, 3.75–5% imiquimod and photodynamic light therapy despite its favorable side effect profile. Finally, traditional photodynamic light therapy, while painful, appears to be of higher efficacy in comparison to its more tolerable counterpart, daylight phototherapy.</jats:p

The Ultraviolet Irradiation of Keratinocytes Induces Ectopic Expression of LINE-1 Retrotransposon Machinery and Leads to Cellular Senescence

Retrotransposons have played an important role in evolution through their transposable activity. The largest and the only currently active human group of mobile DNAs are the LINE-1 retrotransposons. The ectopic expression of LINE-1 has been correlated with genomic instability. Narrow-band ultraviolet B (NB-UVB) and broad-band ultraviolet B (BB-UVB) phototherapy is commonly used for the treatment of dermatological diseases. UVB exposure is carcinogenic and can lead, in keratinocytes, to genomic instability. We hypothesize that LINE-1 reactivation occurs at a high rate in response to UVB exposure on the skin, which significantly contributes to genomic instability and DNA damage leading to cellular senescence and photoaging. Immortalized N/TERT1 and HaCaT human keratinocyte cell lines were irradiated in vitro with either NB-UVB or BB-UVB. Using immunofluorescence and Western blotting, we confirmed UVB-induced protein expression of LINE-1. Using RT-qPCR, we measured the mRNA expression of LINE-1 and senescence markers that were upregulated after several NB-UVB exposures. Selected miRNAs that are known to bind LINE-1 mRNA were measured using RT-qPCR, and the expression of miR-16 was downregulated with UVB exposure. Our findings demonstrate that UVB irradiation induces LINE-1 reactivation and DNA damage in normal keratinocytes along with the associated upregulation of cellular senescence markers and change in miR-16 expression