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17 pages, 7546 KiB

Open AccessFeature PaperArticle

BAP1 Malignant Pleural Mesothelioma Mutations in Caenorhabditis elegans Reveal Synthetic Lethality between ubh-4/BAP1 and the Proteasome Subunit rpn-9/PSMD13

by Carmen Martínez-Fernández, Sweta Jha, Elisabet Aliagas, Carina I. Holmberg, Ernest Nadal and Julián Cerón

Cells 2023, 12(6), 929; https://doi.org/10.3390/cells12060929 - 18 Mar 2023

Cited by 2 | Viewed by 2492

Abstract

The deubiquitinase BAP1 (BRCA1-associated protein 1) is associated with BAP1 tumor predisposition syndrome (TPDS). BAP1 is a tumor suppressor gene whose alterations in cancer are commonly caused by gene mutations leading to protein loss of function. By CRISPR-Cas, we have generated mutations in [...] Read more.

The deubiquitinase BAP1 (BRCA1-associated protein 1) is associated with BAP1 tumor predisposition syndrome (TPDS). BAP1 is a tumor suppressor gene whose alterations in cancer are commonly caused by gene mutations leading to protein loss of function. By CRISPR-Cas, we have generated mutations in ubh-4, the BAP1 ortholog in Caenorhabditis elegans, to model the functional impact of BAP1 mutations. We have found that a mimicked BAP1 cancer missense mutation (UBH-4 A87D; BAP1 A95D) resembles the phenotypes of ubh-4 deletion mutants. Despite ubh-4 being ubiquitously expressed, the gene is not essential for viability and its deletion causes only mild phenotypes without affecting 20S proteasome levels. Such viability facilitated an RNAi screen for ubh-4 genetic interactors that identified rpn-9, the ortholog of human PSMD13, a gene encoding subunit of the regulatory particle of the 26S proteasome. ubh-4[A87D], similarly to ubh-4 deletion, cause a synthetic genetic interaction with rpn-9 inactivation affecting body size, lifespan, and the development of germ cells. Finally, we show how ubh-4 inactivation sensitizes animals to the chemotherapeutic agent Bortezomib, which is a proteasome inhibitor. Thus, we have established a model to study BAP1 cancer-related mutations in C. elegans, and our data points toward vulnerabilities that should be studied to explore therapeutic opportunities within the complexity of BAP1 tumors. Full article

(This article belongs to the Special Issue Research Advances in Invertebrate Experimental Models of Malignant Growth)

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17 pages, 3986 KiB

Open AccessFeature PaperArticle

Transcriptomic Analysis Reveals JAK2/MPL-Independent Effects of Calreticulin Mutations in a C. elegans Model

by Ana Guijarro-Hernández, Laura Eder-Azanza, Cristina Hurtado, David Navarro-Herrera, Begoña Ezcurra, Francisco Javier Novo, Juan Cabello and José Luis Vizmanos

Cells 2023, 12(1), 186; https://doi.org/10.3390/cells12010186 - 2 Jan 2023

Cited by 4 | Viewed by 2386

Abstract

There is growing evidence that Ph-negative myeloproliferative neoplasms (MPNs) are disorders in which multiple molecular mechanisms are significantly disturbed. Since their discovery, CALR driver mutations have been demonstrated to trigger pathogenic mechanisms apart from the well-documented activation of JAK2/MPL-related pathways, but the [...] Read more.

There is growing evidence that Ph-negative myeloproliferative neoplasms (MPNs) are disorders in which multiple molecular mechanisms are significantly disturbed. Since their discovery, CALR driver mutations have been demonstrated to trigger pathogenic mechanisms apart from the well-documented activation of JAK2/MPL-related pathways, but the lack of experimental models harboring CALR mutations in a JAK2/MPL knockout background has hindered the research on these non-canonical mechanisms. In this study, CRISPR/Cas9 was performed to introduce homozygous patient-like calreticulin mutations in a C. elegans model that naturally lacks JAK2 and MPL orthologs. Whole-genome transcriptomic analysis of these worms was conducted, and some of the genes identified to be associated with processes involved in the pathogenesis of MPNs were further validated by qPCR. Some of the transcriptomic alterations corresponded to typically altered genes and processes in cancer and Ph-negative MPN patients that are known to be triggered by mutant calreticulin without the intervention of JAK2/MPL. However, interestingly, we have also found altered other processes described in these diseases that had not been directly attributed to calreticulin mutations without the intervention of JAK2 or MPL. Thus, these results point to a new experimental model for the study of the JAK2/MPL-independent mechanisms of mutant calreticulin that induce these biological alterations, which could be useful to study unknown non-canonical effects of the mutant protein. The comparison with a calreticulin null strain revealed that the alteration of all of these processes seems to be a consequence of a loss of function of mutant calreticulin in the worm, except for the dysregulation of Hedgehog signaling and flh-3. Further analysis of this model could help to delineate these mechanisms, and the verification of these results in mammalian models may unravel new potential therapeutic targets in MPNs. As far as we know, this is the first time that a C. elegans strain with patient-like mutations is proposed as a potential model for leukemia research. Full article

(This article belongs to the Special Issue Research Advances in Invertebrate Experimental Models of Malignant Growth)

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13 pages, 2934 KiB

Open AccessEditor’s ChoiceArticle

Oxidative Stress Is Associated with Overgrowth in Drosophila l(3)mbt Mutant Imaginal Discs

by Paula Climent-Cantó, Cristina Molnar, Paula Santabárbara-Ruiz, Cristina Prieto, Josep F. Abril, Florenci Serras and Cayetano Gonzalez

Cells 2022, 11(16), 2542; https://doi.org/10.3390/cells11162542 - 16 Aug 2022

Viewed by 2363

Abstract

The loss-of-function conditions for an l(3)malignant brain tumour (l(3)mbt) in larvae reared at 29 °C results in malignant brain tumours and hyperplastic imaginal discs. Unlike the former that have been extensively characterised, little is known about the latter. Here we report [...] Read more.

The loss-of-function conditions for an l(3)malignant brain tumour (l(3)mbt) in larvae reared at 29 °C results in malignant brain tumours and hyperplastic imaginal discs. Unlike the former that have been extensively characterised, little is known about the latter. Here we report the results of a study of the hyperplastic l(3)mbt mutant wing imaginal discs. We identify the l(3)mbt wing disc tumour transcriptome and find it to include genes involved in reactive oxygen species (ROS) metabolism. Furthermore, we show the presence of oxidative stress in l(3)mbt hyperplastic discs, even in apoptosis-blocked conditions, but not in l(3)mbt brain tumours. We also find that chemically blocking oxidative stress in l(3)mbt wing discs reduces the incidence of wing disc overgrowths. Our results reveal the involvement of oxidative stress in l(3)mbt wing discs hyperplastic growth. Full article

(This article belongs to the Special Issue Research Advances in Invertebrate Experimental Models of Malignant Growth)

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14 pages, 2136 KiB

Open AccessArticle

Conditional CRISPR-Cas Genome Editing in Drosophila to Generate Intestinal Tumors

by Shivohum Bahuguna, Siamak Redhai, Jun Zhou, Tianyu Wang, Fillip Port and Michael Boutros

Cells 2021, 10(11), 3156; https://doi.org/10.3390/cells10113156 - 13 Nov 2021

Cited by 3 | Viewed by 4256

Abstract

CRISPR-Cas has revolutionized genetics and extensive efforts have been made to enhance its editing efficiency by developing increasingly more elaborate tools. Here, we evaluate the CRISPR-Cas9 system in Drosophila melanogaster to assess its ability to induce stem cell-derived tumors in the intestine. We [...] Read more.

CRISPR-Cas has revolutionized genetics and extensive efforts have been made to enhance its editing efficiency by developing increasingly more elaborate tools. Here, we evaluate the CRISPR-Cas9 system in Drosophila melanogaster to assess its ability to induce stem cell-derived tumors in the intestine. We generated conditional tissue-specific CRISPR knockouts using different Cas9 expression vectors with guide RNAs targeting the BMP, Notch, and JNK pathways in intestinal progenitors such as stem cells (ISCs) and enteroblasts (EBs). Perturbing Notch and BMP signaling increased the proliferation of ISCs/EBs and resulted in the formation of intestinal tumors, albeit with different efficiencies. By assessing both the anterior and posterior regions of the midgut, we observed regional differences in ISC/EB proliferation and tumor formation upon mutagenesis. Surprisingly, high continuous expression of Cas9 in ISCs/EBs blocked age-dependent increase in ISCs/EBs proliferation and when combined with gRNAs targeting tumor suppressors, it prevented tumorigenesis. However, no such effects were seen when temporal parameters of Cas9 were adjusted to regulate its expression levels or with a genetically modified version, which expresses Cas9 at lower levels, suggesting that fine-tuning Cas9 expression is essential to avoid deleterious effects. Our findings suggest that modifications to Cas9 expression results in differences in editing efficiency and careful considerations are required when choosing reagents for CRISPR-Cas9 mutagenesis studies. In summary, Drosophila can serve as a powerful model for context-dependent CRISPR-Cas based perturbations and to test genome-editing systems in vivo. Full article

(This article belongs to the Special Issue Research Advances in Invertebrate Experimental Models of Malignant Growth)

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18 pages, 31735 KiB

Open AccessArticle

Modeling Notch-Induced Tumor Cell Survival in the Drosophila Ovary Identifies Cellular and Transcriptional Response to Nuclear NICD Accumulation

by Allison Jevitt, Yi-Chun Huang, Su-Mei Zhang, Deeptiman Chatterjee, Xian-Feng Wang, Geng-Qiang Xie and Wu-Min Deng

Cells 2021, 10(9), 2222; https://doi.org/10.3390/cells10092222 - 27 Aug 2021

Cited by 10 | Viewed by 4282

Abstract

Notch is a conserved developmental signaling pathway that is dysregulated in many cancer types, most often through constitutive activation. Tumor cells with nuclear accumulation of the active Notch receptor, NICD, generally exhibit enhanced survival while patients experience poorer outcomes. To understand the impact [...] Read more.

Notch is a conserved developmental signaling pathway that is dysregulated in many cancer types, most often through constitutive activation. Tumor cells with nuclear accumulation of the active Notch receptor, NICD, generally exhibit enhanced survival while patients experience poorer outcomes. To understand the impact of NICD accumulation during tumorigenesis, we developed a tumor model using the Drosophila ovarian follicular epithelium. Using this system we demonstrated that NICD accumulation contributed to larger tumor growth, reduced apoptosis, increased nuclear size, and fewer incidents of DNA damage without altering ploidy. Using bulk RNA sequencing we identified key genes involved in both a pre- and post- tumor response to NICD accumulation. Among these are genes involved in regulating double-strand break repair, chromosome organization, metabolism, like raptor, which we experimentally validated contributes to early Notch-induced tumor growth. Finally, using single-cell RNA sequencing we identified follicle cell-specific targets in NICD-overexpressing cells which contribute to DNA repair and negative regulation of apoptosis. This valuable tumor model for nuclear NICD accumulation in adult Drosophila follicle cells has allowed us to better understand the specific contribution of nuclear NICD accumulation to cell survival in tumorigenesis and tumor progression. Full article

(This article belongs to the Special Issue Research Advances in Invertebrate Experimental Models of Malignant Growth)

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12 pages, 3078 KiB

Open AccessArticle

Misshapen Disruption Cooperates with Ras^V12 to Drive Tumorigenesis

by Du Kong, Jin-Yu Lu, Xiaoqin Li, Sihua Zhao, Wenyan Xu, Jinan Fang, Xing Wang and Xianjue Ma

Cells 2021, 10(4), 894; https://doi.org/10.3390/cells10040894 - 14 Apr 2021

Cited by 1 | Viewed by 3569

Abstract

Although RAS family genes play essential roles in tumorigenesis, effective treatments targeting RAS-related tumors are lacking, partly because of an incomplete understanding of the complex signaling crosstalk within RAS-related tumors. Here, we performed a large-scale genetic screen in Drosophila eye imaginal [...] Read more.

Although RAS family genes play essential roles in tumorigenesis, effective treatments targeting RAS-related tumors are lacking, partly because of an incomplete understanding of the complex signaling crosstalk within RAS-related tumors. Here, we performed a large-scale genetic screen in Drosophila eye imaginal discs and identified Misshapen (Msn) as a tumor suppressor that synergizes with oncogenic Ras (Ras^V12) to induce c-Jun N-terminal kinase (JNK) activation and Hippo inactivation, then subsequently leads to tumor overgrowth and invasion. Moreover, ectopic Msn expression activates Hippo signaling pathway and suppresses Hippo signaling disruption-induced overgrowth. Importantly, we further found that Msn acts downstream of protocadherin Fat (Ft) to regulate Hippo signaling. Finally, we identified msn as a Yki/Sd target gene that regulates Hippo pathway in a negative feedback manner. Together, our findings identified Msn as a tumor suppressor and provide a novel insight into RAS-related tumorigenesis that may be relevant to human cancer biology. Full article

(This article belongs to the Special Issue Research Advances in Invertebrate Experimental Models of Malignant Growth)

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20 pages, 4518 KiB

Open AccessReview

Modelling Cancer Metastasis in Drosophila melanogaster

by Joanne L. Sharpe, Jason Morgan, Nicholas Nisbet, Kyra Campbell and Andreu Casali

Cells 2023, 12(5), 677; https://doi.org/10.3390/cells12050677 - 21 Feb 2023

Cited by 1 | Viewed by 4080

Abstract

Cancer metastasis, the process by which tumour cells spread throughout the body and form secondary tumours at distant sites, is the leading cause of cancer-related deaths. The metastatic cascade is a highly complex process encompassing initial dissemination from the primary tumour, travel through [...] Read more.

Cancer metastasis, the process by which tumour cells spread throughout the body and form secondary tumours at distant sites, is the leading cause of cancer-related deaths. The metastatic cascade is a highly complex process encompassing initial dissemination from the primary tumour, travel through the blood stream or lymphatic system, and the colonisation of distant organs. However, the factors enabling cells to survive this stressful process and adapt to new microenvironments are not fully characterised. Drosophila have proven a powerful system in which to study this process, despite important caveats such as their open circulatory system and lack of adaptive immune system. Historically, larvae have been used to model cancer due to the presence of pools of proliferating cells in which tumours can be induced, and transplanting these larval tumours into adult hosts has enabled tumour growth to be monitored over longer periods. More recently, thanks largely to the discovery that there are stem cells in the adult midgut, adult models have been developed. We focus this review on the development of different Drosophila models of metastasis and how they have contributed to our understanding of important factors determining metastatic potential, including signalling pathways, the immune system and the microenvironment. Full article

(This article belongs to the Special Issue Research Advances in Invertebrate Experimental Models of Malignant Growth)

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15 pages, 840 KiB

Open AccessReview

A Blueprint for Cancer-Related Inflammation and Host Innate Immunity

by Lucia García-López, Isabel Adrados, Dolors Ferres-Marco and Maria Dominguez

Cells 2021, 10(11), 3211; https://doi.org/10.3390/cells10113211 - 17 Nov 2021

Cited by 9 | Viewed by 3876

Abstract

Both in situ and allograft models of cancer in juvenile and adult Drosophila melanogaster fruit flies offer a powerful means for unravelling cancer gene networks and cancer–host interactions. They can also be used as tools for cost-effective drug discovery and repurposing. Moreover, in [...] Read more.

Both in situ and allograft models of cancer in juvenile and adult Drosophila melanogaster fruit flies offer a powerful means for unravelling cancer gene networks and cancer–host interactions. They can also be used as tools for cost-effective drug discovery and repurposing. Moreover, in situ modeling of emerging tumors makes it possible to address cancer initiating events—a black box in cancer research, tackle the innate antitumor immune responses to incipient preneoplastic cells and recurrent growing tumors, and decipher the initiation and evolution of inflammation. These studies in Drosophila melanogaster can serve as a blueprint for studies in more complex organisms and help in the design of mechanism-based therapies for the individualized treatment of cancer diseases in humans. This review focuses on new discoveries in Drosophila related to the diverse innate immune responses to cancer-related inflammation and the systemic effects that are so detrimental to the host. Full article

(This article belongs to the Special Issue Research Advances in Invertebrate Experimental Models of Malignant Growth)

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