Spontaneous Squamous Cell Carcinoma Research Articles

Anti-PD-1 immunotherapy with adjuvant ablative fractional laser displays increased tumour clearance of squamous cell carcinoma, a murine study.

PD-1 checkpoint inhibitors are used as systemic immunotherapy for locally advanced and metastatic cutaneous squamous cell carcinoma (SCC); however, improved treatment efficacy is urgently needed. In this study, we aimed to investigate the effect of combining systemic anti-PD-1 treatment with adjuvant ablative fractional laser (AFL) in a spontaneous SCC mouse model. Tumours induced by ultraviolet radiation in the strain C3.Cg-Hrhr /TifBomTac were divided into four groups: anti-PD-1-antibody+AFL (n = 33), AFL alone (n = 22) anti-PD-1-antibody alone (n = 31) and untreated controls (n = 46). AFL was given at Day 0 (100 mJ/mb, 5% density), while anti-PD-1-antibody (ip, 200 μg) at Days 0, 2, 4, 6 and 8. Response to treatment was evaluated by tumour growth, survival time and by dividing response to treatment into complete responders (clinically cleared tumours), partial responders (reduced tumour growth rate compared to untreated controls) and non-responders (no decrease in tumour growth rate compared to untreated controls). The strongest tumour response was observed following the combination of systemic anti-PD-1 treatment combined with laser exposure, resulting in the highest percentage of complete responders (24%) compared with untreated controls (0%, p < 0.01), AFL monotherapy (13%, p > 0.05) and anti-PD-1-antibody monotherapy (3%, p > 0.05). Moreover, all three treatment interventions demonstrated significantly reduced tumour growth rates compared with untreated controls (p < 0.01), and the mice had significantly longer survival times (p < 0.01). In conclusion, the combination treatment revealed an improved treatment effect that significantly enhanced the complete tumour clearance not observed with the monotherapies, indicating a possible additive effect of anti-PD-1 with adjuvant AFL in treatment of SCC.

Updating the biologically based dose-response model for the nasal carcinogenicity of inhaled formaldehyde in the F344 rat.

Chronic inhalation of formaldehyde by F344 rats causes nasal squamous cell carcinoma (SCC). This outcome is well-characterized: including dose-response and time course data for SCC, mechanistic endpoints, and nasal dosimetry. Conolly et al. (Toxicol. Sci. 75, 432-447, 2003) used these resources to develop a biologically based dose-response (BBDR) model for SCC in F344 rats. This model, scaled up to humans, has informed dose-response conclusions reached by several international regulatory agencies. However, USEPA concluded that uncertainties precluded its use for cancer risk assessment. Here, we describe an updated BBDR model that addresses uncertainties through refined dosimetry modeling, revised analysis of labeling index data, and an extended dataset where both inhaled (exogenous) and endogenous formaldehyde (exogF, endoF) form DNA adducts. Further, since Conolly et al. (ibid) was published, it has become clear that, when controls from all F344 inhalation bioassays are considered, accounting for over 4000 rats, at most one nasal SCC occurred. This low spontaneous incidence constrains possible contribution of endoF to the formation of nasal SCC via DNA reactivity. Further, since both exogF and endoF form DNA adducts, this constraint also applies to exogF. The revised BBDR model therefore drives SCC formation through the cytotoxicity of high concentration exogF. An option for direct mutagenicity associated with DNA adducts is retained to allow estimation of an upper bound on adduct mutagenicity consistent with the lack of a spontaneous SCC incidence. These updates represent an iterative refinement of the 2003 model, incorporating new data and insights to reduce identified model uncertainties.

LB882 Dissecting mechanisms of responsiveness to the combination therapy of radiation and anti-PD-L1/anti-TGFb treatment in murine squamous cell carcinoma models

Squamous cell carcinoma (SCC) is often marked by an immunosuppressive tumor microenvironment, particularly in metastatic/recurrent disease. The purpose of this study is to evaluate the anti-tumor efficacy of and mechanisms of response to the combination therapy of radiotherapy (RT) and anti-PD-L1/anti-TGFb treatment using SCC models. We transplanted tumor cells derived from either carcinogen-induced SCCs or spontaneous SCCs of K15.Kras12D/Smad4-/- mice to syngeneic mouse recipients and subjected them to RT and anti-PD-L1/anti-TGFb therapy.

Abstract 1339: Interleukin 4 Producing CD4 T cells initiate a crucial event for lung cancer development

Abstract Lung cancer remains one of the leading causes of mortality of cancer patients. Kinase-dead Ikkα knock-in (KA/KA) mice develop CD4 T cell-derived autoimmune disease. Previously, we reported spontaneous lung squamous cell carcinoma (SCC) associated with increase macrophage and CD4 T cell numbers in KA/KA mice. Depleting macrophages dampens lung SCC development in KA/KA mice, but a lot of CD4 T cells remain in the lungs. The activity of CD4 T cells in lung SCC development is unknown. In this study, we attempted to investigate the role of CD4 T cells in lung SCC development. We performed adoptive CD4 T cell transfer experiment and found that KA/KA CD4 T cell transfer, not WT CD4 T cells, recruited marked macrophages to the lungs and induced lung SCC in KA/KA;Rag1-/- mice. The CD4 T cells isolated from the lungs of KA/KA mice and KA/KA;Rag1-/- mice with KA/KA CD4 T cell transfer expressed elevated PD-1, IL-4, IL-5, Il-13, and Il-21 levels compared to WT mice. We found that Il4 ablation, not Il5 deletion, reduced macrophage recruitment and blocked SCC development in KA/KA;Il4-/- mice as well as unlike KA/KA CD4 T cells, KA/KA;Il4-/- CD4 T cells did not induce spontaneous lung SCC in KA/KA;Rag1-/- mice with KA/KA;Il4-/- CD4 T cell injections. Taken together, the finding highlights a pathogenic role of IL-4-producing CD4 T cells in lung carcinogenesis. Citation Format: Gajendra Jogdand, Sayantan Banerjee, Gongping Shi, Jami Willet Brown, Amit Singh, Yongmei Zhao, Jyoti Shetty, Bao Tran, Yinling Hu. Interleukin 4 Producing CD4 T cells initiate a crucial event for lung cancer development [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 1339.

VEGF Expression, Cellular Infiltration, and Intratumoral Collagen Levels after Electroporation-Based Treatment of Dogs with Cutaneous Squamous Cell Carcinoma.

Canine cutaneous squamous cell carcinoma (SCC) is the most common type of skin cancer in tropical countries and is generally associated with exposure to solar ultraviolet light. It has a low metastatic rate, and local treatments, such as electrochemotherapy (ECT), promote long-term control or even complete remission. This study aimed to evaluate pre- and post-ECT treatment expression levels of vascular endothelial growth factor (VEGF) and CD31, cellular infiltration, and intratumoral collagen levels in dogs with cutaneous SCC. A prospective nonrandomized clinical study was performed using dogs with spontaneous SCC treated with ECT. Eighteen lesions from 11 dogs were included in the study. The expression levels of VEGF and CD31; cellular infiltration; and intratumoral collagen levels, as determined by Masson’s trichrome staining, were not significantly different from pre-treatment measurements on day 21 (p > 0.05). However, among cellular infiltration, the mixed subtype was correlated with better overall survival time when compared to lymphoplasmacytic and neutrophilic infiltration (p < 0.05). In conclusion, ECT had no effect on VEGF expression, cellular infiltration, or intratumoral collagen levels in dogs with cutaneous SCC at the time of evaluation, suggesting that early and late post-ECT-treatment phases should be considered.

Regulation of 5-Hydroxymethylcytosine by TET2 Contributes to Squamous Cell Carcinoma Tumorigenesis

DNA methylation is a key regulatory event controlling a variety of physiological processes and can have dramatic effects on gene transcription. Methylated cytosine (5-methylcytosine) can be oxidized by the TET family of enzymes to 5-hydroxymethylcytosine (5-hmC), a key intermediate in the demethylation cycle, and 5-hmC levels are reduced in malignancies such as acute myeloid leukemia and melanoma. We constructed a tissue microarray of human cutaneous squamous cell carcinoma tumors and found a global reduction in 5-hmC levels compared with that in the adjacent skin. Using a murine K14-CreER system, we have found that loss of Tet2 promotes carcinogen-induced squamous cell carcinoma and cooperates with loss of Tp53 to drive spontaneous squamous cell carcinoma tumors in epithelial tissues. Analysis of changes in 5-hmC and gene expression after loss of Tet2 in the epidermis revealed focal alterations in 5-hmC levels and an increase in hair follicle transient amplifying cell genes along with a reduction in epidermal differentiation genes. These results show a role for TET2 in epidermal lineage specification, consistent with reported roles for TET enzymes in controlling lineage commitment in hematopoietic stem cells and embryonic stem cells and establishing TET2 as a bone fide tumor suppressor in squamous cell carcinoma.

Cutaneous squamous cell carcinomas in domestic rabbits (Oryctolagus cuniculus): 39 cases (1998-2019)

Domestic rabbits ( Oryctolagus cuniculi ) can develop a variety of cutaneous neoplasms, including squamous cell carcinoma (SCC). A detailed review of gross and microscopic pathology and response to treatment of spontaneously arising SCCs in domestic rabbits has not been published. A retrospective survey study of the clinical characteristics and response to treatment in 39 cases of spontaneous SCC in pet rabbits was performed in an attempt to better characterize the typical presentation, prognosis, and therapeutic response of SCCs in domestic rabbits. Sixteen of these cases were also selected for papillomavirus testing using a generic polymerase chain reaction. SCC was identified in rabbits between 2 and 10 years of age, with a median age of 7 years. The neoplasm has a predilection for ears and feet and the conventional subtype is most commonly diagnosed microscopically. Lighter colored rabbits may be predisposed to developing SCC. The majority of cases examined were found in rabbits housed primarily indoors. Only one SCC tested positive for papillomavirus and was located in the oral cavity. Sequencing of the detected PCR product detected 98.75% similarity to human papillomavirus type 120. The significance of this virus for tumorigenesis is unknown. Aggressive surgical resection provided the most successful therapeutic option and proved curative in 12 of 23 rabbits. Papillomavirus likely does not play a major role in the development of spontaneous SCCs in pet rabbits. More research is needed to investigate the use of adjunctive therapies in treatment of this neoplasm in pet rabbits.

Deletion of p53 and Hyper-Activation of PIK3CA in Keratin-15+ Stem Cells Lead to the Development of Spontaneous Squamous Cell Carcinoma.

Squamous cell carcinoma (SCC) is the second commonest type of skin cancer, and SCCs make up about 90% of head and neck cancers (HNSCCs). HNSCCs harbor two frequent molecular alterations, namely, gain-of-function alterations of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and loss-of-function mutations of tumor protein p53 (TP53). However, it remains poorly understood whether HNSCCs harboring different genetic alterations exhibit differential immune tumor microenvironments (TME). It also remains unknown whether PIK3CA hyperactivation and TP53 deletion can lead to SCC development spontaneously. Here, we analyzed the Cancer Genome Atlas (TCGA) datasets of HNSCCs and found that patients with both PIK3CA and TP53 alterations exhibited worse survival, significantly lower CD8 tumor infiltrating lymphocytes (TILs) and higher M0 macrophages than other controls. To better model human tumorigenesis, we deleted TP53 and constitutively activated PIK3CA in mouse keratin-15-expressing stem cells, which leads to the spontaneous development of multilineage tumors including SCCs, termed Keratin-15-p53-PIK3CA (KPPA) tumors. KPPA tumors were heavily infiltrated with myeloid-derived suppressor cells (MDSCs), with a drastically increased ratio of polymorphonuclear-MDSC (PMN-MDSC) versus monocytic-MDSC (M-MDSC). CD8 TILs expressed more PD-1 and reduced their polyfunctionality. Overall, we established a genetic model to mimic human HNSCC pathogenesis, manifested with an immunosuppressive TME, which may help further elucidate immune evasion mechanisms and develop more effective immunotherapies for HNSCCs.

Abstract 2355: IKKa serves as a molecular switch linking miRNA biogenesis and inflammation in skin tumorigenesis

Abstract Our previous studies have shown that the absence of tumor suppressor IKKα leads to development of spontaneous mouse squamous cell carcinomas (SCCs) in the skin, lungs, and esophagus, which is partially mediated by aberrant epigenetic alterations and inflammation. However, it remains unknown whether there is a crosstalk between the above-mentioned mechanisms. As a part of the epigenetic machinery, miRNAs regulate many genes post-transcriptionally. Here we show that IKKα deletion in the mouse epidermis results in impaired miRNA processing with markedly reduced expression of ribonuclease Dicer, an enzyme required for miRNA maturation. We found that the lack of IKKα causes Dicer gene silencing through DNA methylation. As a result, the maturation of miR-203-3p, which is the most abundant miRNA in keratinocytes, is blocked in Ikkα-null keratinocytes. Next, we identified Myd88, an important component of innate immunity and inflammation, as one of miR-203-3p targets. The level of Myd88 expression is robustly increased in Ikkα-null keratinocytes due to the lack of miR-203-3p. We also observed increased activity of caspase 1 cleavage, an indicator of inflammasome activation, in Ikkα-null keratinocytes. Interestingly, Myd88 deletion or 5-aza-2’-deoxycytidine (DNA methyltransferase inhibitor) treatment rescued the survival of Ikkαf/f;K5Cre mice, which usually die within three weeks after birth. Ikkαf/f;Myd88f/f;K5Cre mice also develop much fewer spontaneous skin tumors. By analyzing a cohort of human head and neck SCCs from TCGA database, we found that Dicer expression is concurrently downregulated among 32% of patients with low IKKα expression. Taken together, our finding illustrated how IKKα loss mediates Dicer gene silencing, which turns off miRNA processing, including miR-203-3p maturation, and eventually triggers Myd88-dependent inflammation. Thus, our study revealed a novel IKKα/Dicer/miR-203-3p/Myd88 signaling loop, in which IKKα serves as a molecular switch linking microRNA biogenesis and inflammation in skin tumorigenesis. Citation Format: Feng Zhu, Jian Zhang, Jami Willette-Brown, Ling Su, Xiaolin Wu, Yinling Hu. IKKa serves as a molecular switch linking miRNA biogenesis and inflammation in skin tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2355.

Abstract 1491: Immune resistance emerges from tumor-initiating stem cells

Abstract The remarkable capacity of the long-lived stem cells-like population to self-renew, initiate and propagate a tumor placed these cancer stem cells (CSC) at the root of tumorigenesis. Many unique molecular features of stem cells endow these key population with specialized properties to drive tumor formation. However, in order to achieve these functions, the neoplastic stem cells must first overcome the immune barrier. Although it has long been postulated that, as the core of a tumor, CSCs must be superior at evading the immune detection, direct evidence is still lacking to demonstrate this stem cell trait. Another important feature of CSCs is that these cells are programed to resist traditional cancer treatments, including chemotherapy and radiation. Yet, it is still unexplored how CSCs react to the immunotherapy, which just start to revolutionize the cancer treatments. In order to address these fascinating questions, we have designed a spontaneous skin squamous cell carcinoma (SCC) mouse model that can be effectively targeted by adoptive cell transfer (ACT)-based immunotherapy, without losing the targeted tumor antigen. Employing this model, we have lineage traced the fate of tumor-initiating stem cells during and after ACT treatment. In this study, we will present data demonstrating that a subset of TGFβ-responding CSCs, residing at the tumor-stromal interface, are always able to survive from the injected CD8+ T cell treatment and give rise to the relapsed tumor. Furthermore, the extensive profiling of ACT surviving stem cells revealed that multiple immune resistant pathways are highly up-regulated in these stem cells. In addition, TGFβ appears to orchestrate these immune resistant processes in the tumor-initiating stem cells. This study highlights CSCs as the core to mediate immune evasion. Citation Format: Yuxuan Miao. Immune resistance emerges from tumor-initiating stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1491.

Paradoxical roles of TGF-β signaling in suppressing and promoting squamous cell carcinoma.

Transforming growth factor β (TGF-β) signaling either promotes or inhibits tumor formation and/or progression of many cancer types including squamous cell carcinoma (SCC). Canonical TGF-β signaling is mediated by a number of downstream proteins including Smad family proteins. Alterations in either TGF-β or Smad signaling can impact cancer. For instance, defects in TGF-β type I and type II receptors (TGF-βRI and TGF-βRII) and in Smad2/3/4 could promote tumor development. Conversely, increased TGF-β1 and activated TGF-βRI and Smad3 have all been shown to have tumor-promoting effects in experimental systems of human and mouse SCCs. Among TGF-β/Smad signaling, only TGF-βRII or Smad4 deletion in mouse epithelium causes spontaneous SCC in the mouse model, highlighting the critical roles of TGF-βRII and Smad4 in tumor suppression. Herein, we review the dual roles of the TGF-β/Smad signaling pathway and related mechanisms in SCC, highlighting the potential benefits and challenges of TGF-β/Smad-targeted therapies.

Paradoxical roles of TGF-β signaling in suppressing and promoting squamous cell carcinoma.

Transforming growth factor β (TGF-β) signaling either promotes or inhibits tumor formation and/or progression of many cancer types including squamous cell carcinoma (SCC). Canonical TGF-β signaling is mediated by a number of downstream proteins including Smad family proteins. Alterations in either TGF-β or Smad signaling can impact cancer. For instance, defects in TGF-β type I and type II receptors (TGF-βRI and TGF-βRII) and in Smad2/3/4 could promote tumor development. Conversely, increased TGF-β1 and activated TGF-βRI and Smad3 have all been shown to have tumor-promoting effects in experimental systems of human and mouse SCCs. Among TGF-β/Smad signaling, only TGF-βRII or Smad4 deletion in mouse epithelium causes spontaneous SCC in the mouse model, highlighting the critical roles of TGF-βRII and Smad4 in tumor suppression. Herein, we review the dual roles of the TGF-β/Smad signaling pathway and related mechanisms in SCC, highlighting the potential benefits and challenges of TGF-β/Smad-targeted therapies.

Science Signaling Podcast for 21 June 2016: MET and skin cancer.

This Podcast features an interview with Stuart Yuspa, senior author of a Research Article that appears in the 21 June 2016 issue of Science Signaling, about how activation of the receptor tyrosine kinase MET stimulates the formation of squamous cell carcinoma in the skin. Hepatocyte growth factor (HGF) is produced by mesenchymal cells and stimulates MET, which is present on the surface of epithelial cells. HGF-MET signaling directs the proliferation and migration of epithelial cells during the development of various organs and is important during wound healing. Aberrant MET activation has been implicated in several types of cancer, including squamous cell carcinoma. Using a model in which mice overexpressing HGF develop spontaneous squamous cell carcinomas in the skin, Cataisson et al found that MET promoted the development of squamous tumors by stimulating the synthesis and release of ligands that activate the epidermal growth factor receptor (EGFR). This mechanism was similar to that through which oncogenic RAS promotes skin tumors. Blocking EGFR signaling caused HGF-induced squamous tumors to regress, suggesting that EGFR inhibitors might be useful for treating squamous cell carcinomas.Listen to Podcast.

Abstract 5147: Canine spontaneous head and neck squamous cell carcinomas represent their human counterparts at the molecular level

Abstract Spontaneous canine head and neck squamous cell carcinoma (HNSCC) represents an excellent model of human HNSCC but is greatly understudied. To better utilize this valuable resource, we performed its first genome-wide characterization by investigating 12 canine HNSCC cases, of which 9 are oral, via high density array comparative genomic hybridization and RNA-seq. The analyses reveal that these canine cancers faithfully recapitulate many key molecular features of human HNSCC. These include similar genomic copy number abnormality landscapes, analogous sequence mutation patterns, and recurrent alteration of known HNSCC genes (e.g., MYC, CDKN2A) and pathways (e.g., cell cycle, mitogenic signaling, TGFβ signaling). Amplification or overexpression of protein kinase genes, matrix metalloproteinase genes, and epithelial-mesenchymal transition genes TWIST1 and SNAI are also prominent in these canine tumors. The strong dog-human molecular homology fundamentally justifies the use of our novel dog-human comparison strategy for driver-passenger discrimination for HNSCC. This cross-species comparison will significantly advance beyond the current findings with the massive genomic data of the cancer genome atlas (TCGA), as exemplified by our pilot study on human 8q, a recurrently amplified site in human HNSCC. HNSCC represents the sixth leading cancer by incidence in humans; thus developing effective therapeutic interventions is important. While our understanding of the biology of HNSCC has been greatly advanced over past decades, translating these findings into clinical success has been frustratingly slow. A significant obstacle is the lack of translational model that can bridge the gap between preclinical research and human clinical trials. Our study indicates that spontaneous canine HSCC can effectively bridge this gap, significantly accelerating anti-HNSCC drug discovery. Note: This abstract was not presented at the meeting. Citation Format: Deli Liu, Huan Xiong, Angela E. Ellis, Nicole C. Northrup, Dong M. Shin, Shaying Zhao. Canine spontaneous head and neck squamous cell carcinomas represent their human counterparts at the molecular level. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5147. doi:10.1158/1538-7445.AM2015-5147

Pluripotency factor Nanog is tumorigenic by deregulating DNA damage response in somatic cells.

The pluripotency gene Nanog is not expressed in normal adult tissues but is overexpressed in some human cancers. However, the tumorigenic roles of Nanog remain unclear. The ectopic expression of Nanog is not sufficient to induce spontaneous tumors in mice but can promote metastasis of established tumors by activating the expression of metastatic genes. The expression of Nanog in mouse skin activates tumor suppressor p53, leading to the differentiation of epidermal stem cells. In the absence of p53, Nanog induces spontaneous squamous cell carcinoma, identifying a novel role of Nanog in tumorigenesis. Therefore, the induction of p53 and differentiation in Nanog-expressing skin suppresses the tumorigenic activities of Nanog, which include the induction of DNA double-stranded break damage. Notably, Nanog interacts with the KRAB-associated protein 1 (KAP1) and inhibits its sumoylation activity, impairing KAP1-mediated chromatin remodeling, which is important for efficiently activating DNA damage response. In summary, Nanog is an oncogene with multiple roles in promoting tumorigenesis and metastasis.

Abstract 603: TNFR1 is indispensable in the development of spontaneous lung squamous cell carcinomas

Abstract We recently reported that kinase-dead IKKα knockin mice, in which an ATP binding site of IKKα, Lys 44 was replaced by alanine (IkkαKA/KA), develop spontaneous lung SCCs that were closely associated with lung inflammatory microenvironment. This mouse model resembles human lung SCCs, sheds light on the mechanisms underlying lung malignancy development, and identifies targets for therapy of lung SCCs (Xiao et al. Cancer Cell 23(4):527-40, 2013). To further elucidate what and how the inflammatory stimuli contribute to the tumorigenesis, we generated bitransgenic mice harboring IkkαKA/KA and tnfrsf1-/- (KALTFR). It was identified that the KALTFR mice display lung tumor-free even the lung inflammations are still severe, while almost all of the littermate IkkαKA/KA mice developed lung SCCs. Interestingly, we found a noticeably increased TNFR1 expression in the lung SCCs of IkkαKA/KA mice compared to normal lung tissues. It has also been detected that the TNFR1 expressions are significantly higher in both transcript and protein levels in human lung SCCs with the compare to their adjacent tissues. These findings indicate that TNFR1 may play a crucial role in the development of lung SCC in either IkkαKA/KA mice or humans. Further investigation demonstrated that TNFR1 is a critical factor to regulate nuclear transcriptional factors and oncogenes including C-Myc, through one bromodomain (BRD) proteins- containing complex. These findings broaden our understanding about how inflammatory microenvironment contributes to tumorigenesis through TNFα-TNFRs engagements. Citation Format: Zuoxiang Xiao, Jami Willette-Brown, Yinling Hu, Sichuan Xi. TNFR1 is indispensable in the development of spontaneous lung squamous cell carcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 603. doi:10.1158/1538-7445.AM2014-603

Abstract 931: Role of IKKα in K-ras-driven lung carcinogenesis

Abstract IKKα, one of subunits of the IKK complex, is required for the epidermal homeostasis. We previously demonstrated that keratin 5-specific deletion of IKKα induces spontaneous skin squamous cell carcinomas (SCCs) in mice. Interestingly, IKKα kinase dead knockin (IKKαK44A/K44A, KA) mice spontaneously develop lung SCCs which is associated with increased inflammation and IKKα reduction. Thus, IKKα downregulation is crucial for the SCC formation in skin and lung. Here, we attempted to verify whether IKKα inactivation plays a role in the development of lung adenocarcinomas (ADCs) as well. We have crossed between KA mice and K-rasG12D oncogenic mutant (Kras) mice. Then, these IKKαK44A/K44A;K-rasG12D (KA;Kras) mice were intratracheally treated with adenovirus carrying the cre recombinase gene to activate K-ras oncogene. At 4.5 months after intratracheal injection, the lung weight was significantly increased in KA;Kras compared to Kras mice. Moreover, the lungs of KA;Kras mice showed increased bronchial epithelial cells and Ki67-positive cells in the bronchial regions. At 7 months after treatment, lung tumors in KA;Kras mice were larger compared to tumors in Kras mice. Finally, KA;Kras mice had shorter life span due to breathing problems. KA;Kras mice started to die at around 20 weeks after injection, while KR mice started to die from around 30 weeks. To confirm whether IKKα reduction promotes K-ras-driven lung carcinogenesis, we generated IKKαf/f (FF) mice crossed with Kras (FF;Kras) mice. Similar to KA;Kras, FF;Kras mice showed significant increase of lung weight, hyperproliferative bronchial abnormalities, Ki-67-positive cells in the bronchial regions and the number of large tumors at 4 months following treatment compared with Kras only group. Taken together, these data suggest that IKKα downregulation promotes K-ras-driven lung tumorigenesis and plays an important role in lung ADCs as well as in SCCs. The underlying molecular mechanism needs to be further investigated. Citation Format: Na-Young Song, Jami Willette-Brown, Mahesh Dalta, Yinling Hu. Role of IKKα in K-ras-driven lung carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 931. doi:10.1158/1538-7445.AM2014-931

High-affinity Cyclic Peptide Matriptase Inhibitors

Sunflower trypsin inhibitor-1 (SFTI-1) and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) are potent protease inhibitors comprising a cyclic backbone. Elucidation of structure-activity relationships for SFTI-1 and MCoTI-II was used to design inhibitors with enhanced inhibitory activity. An analog of MCoTI-II is one of the most potent inhibitors of matriptase. These results provide a solid basis for the design of selective peptide inhibitors of matriptase with therapeutic potential. The type II transmembrane serine protease matriptase is a key activator of multiple signaling pathways associated with cell proliferation and modification of the extracellular matrix. Deregulated matriptase activity correlates with a number of diseases, including cancer and hence highly selective matriptase inhibitors may have therapeutic potential. The plant-derived cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), is a promising drug scaffold with potent matriptase inhibitory activity. In the current study we have analyzed the structure-activity relationships of SFTI-1 and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II), a structurally divergent trypsin inhibitor from Momordica cochinchinensis that also contains a cyclic backbone. We show that MCoTI-II is a significantly more potent matriptase inhibitor than SFTI-1 and that all alanine mutants of both peptides, generated using positional scanning mutagenesis, have decreased trypsin affinity, whereas several mutations either maintain or result in enhanced matriptase inhibitory activity. These intriguing results were used to design one of the most potent matriptase inhibitors known to date with a 290 pm equilibrium dissociation constant, and provide the first indication on how to modulate affinity for matriptase over trypsin in cyclic peptides. This information might be useful for the design of more selective and therapeutically relevant inhibitors of matriptase.

Abstract 1083: The pivotal role of IKKalpha in the development of spontaneous lung squamous cell carcinomas.

Abstract During the last decades lung cancer has become the leading cause of cancer deaths in the world, and the need to develop better diagnostic techniques and therapies is urgent. To advance the understanding of this disease, various genetically engineered and chemical induced mouse models have been established. However, there are no robust animal models of lung squamous cell carcinomas (SCCs), one of the major types of lung cancer. Here, we generated Ikkα-KA/KA knock-in mice (KA/KA) in which an ATP binding site of IKKα, Lys 44 was replaced by alanine. All the knock-in mice at 4 to 6 months of age developed spontaneous lethal lung SCCs associated with markedly increased leukocyte infiltration and expression of cytokines, and chemokines in the lungs. Furthermore, we identified deregulated c-myc, Nanog, Oct3/4, p53, Rb, EGFR, MAPK, Jun-B, p63, Trim29, Rhov, CDK1, and IGF1 in mouse lung SCCs, and identified reduced IKKα and IκBα and increased ROS1 in mutant lungs and SCCs, some of which were found in human lung SCCs. Lung cancers were prevented by reintroducing epithelial-cell IKKα, depleting macrophages or depleting lymphocytes. This study not only provides a novel model for studying the pathogenesis, treatment, early detection, and prevention of human lung SCCs, but also demonstrates how a single mutation in IKKα elicits malignancy through the combined epithelial-cell-autonomous and immune mechanisms. Citation Format: Zuoxiang Xiao, Qun Jiang, Jami Willette-Brown, Feng Zhu, Sichuan Xi, Sandra Burkett, Fanching Lin, Timothy Back, Mahesh Datla, Zhonghe Sun, Romina Goldszmid, Xiaolin Wu, David Schrump, Howard Young, Georgio Trinchieri, Robert Wiltrout, Yinling Hu. The pivotal role of IKKalpha in the development of spontaneous lung squamous cell carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1083. doi:10.1158/1538-7445.AM2013-1083

Mouse Genetic Models Reveal Surprising Functions of IkB Kinase Alpha in Skin Development and Skin Carcinogenesis

Gene knockout studies unexpectedly reveal a pivotal role for IκB kinase alpha (IKKα) in mouse embryonic skin development. Skin carcinogenesis experiments show that Ikkα heterozygous mice are highly susceptible to chemical carcinogen or ultraviolet B light (UVB) induced benign and malignant skin tumors in comparison to wild-type mice. IKKα deletion mediated by keratin 5 (K5).Cre or K15.Cre in keratinocytes induces epidermal hyperplasia and spontaneous skin squamous cell carcinomas (SCCs) in Ikkα floxed mice. On the other hand, transgenic mice overexpressing IKKα in the epidermis, under the control of a truncated loricrin promoter or K5 promoter, develop normal skin and show no defects in the formation of the epidermis and other epithelial organs, and the transgenic IKKα represses chemical carcinogen or UVB induced skin carcinogenesis. Moreover, IKKα deletion mediated by a mutation, which generates a stop codon in the Ikkα gene, has been reported in a human autosomal recessive lethal syndrome. Downregulated IKKα and Ikkα mutations and deletions are found in human skin SCCs. The collective evidence not only highlights the importance of IKKα in skin development, maintaining skin homeostasis, and preventing skin carcinogenesis, but also demonstrates that mouse models are extremely valuable tools for revealing the mechanisms underlying these biological events, leading our studies from bench side to bedside.