Novel Mouse Cell Lines and In Vivo Models for Human High-Grade Neuroendocrine Lung Carcinoma, Small Cell Lung Carcinoma (SCLC), and Large Cell Neuroendocrine Carcinoma (LCNEC).

There is a clear need to expand the toolkit of adequate mouse models and cell lines available for preclinical studies of high-grade neuroendocrine lung carcinoma (small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC)). SCLC and LCNEC are two highly aggressive tumor types with dismal prognoses and few therapeutic options. Currently, there is an extreme paucity of material, particularly in the case of LCNEC. Given the lack of murine cell lines and transplant models of LCNEC, the need is imperative. In this study, we generated and examined new models of LCNEC and SCLC transplantable cell lines derived from our previously developed primary mouse LCNEC and SCLC tumors. RNA-seq analysis demonstrated that our cell lines and syngeneic tumors maintained the transcriptome program from the original transgenic primary tumor and displayed strong similarities to human SCLC or LCNEC. Importantly, the SCLC transplanted cell lines showed the ability to metastasize and mimic this characteristic of the human condition. In summary, we generated mouse cell line tools that allow further basic and translational research as well as preclinical testing of new treatment strategies for SCLC and LCNEC. These tools retain important features of their human counterparts and address the lack of LCNEC disease models.

Pten and p53 Loss in the Mouse Lung Causes Adenocarcinoma and Sarcomatoid Carcinoma.

Lung cancer remains the leading cause of cancer deaths worldwide. Among the Non-Small Cell Carcinoma (NSCLC) category, Adenocarcinoma (ADC) represents the most common type, with different reported driver mutations, a bunch of models described and therapeutic options. Meanwhile, Pulmonary Sarcomatoid Carcinoma (PSC) is one of the rarest, with very poor outcomes, scarce availability of patient material, no effective therapies and no models available for preclinical research. Here, we describe that the combined deletion of Pten and Trp53 in the lungs of adult conditional mice leads to the development of both ADC and PSC irrespective of the lung targeted cell type after naphthalene induced airway epithelial regeneration. Although this model shows long latency periods and incomplete penetrance for tumor development, it is the first PSC mouse model reported so far, and sheds light on the relationships between ADC and PSC and their cells of origin. Moreover, human ADC show strong transcriptomic similarities to the mouse PSC, providing a link between both tumor types and the human ADC.

Neuroendocrine Lung Cancer Mouse Models: An Overview.

Neuroendocrine lung tumors comprise a range of malignancies that extend from benign tumorlets to the most prevalent and aggressive Small Cell Lung Carcinoma (SCLC). They also include low-grade Typical Carcinoids (TC), intermediate-grade Atypical Carcinoids (AC) and high-grade Large Cell Neuroendocrine Carcinoma (LCNEC). Optimal treatment options have not been adequately established: surgical resection when possible is the choice for AC and TC, and for SCLC chemotherapy and very recently, immune checkpoint inhibitors. Some mouse models have been generated based on the molecular alterations identified in genomic analyses of human tumors. With the exception of SCLC, there is a limited availability of (preclinical) models making their development an unmet need for the understanding of the molecular mechanisms underlying these diseases. For SCLC, these models are crucial for translational research and novel drug testing, given the paucity of human material from surgery. The lack of early detection systems for lung cancer point them out as suitable frameworks for the identification of biomarkers at the initial stages of tumor development and for testing molecular imaging methods based on somatostatin receptors. Here, we review the relevant models reported to date, their impact on the understanding of the biology of the tumor subtypes and their relationships, as well as the effect of the analyses of the genetic landscape of the human tumors and molecular imaging tools in their development.

Development and long-term evaluation of a new 68Ge/68Ga generator based on nano-SnO2 for PET imaging.

Radionuclide generator systems can routinely provide radionuclides on demand such as 68Ga produced by a 68Ge/68Ga generator without the availability of an on-site accelerator or a research reactor. Thus, in this work nano-SnO2 was used to develop a new 68Ge/68Ga generator which was evaluated over a period of 17 months and 305 elution cycles. The elution yield was 91.1 ± 1.8% in the first 7 mL (1 M HCl as eluent) when the generator was new and then it decreased with time and use to 73.8 ± 1.9%. Around 80% of the elutable 68Ga activity was obtained in 1 mL and the 68Ge content in the eluate did not exceed 1 × 10-4% over the investigation period when it was eluted regularly. The described generator provided adequate results for radiolabelling of DOTA-TOC with direct use of eluate. In addition, [68Ga]Ga-DOTA-TOC was tested satisfactorily for in vivo tumor detection by microPET/CT imaging in a lung cancer mouse model.

New models of large-cell neuroendocrine carcinoma and small-cell lung carcinoma.

High-grade neuroendocrine lung carcinomas (LCNEC, SCLC) are recalcitrant cancers for which no optimal management has been achieved. We have recently described two models of LCNEC and SCLC developed upon inactivation of 4 tumor suppressors genes (Rb1 (RB transcriptional corepressor 1), Rbl1 (RB transcriptional corepressor like 1), Pten (phosphatase and tensin homolog), Trp53 (transformation-related protein 53), which provide a suitable frame for preclinical intervention. A defined model for LCNEC had not been previously reported.

Differential development of large-cell neuroendocrine or small-cell lung carcinoma upon inactivation of 4 tumor suppressor genes.

High-grade neuroendocrine lung malignancies (large-cell neuroendocrine cell carcinoma, LCNEC, and small-cell lung carcinoma, SCLC) are among the most deadly lung cancer conditions with no optimal clinical management. The biological relationships between SCLC and LCNEC are still largely unknown and a current matter of debate as growing molecular data reveal high heterogeneity with potential therapeutic consequences. Here we describe murine models of high-grade neuroendocrine lung carcinomas generated by the loss of 4 tumor suppressors. In an Rbl1-null background, deletion of Rb1, Pten, and Trp53 floxed alleles after Ad-CMVcre infection in a wide variety of lung epithelial cells produces LCNEC. Meanwhile, inactivation of these genes using Ad-K5cre in basal cells leads to the development of SCLC, thus differentially influencing the lung cancer type developed. So far, a defined model of LCNEC has not been reported. Molecular and transcriptomic analyses of both models revealed strong similarities to their human counterparts. In addition, a 68Ga-DOTATOC-based molecular-imaging method provides a tool for detection and monitoring the progression of the cancer. These data offer insight into the biology of SCLC and LCNEC, providing a useful framework for development of compounds and preclinical investigations in accurate immunocompetent models.

CDK4/6 Inhibitor as a Novel Therapeutic Approach for Advanced Bladder Cancer Independently of RB1 Status.

PURPOSE: Bladder cancer is a clinical and social problem due to its high incidence and recurrence rates. It frequently appears in elderly patients showing other medical comorbidities that hamper the use of standard chemotherapy. We evaluated the activity of CDK4/6 inhibitor as a new therapy for patients unfit for cisplatin (CDDP). EXPERIMENTAL DESIGN: Bladder cancer cell lines were tested for in vitro sensitivity to CDK4/6 inhibition. A novel metastatic bladder cancer mouse model was developed and used to test its in vivo activity. RESULTS: Cell lines tested were sensitive to CDK4/6 inhibition, independent on RB1 gene status. Transcriptome analyses and knockdown experiments revealed a major role for FOXM1 in this response. CDK4/6 inhibition resulted in reduced FOXM1 phosphorylation in vitro and in vivo and showed synergy with CDDP, allowing a significant tumor regression. FOXM1 exerted important oncogenic roles in bladder cancer. CONCLUSIONS: CDK4/6 inhibitors, alone or in combination, are a novel therapeutic strategy for patients with advanced bladder cancer previously classified as unfit for current treatment options.

Ablating all three retinoblastoma family members in mouse lung leads to neuroendocrine tumor formation.

Lung cancer is a deadly disease with increasing cases diagnosed worldwide and still a very poor prognosis. While mutations in the retinoblastoma (RB1) tumor suppressor have been reported in lung cancer, mainly in small cell lung carcinoma, the tumor suppressive role of its relatives p107 and p130 is still a matter of debate. To begin to investigate the role of these two Rb family proteins in lung tumorigenesis, we have generated a conditional triple knockout mouse model (TKO) in which the three Rb family members can be inactivated in adult mice. We found that ablation of all three family members in the lung of mice induces tumorlets, benign neuroendocrine tumors that are remarkably similar to their human counterparts. Upon chemical carcinogenesis, DHPN and urethane accelerate tumor development; the TKO model displays increased sensitivity to DHPN, and urethane increases malignancy of tumors. All the tumors developing in TKO mice (spontaneous and chemically induced) have neuroendocrine features but do not progress to fully malignant tumors. Thus, loss of Rb and its family members confers partial tumor susceptibility in neuroendocrine lineages in the lungs of mice. Our data also imply the requirement of other oncogenic signaling pathways to achieve full transformation in neuroendocrine lung lesions mutant for the Rb family.

Deregulation of the pRb-E2F4 axis alters epidermal homeostasis and favors tumor development.

E2F/RB activity is altered in most human tumors. The retinoblastoma family of proteins plays a key role in regulating the progression of the cell cycle from the G1 to S phases. This is achieved through negative regulation of E2F transcription factors, important positive regulators of cell cycle entry. E2F family members are divided into two groups: activators (E2F1-E2F3a) and repressors (E2F3b-E2F8). E2F4 accounts for a large part of the E2F activity and is a main E2F repressor member in vivo. Perturbations in the balance from quiescence towards proliferation contribute to increased mitotic gene expression levels frequently observed in cancer. We have previously reported that combined Rb1-Rbl1 or Rb1-E2f1 ablation in epidermis produces important alterations in epidermal proliferation and differentiation, leading to tumor development. However, the possible roles of E2F4 in this context are still to be determined. Here, we show the absence of any discernible phenotype in the skin of mice lacking of E2f4. In contrast, the inducible loss of Rb1 in the epidermis of E2F4-null mice produced multiple skin abnormalities including altered differentiation and proliferation, spontaneous wounds, carcinoma in situ development and stem cell perturbations. All these phenotypic alterations are associated with extensive gene expression changes, the induction of c-myc and the Akt activation. Moreover the whole transcriptome analyses in comparison with previous models generated also revealed extensive changes in multiple repressive complexes and in transcription factor activity. These results point to E2F4 as a master regulator in multiple steps of epidermal homeostasis in Rb1 absence.

The downregulation of ΔNp63 in p53-deficient mouse epidermal tumors favors metastatic behavior.

The TP63 gene codes for two major isoform types, TAp63 and ΔNp63, with probable opposite roles in tumorigenesis. The ΔNp63α protein is frequently amplified and overexpressed in different epithelial tumors. Accordingly, it has been considered a potential oncogene. Nonetheless, a possible metastatic suppressor activity has also been suggested based on the experimental observation that its expression is reduced or even absent in advanced invasive tumors. Such metastatic suppressor activities are often related to tumors bearing point mutated TP53 gene. However, its potential roles in TP53-deficient tumors are poorly characterized. Here we show that in spontaneous tumors, induced by the epidermal-specific Trp53 ablation, the reduction of ΔNp63 expression is an early event, whereas it is re-expressed in the lung metastatic lesions. Using knock down and ectopic expression approaches, we show that ΔNp63 expression opposes the epithelial-mesenchymal transition and reduces the metastatic potential of the cells. This process occurs through the modulation of ΔNp63-dependent downstream targets (including transcription factors and microRNAs) likely to play metastatic roles. Further, ΔNp63 also favors the expression of factors involved in iPS reprogramming, thus suggesting that it can also modulate specific stem cell traits in mouse epidermal tumor cells. Overall, our data assign antimetastatic roles to ΔNp63 in the context of p53 deficiency and epidermis.

In vivo disruption of an Rb-E2F-Ezh2 signaling loop causes bladder cancer.

Bladder cancer is a highly prevalent human disease in which retinoblastoma (Rb) pathway inactivation and epigenetic alterations are common events. However, the connection between these two processes is still poorly understood. Here, we show that the in vivo inactivation of all Rb family genes in the mouse urothelium is sufficient to initiate bladder cancer development. The characterization of the mouse tumors revealed multiple molecular features of human bladder cancer, including the activation of E2F transcription factor and subsequent Ezh2 expression and the activation of several signaling pathways previously identified as highly relevant in urothelial tumors. These mice represent a genetically defined model for human high-grade superficial bladder cancer. Whole transcriptional characterizations of mouse and human bladder tumors revealed a significant overlap and confirmed the predominant role for Ezh2 in the downregulation of gene expression programs. Importantly, the increased tumor recurrence and progression in human patients with superficial bladder cancer is associated with increased E2F and Ezh2 expression and Ezh2-mediated gene expression repression. Collectively, our studies provide a genetically defined model for human high-grade superficial bladder cancer and demonstrate the existence of an Rb-E2F-Ezh2 axis in bladder whose disruption can promote tumor development.

Akt signaling leads to stem cell activation and promotes tumor development in epidermis.

Hair follicle stem cells (HF-SCs) alternate between periods of quiescence and proliferation, to finally differentiate into all the cell types that constitute the hair follicle. Also, they have been recently identified as cells of origin in skin cancer. HF-SCs localize in a precise region of the hair follicle, the bulge, and molecular markers for this population have been established. Thus, HF-SCs are good model to study the potential role of oncogenic activations on SC physiology. Expression of a permanently active form of Akt (myrAkt) in basal cells leads to Akt hyperactivation specifically in the CD34(+)Itga6(H) population. This activation causes bulge stem cells to exit from quiescence increasing their response to proliferative stimuli and affecting some functions such as cell migration. HF-SC identity upon Akt activation is preserved; in this sense, increased proliferation does not result in stem cell exhaustion with age suggesting that Akt activation does not affect self-renewal an important aspect for normal tissue maintenance and cancer development. Genome-wide transcriptome analysis of HF-SC isolated from myrAkt and wild-type epidermis underscores changes in metabolic pathways characteristic of cancer cells. These differences manifest during a two-step carcinogenesis protocol in which Akt activation in HF-SCs results in increased tumor development and malignant transformation.

p21 suppresses inflammation and tumorigenesis on pRB-deficient stratified epithelia.

The retinoblastoma gene product (pRb) controls proliferation and differentiation processes in stratified epithelia. Importantly, and in contrast to other tissues, Rb deficiency does not lead to spontaneous skin tumor formation. As the cyclin-dependent kinase inhibitor p21 regulates proliferation and differentiation in the absence of pRb, we analyzed the consequences of deleting p21 in pRb-ablated stratified epithelia (hereafter pRb(ΔEpi);p21-/-). These mice display an enhancement of the phenotypic abnormalities observed in pRb(ΔEpi) animals, indicating that p21 partially compensates pRb absence. Remarkably, pRb(ΔEpi);p21-/- mice show an acute skin inflammatory phenotype and develop spontaneous epithelial tumors, particularly affecting tongue and oral tissues. Biochemical analyses and transcriptome studies reveal changes affecting multiple pathways, including DNA damage and p53-dependent signaling responses. Comparative metagenomic analyses, together with the histopathological profiles, indicate that these mice constitute a faithful model for human head and neck squamous cell carcinomas. Collectively, our findings demonstrate that p21, in conjunction with pRb, has a central role in regulating multiple epithelial processes and orchestrating specific tumor suppressor functions.

Skin Tumors Rb(eing) Uncovered.

The Rb1 gene was the first bona fide tumor suppressor identified and cloned more than 25 years ago. Since then, a plethora of studies have revealed the functions of pRb and the existence of a sophisticated and strictly regulated pathway that modulates such functional roles. An emerging paradox affecting Rb1 in cancer connects the relatively low number of mutations affecting Rb1 gene in specific human tumors, compared with the widely functional inactivation of pRb in most, if not in all, human cancers. The existence of a retinoblastoma family of proteins pRb, p107, and p130 and their potential unique and overlapping functions as master regulators of cell cycle progression and transcriptional modulation by similar processes, may provide potential clues to explain such conundrum. Here, we will review the development of different genetically engineered mouse models, in particular those affecting stratified epithelia, and how they have offered new avenues to understand the roles of the Rb family members and their targets in the context of tumor development and progression.

A novel tumor suppressor network in squamous malignancies.

The specific ablation of Rb1 gene in stratified epithelia (Rb(F/F);K14cre) promotes proliferation and altered differentiation but is insufficient to produce spontaneous tumors. The pRb relative, p107, compensates some of the functions of pRb in these tissues; however, Rb(F/F);K14cre;p107(-/-) mice die postnatally. Here we show, using an inducible mouse model (Rb(F/F);K14creER(TM)), that p107 exerts specific tumor suppressor functions in the absence of pRb in stratified epithelia. The simultaneous absence of pRb and p107 produces impaired p53 transcriptional functions and reduction of Pten expression, allowing spontaneous squamous carcinoma development. These tumors display significant overlap with human squamous carcinomas, supporting that Rb(F/F);K14creER(TM);p107(-/-) mice might constitute a new model for these malignancies. Remarkably tumor development in vivo is partially alleviated by mTOR inhibition. These data demonstrate the existence of a previously unreported functional connection between pRb, Pten and p53 tumor suppressors, through p107, of a particular relevance in squamous tumor development.

Mouse p53-deficient cancer models as platforms for obtaining genomic predictors of human cancer clinical outcomes.

Mutations in the TP53 gene are very common in human cancers, and are associated with poor clinical outcome. Transgenic mouse models lacking the Trp53 gene or that express mutant Trp53 transgenes produce tumours with malignant features in many organs. We previously showed the transcriptome of a p53-deficient mouse skin carcinoma model to be similar to those of human cancers with TP53 mutations and associated with poor clinical outcomes. This report shows that much of the 682-gene signature of this murine skin carcinoma transcriptome is also present in breast and lung cancer mouse models in which p53 is inhibited. Further, we report validated gene-expression-based tests for predicting the clinical outcome of human breast and lung adenocarcinoma. It was found that human patients with cancer could be stratified based on the similarity of their transcriptome with the mouse skin carcinoma 682-gene signature. The results also provide new targets for the treatment of p53-defective tumours.

EMT and induction of miR-21 mediate metastasis development in Trp53-deficient tumours.

Missense mutations in TP53 gene promote metastasis in human tumours. However, little is known about the complete loss of function of p53 in tumour metastasis. Here we show that squamous cell carcinomas generated by the specific ablation of Trp53 gene in mouse epidermis are highly metastatic. Biochemical and genome-wide mRNA and miRNA analyses demonstrated that metastases are associated with the early induction of epithelial-mesenchymal transition (EMT) and deregulated miRNA expression in primary tumours. Increased expression of miR-21 was observed in undifferentiated, prometastatic mouse tumours and in human tumours characterized by p53 mutations and distant metastasis. The augmented expression of miR-21, mediated by active mTOR and Stat3 signalling, conferred increased invasive properties to mouse keratinocytes in vitro and in vivo, whereas blockade of miR-21 in a metastatic spindle cell line inhibits metastasis development. Collectively these data identify novel molecular mechanisms leading to metastasis in vivo originated by p53 loss in epithelia.

Gene expression profiling of mouse p53-deficient epidermal carcinoma defines molecular determinants of human cancer malignancy.

BACKGROUND: The epidermal specific ablation of Trp53 gene leads to the spontaneous development of aggressive tumors in mice through a process that is accelerated by the simultaneous ablation of Rb gene. Since alterations of p53-dependent pathway are common hallmarks of aggressive, poor prognostic human cancers, these mouse models can recapitulate the molecular features of some of these human malignancies. RESULTS: To evaluate this possibility, gene expression microarray analysis was performed in mouse samples. The mouse tumors display increased expression of cell cycle and chromosomal instability associated genes. Remarkably, they are also enriched in human embryonic stem cell gene signatures, a characteristic feature of human aggressive tumors. Using cross-species comparison and meta-analytical approaches, we also observed that spontaneous mouse tumors display robust similarities with gene expression profiles of human tumors bearing mutated TP53, or displaying poor prognostic outcome, from multiple body tissues. We have obtained a 20-gene signature whose genes are overexpressed in mouse tumors and can identify human tumors with poor outcome from breast cancer, astrocytoma and multiple myeloma. This signature was consistently overexpressed in additional mouse tumors using microarray analysis. Two of the genes of this signature, AURKA and UBE2C, were validated in human breast and cervical cancer as potential biomarkers of malignancy. CONCLUSIONS: Our analyses demonstrate that these mouse models are promising preclinical tools aimed to search for malignancy biomarkers and to test targeted therapies of prospective use in human aggressive tumors and/or with p53 mutation or inactivation.