ex-vivo-organoid-model-adenovirus-cre-mediated-gene-deletions-mouse

<em>Ex Vivo</em> Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

generation-tumor-organoids-from-genetically-engineered-mouse-models

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

Roswell Park Comprehensive Cancer Center

The retinoblastoma tumor suppressor gene (RB1) is currently the only known gene whose mutation is necessary and sufficient for the development of a human cancer. Mutation or deregulation of RB1 is observed so frequently in other tumor types that compromising RB1 function may be a prerequisite for malignant transformation. Identifying the molecular mechanisms that provide the basis for RB1-mediated tumor suppression has become an important goal in the quest to understand and treat cancer. The lion's share of research on these mechanisms has focused on the carboxy-terminal half of the RB1 encoded protein (pRB). This focus is with good reason since this part of the protein, now called the "large pocket," is required for most of its known activities identified in vitro and in vivo. Large pocket mediated mechanisms alone, however, cannot account for all observed properties of pRB. The thesis presented here is that the relatively uncharacterized amino-terminal half of the protein makes important contributions to pRB-mediated tumor suppression. The goals of this review are to summarize evidence indicating that an amino-terminal structural domain is important for pRB function and to suggest a general hypothesis as to how this domain can be integrated with current models of pRB function.

How the other half lives, the amino-terminal domain of the retinoblastoma tumor suppressor protein.

The retinoblastoma tumor-suppressor gene (Rb1) is centrally important in cancer research. Mutational inactivation of Rb1 causes the pediatric cancer retinoblastoma, while deregulation of the pathway in which it functions is common in most types of human cancer. The Rb1-encoded protein (pRb) is well known as a general cell cycle regulator, and this activity is critical for pRb-mediated tumor suppression. The main focus of this review, however, is on more recent evidence demonstrating the existence of additional, cell type-specific pRb functions in cellular differentiation and survival. These additional functions are relevant to carcinogenesis suggesting that the net effect of Rb1 loss on the behavior of resulting tumors is highly dependent on biological context. The molecular mechanisms underlying pRb functions are based on the cellular proteins it interacts with and the functional consequences of those interactions. Better insight into pRb-mediated tumor suppression and clinical exploitation of pRb as a therapeutic target will require a global view of the complex, interdependent network of pocket protein complexes that function simultaneously within given tissues.

The retinoblastoma tumor-suppressor gene, the exception that proves the rule.

The majority of newly diagnosed prostate cancers will remain indolent, but distinguishing between aggressive and indolent disease is imprecise. This has led to the important clinical problem of overtreatment. THOC1 encodes a nuclear ribonucleoprotein whose expression is higher in some cancers than in normal tissue. The hypothesis that THOC1 may be a functionally relevant biomarker that can improve the identification of aggressive prostate cancer has not been tested.

The Thoc1 ribonucleoprotein and prostate cancer progression.

The Rb1 tumor suppressor protein is a molecular adaptor that physically links transcription factors like E2f with various proteins acting on DNA or RNA to repress gene expression. Loss of Rb1 liberates E2f to activate the expression of genes mediating resulting phenotypes. Most Rb1 binding proteins, including E2f, interact through carboxyl-terminal protein interaction domains, but genetic evidence suggests that an amino-terminal protein interaction domain is also important. One protein that binds Rb1 through the amino-terminal domain is encoded by Thoc1, a required component of the THO ribonucleoprotein complex important for RNA processing and transport. The physiological relevance of this interaction is unknown. Here we tested whether Thoc1 mediates effects of Rb1 loss on mouse embryonic development. We found that Thoc1 deficiency delays embryo death, and this delay correlates with reduced apoptosis in the brain. E2f protein levels are reduced in Rb1:Thoc1-deficient brain tissue. Expression of apoptotic regulatory genes regulated by E2f, like Apaf1 and Bak1, is also reduced. These observations suggest that Thoc1 is required to support increased expression of E2f and apoptotic regulatory genes that trigger apoptosis upon Rb1 loss. These findings implicate Rb1 in the regulation of the THO ribonucleoprotein complex.

Evaluating Effects of Hypomorphic Thoc1 Alleles on Embryonic Development in Rb1 Null Mice.

Prostate cancer relapsing from antiandrogen therapies can exhibit variant histology with altered lineage marker expression, suggesting that lineage plasticity facilitates therapeutic resistance. The mechanisms underlying prostate cancer lineage plasticity are incompletely understood. Studying mouse models, we demonstrate that Rb1 loss facilitates lineage plasticity and metastasis of prostate adenocarcinoma initiated by Pten mutation. Additional loss of Trp53 causes resistance to antiandrogen therapy. Gene expression profiling indicates that mouse tumors resemble human prostate cancer neuroendocrine variants; both mouse and human tumors exhibit increased expression of epigenetic reprogramming factors such as Ezh2 and Sox2. Clinically relevant Ezh2 inhibitors restore androgen receptor expression and sensitivity to antiandrogen therapy. These findings uncover genetic mutations that enable prostate cancer progression; identify mouse models for studying prostate cancer lineage plasticity; and suggest an epigenetic approach for extending clinical responses to antiandrogen therapy.

Rb1 and Trp53 cooperate to suppress prostate cancer lineage plasticity, metastasis, and antiandrogen resistance.

Significant advances in our understanding of normal development and disease have been facilitated by engineered mice in which genes can be altered in a spatially, temporally, or cell type restricted manner using site specific recombinase systems like Cre-loxP or Flp-frt. In many circumstances it is important to understand how interactions between multiple genes influence a given phenotype. Robust approaches for precisely controlling multiple genetic alterations independently are limited, however, thus the impact of mutation order and timing on phenotype is generally unknown. Here we describe and validate a novel Gt(ROSA)26Sor targeted transgene allowing precise control over the order and timing of multiple genetic mutations in the mouse. The transgene expresses an optimized, Flp-estrogen receptor fusion protein (Flpo-ERT2) under the control of a loxP-stop-loxP cassette. In this system, genes modified by loxP sites are altered first upon expression of Cre. Cre also eliminates the loxP-stop-loxP cassette, permitting widespread expression of Flpo-ERT2. Because of the estrogen receptor fusion, Flp activity remains inert until administration of tamoxifen, allowing genes modified by frt sites to be modified subsequently with controllable timing. This mouse transgene will be useful in a wide variety of applications where independent control of different mutations in the mouse is desirable.

An approach for controlling the timing and order of engineered mutations in mice.

Prostate cancer (PCa) is diagnosed at the highest rate of all non-cutaneous male cancers in the United States. The androgen-dependent (AD) transcription factor, androgen receptor (AR), drives PCa-but inhibiting AR or androgen biosynthesis induces remission for only a short time. At which point, patients acquire more aggressive castration-resistant (CR) disease with re-activated AR-dependent signaling. To combat treatment resistance, down-regulating AR protein expression has been considered as a potential treatment strategy for CR-PCa.

Riluzole induces AR degradation via endoplasmic reticulum stress pathway in androgen-dependent and castration-resistant prostate cancer cells.

Recent studies highlight the importance of the RB1 tumor suppressor as a target for cancer therapy. Canonically, RB1 regulates cell cycle progression and represents the downstream target for cyclin-dependent kinase (CDK) 4/6 inhibitors that are in clinical use. However, newly discovered features of the RB1 pathway suggest new therapeutic strategies to counter resistance and improve precision medicine. These therapeutic strategies include deepening cell cycle exit with CDK4/6 inhibitor combinations, selectively targeting tumors that have lost RB1, and expanding therapeutic index by mitigating therapy-associated adverse effects. In addition, RB1 impacts immunological features of tumors and the microenvironment that can enhance sensitivity to immunotherapy. Lastly, RB1 specifies epigenetically determined cell lineage states that are disrupted during therapy resistance and could be re-installed through the direct use of epigenetic therapies. Thus, new opportunities are emerging to improve cancer therapy by exploiting the RB1 pathway.

Cell Cycle and Beyond: Exploiting New RB1 Controlled Mechanisms for Cancer Therapy.

EGFR tyrosine kinase inhibitors (EGFR TKIs) are the standard of care treatment for patients with -mutant lung adenocarcinoma (LUAD). Although initially effective, EGFR TKIs are not curative. Disease inevitably relapses due to acquired drug resistance. We hypothesized that vitamin D metabolites could be used with EGFR TKIs to prevent therapeutic failure. To test this idea, we investigated the link between serum 25-hydroxyvitamin D3 (25(OH)D3) and progression-free survival (PFS) in patients with -mutant LUAD that received EGFR TKIs (erlotinib n = 20 and afatinib n = 1). Patients who were 25(OH)D3-sufficient experienced significantly longer benefit from EGFR TKI therapy (mean 14.5 months) than those with 25(OH)D3 insufficiency (mean 10.6 months,  = 0.026). In contrast, 25(OH)D3 had no prognostic value in patients with -mutant LUAD that received cytotoxic chemotherapy. To gain mechanistic insights, we tested 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) activity in vitro. 1,25(OH)2D3 promoted epithelial differentiation and restored EGFR TKI sensitivity in models of EGFR TKI resistance that were associated with epithelial-mesenchymal transition (EMT). 1,25(OH)2D3 was ineffective in a non-EMT model of resistance. We conclude that vitamin D sufficiency portends increased PFS among -mutant LUAD patients that receive EGFR TKIs, and that vitamin D signaling maintains drug efficacy in this specific patient subset by opposing EMT.

Vitamin D3 Metabolites Demonstrate Prognostic Value in -Mutant Lung Adenocarcinoma and Can be Deployed to Oppose Acquired Therapeutic Resistance.

The retinoblastoma tumor suppressor gene (RB1) plays a critical role in coordinating multiple pathways that impact cancer initiation, disease progression, and therapeutic responses. Here we probed molecular features associated with the RB-pathway across 31 tumor-types. While the RB-pathway has been purported to exhibit multiple mutually exclusive genetic events, only RB1 alteration is mutually exclusive with deregulation of CDK4/6 activity. An ER+ breast cancer model with targeted RB1 deletion was used to identify signatures of CDK4/6 activity and RB-dependency (CDK4/6-RB integrated signature). This signature was prognostic in tumor-types with gene expression features indicative of slower growth. Single copy loss on chromosome 13q encompassing the RB1 locus is prevalent in many cancers, yielding reduced expression of multiple genes in cis, and is inversely related to the CDK4/6-RB integrated signature supporting a cause-effect relationship. Genes that are positively and inversely correlated with the CDK4/6-RB integrated signature define new tumor-specific pathways associated with RB-pathway activity.

Pan-cancer molecular analysis of the RB tumor suppressor pathway.

Somatic alterations in the epidermal growth factor receptor gene () result in aberrant activation of kinase signaling and occur in ∼15% of non-small cell lung cancers (NSCLC). Patients diagnosed with -mutant NSCLC have good initial clinical response to EGFR tyrosine kinase inhibitors (EGFR TKIs), yet tumor recurrence is common and quick to develop. Mechanisms of acquired resistance to EGFR TKIs have been studied extensively over the past decade. Great progress has been made in understanding two major routes of therapeutic failure: additional genomic alterations in the  gene and activation of alternative kinase signaling (so-called "bypass activation"). Several pharmacological agents aimed at overcoming these modes of EGFR TKI resistance are FDA-approved or under clinical development. Phenotypic transformation, a less common and less well understood mechanism of EGFR TKI resistance is yet to be addressed in the clinic. In the context of acquired EGFR TKI resistance, phenotypic transformation encompasses epithelial to mesenchymal transition (EMT), transformation of adenocarcinoma of the lung (LUAD) to squamous cell carcinoma (SCC) or small cell lung cancer (SCLC). SCLC transformation, or neuroendocrine differentiation, has been linked to inactivation of TP53 and RB1 signaling. However, the exact mechanism that permits lineage switching needs further investigation. Recent reports indicate that LUAD and SCLC have a common cell of origin, and that trans-differentiation occurs under the right conditions. Options for therapeutic targeting of -mutant SCLC are limited currently to conventional genotoxic chemotherapy. Similarly, the basis of EMT-associated resistance is not clear. EMT is a complex process that can be characterized by a spectrum of intermediate states with diverse expression of epithelial and mesenchymal factors. In the context of acquired resistance to EGFR TKIs, EMT frequently co-occurs with bypass activation, making it challenging to determine the exact contribution of EMT to therapeutic failure. Reversibility of EMT-associated resistance points toward its epigenetic origin, with additional adjustments, such as genetic alterations and bypass activation, occurring later during disease progression. This review will discuss the mechanistic basis for EGFR TKI resistance linked to phenotypic transformation, as well as challenges and opportunities in addressing this type of targeted therapy resistance in -mutant NSCLC.

Understanding Lineage Plasticity as a Path to Targeted Therapy Failure in -Mutant Non-small Cell Lung Cancer.

Neuroendocrine prostate cancer (NEPrCa) arises de novo or after accumulation of genomic alterations in pre-existing adenocarcinoma tumors in response to androgen deprivation therapies. We have provided evidence that small extracellular vesicles released by PrCa cells and containing the αVβ3 integrin promote neuroendocrine differentiation of PrCa in vivo and in vitro. Here, we examined αVβ3 integrin expression in three murine models carrying a deletion of PTEN (SKO), PTEN and RB1 (DKO), or PTEN, RB1 and TRP53 (TKO) genes in the prostatic epithelium; of these three models, the DKO and TKO tumors develop NEPrCa with a gene signature comparable to those of human NEPrCa. Immunostaining analysis of SKO, DKO and TKO tumors shows that αVβ3 integrin expression is increased in DKO and TKO primary tumors and metastatic lesions, but absent in SKO primary tumors. On the other hand, SKO tumors show higher levels of a different αV integrin, αVβ6, as compared to DKO and TKO tumors. These results are confirmed by RNA-sequencing analysis. Moreover, TRAMP mice, which carry NEPrCa and adenocarcinoma of the prostate, also have increased levels of αVβ3 in their NEPrCa primary tumors. In contrast, the αVβ6 integrin is only detectable in the adenocarcinoma areas. Finally, analysis of 42 LuCaP patient-derived xenografts and primary adenocarcinoma samples shows a positive correlation between αVβ3, but not αVβ6, and the neuronal marker synaptophysin; it also demonstrates that αVβ3 is absent in prostatic adenocarcinomas. In summary, we demonstrate that αVβ3 integrin is upregulated in NEPrCa primary and metastatic lesions; in contrast, the αVβ6 integrin is confined to adenocarcinoma of the prostate. Our findings suggest that the αVβ3 integrin, but not αVβ6, may promote a shift in lineage plasticity towards a NE phenotype and might serve as an informative biomarker for the early detection of NE differentiation in prostate cancer.

Differential expression of αVβ3 and αVβ6 integrins in prostate cancer progression.

Forkhead box protein A1 (FOXA1) is essential for androgen-dependent prostate cancer (PCa) growth. However, how FOXA1 levels are regulated remains elusive and its therapeutic targeting proven challenging. Here, we report FOXA1 as a nonhistone substrate of enhancer of zeste homolog 2 (EZH2), which methylates FOXA1 at lysine-295. This methylation is recognized by WD40 repeat protein BUB3, which subsequently recruits ubiquitin-specific protease 7 (USP7) to remove ubiquitination and enhance FOXA1 protein stability. They functionally converge in regulating cell cycle genes and promoting PCa growth. FOXA1 is a major therapeutic target of the inhibitors of EZH2 methyltransferase activities in PCa. FOXA1-driven PCa growth can be effectively mitigated by EZH2 enzymatic inhibitors, either alone or in combination with USP7 inhibitors. Together, our study reports EZH2-catalyzed methylation as a key mechanism to FOXA1 protein stability, which may be leveraged to enhance therapeutic targeting of PCa using enzymatic EZH2 inhibitors.

Posttranslational regulation of FOXA1 by Polycomb and BUB3/USP7 deubiquitin complex in prostate cancer.

Cancer heterogeneity impacts therapeutic response, driving efforts to discover over-arching rules that supersede variability. Here, we define pan-cancer binary classes based on distinct expression of YAP and YAP-responsive adhesion regulators. Combining informatics with in vivo and in vitro gain- and loss-of-function studies across multiple murine and human tumor types, we show that opposite pro- or anti-cancer YAP activity functionally defines binary YAP or YAP cancer classes that express or silence YAP, respectively. YAP solid cancers are neural/neuroendocrine and frequently RB1, such as retinoblastoma, small cell lung cancer, and neuroendocrine prostate cancer. YAP silencing is intrinsic to the cell of origin, or acquired with lineage switching and drug resistance. The binary cancer groups exhibit distinct YAP-dependent adhesive behavior and pharmaceutical vulnerabilities, underscoring clinical relevance. Mechanistically, distinct YAP/TEAD enhancers in YAP or YAP cancers deploy anti-cancer integrin or pro-cancer proliferative programs, respectively. YAP is thus pivotal across cancer, but in opposite ways, with therapeutic implications.

Binary pan-cancer classes with distinct vulnerabilities defined by pro- or anti-cancer YAP/TEAD activity.

Neoadjuvant chemotherapy (NAC) followed by radical cystectomy is the standard-of-care for patients with muscle-invasive bladder cancer (MIBC). Defects in nucleotide excision repair (NER) are associated with improved responses to NAC. Excision Repair Cross-Complementation group 3 (ERCC3) is a key component of NER process. No NER inhibitors are available for treating patients with bladder cancer. We have developed an ex vivo cell-based assay of 6-4 pyrimidine-pyrimidinone (6-4PP) removal as a surrogate measure of NER capacity in human bladder cancer cell lines. The protein expression of ERCC3 was examined in human MIBC specimens and cell lines. Small molecule inhibitors were screened for NER inhibition in bladder cancer cell lines. Spironolactone was identified as a potent NER inhibitor. Combined effects of spironolactone with chemo-drugs were evaluated in vitro and in vivo. The efficacy between platinum and spironolactone on cytotoxicity was determined by combination index. A correlation between NER capacity and cisplatin sensitivity was demonstrated in a series of bladder cancer cell lines. Further, siRNA-mediated knockdown of ERCC3 abrogated NER capacity and enhanced cisplatin cytotoxicity. Spironolactone inhibited ERCC3 protein expression, abrogated NER capacity, and increased platinum-induced cytotoxicity in bladder cancer cells in vivo and in patient-derived organoids. Moreover, spironolactone exhibited the potential synergism effects with other clinical chemotherapy regimens in bladder cancer cell lines. Our data support the notion of repurposing spironolactone for improving the chemotherapy response of NAC in patients with MIBC. Further clinical trials are warranted to determine the safety and efficacy of spironolactone in combination with chemotherapy.

A Preclinical Study to Repurpose Spironolactone for Enhancing Chemotherapy Response in Bladder Cancer.

The retinoblastoma susceptibility gene () is the first tumor suppressor gene discovered and a prototype for understanding regulatory networks that function in opposition to oncogenic stimuli. More than 3 decades of research has firmly established a widespread and prominent role for  in human cancer. Yet, this gene encodes but one of three structurally and functionally related proteins that comprise the pocket protein family. A central question in the field is whether the additional genes in this family,  and , are important tumor suppressor genes. If so, how does their tumor suppressor activity overlap or differ from . Here we revisit these questions by reviewing relevant data from human cancer genome sequencing studies that have been rapidly accumulating in recent years as well as pertinent functional studies in genetically engineered mice. We conclude that  and  do have important tumor suppressor activity in some contexts, but  remains the dominant tumor suppressor in the family. Given their similarities, we speculate on why  tumor suppressor activity is unique.

ArticleTotal : 2	Year
Publication title Cited by 2	2022
Publication title Cited by 5	2019

Article	Year
How the other half lives, the amino-terminal domain of the retinoblastoma tumor suppressor protein. Journal of cellular physiology\| PubMed ID: 14502556	2003
The retinoblastoma tumor-suppressor gene, the exception that proves the rule. Oncogene\| PubMed ID: 16936742	2006
The Thoc1 ribonucleoprotein and prostate cancer progression. Journal of the National Cancer Institute\| PubMed ID: 25296641	2014
Evaluating Effects of Hypomorphic Thoc1 Alleles on Embryonic Development in Rb1 Null Mice. Molecular and cellular biology\| PubMed ID: 27001308	2016
Rb1 and Trp53 cooperate to suppress prostate cancer lineage plasticity, metastasis, and antiandrogen resistance. Science (New York, N.Y.)\| PubMed ID: 28059767	2017
An approach for controlling the timing and order of engineered mutations in mice. Genesis (New York, N.Y. : 2000)\| PubMed ID: 30113769	2018
Riluzole induces AR degradation via endoplasmic reticulum stress pathway in androgen-dependent and castration-resistant prostate cancer cells. The Prostate\| PubMed ID: 30280407	2019
Cell Cycle and Beyond: Exploiting New RB1 Controlled Mechanisms for Cancer Therapy. Trends in cancer\| PubMed ID: 31174843	2019
Vitamin D3 Metabolites Demonstrate Prognostic Value in -Mutant Lung Adenocarcinoma and Can be Deployed to Oppose Acquired Therapeutic Resistance. Cancers\| PubMed ID: 32183160	2020
Pan-cancer molecular analysis of the RB tumor suppressor pathway. Communications biology\| PubMed ID: 32242058	2020
Understanding Lineage Plasticity as a Path to Targeted Therapy Failure in -Mutant Non-small Cell Lung Cancer. Frontiers in genetics\| PubMed ID: 32292420	2020
Differential expression of αVβ3 and αVβ6 integrins in prostate cancer progression. PloS one\| PubMed ID: 33481853	2021
Posttranslational regulation of FOXA1 by Polycomb and BUB3/USP7 deubiquitin complex in prostate cancer. Science advances\| PubMed ID: 33827814	2021
Binary pan-cancer classes with distinct vulnerabilities defined by pro- or anti-cancer YAP/TEAD activity. Cancer cell\| PubMed ID: 34270926	2021
A Preclinical Study to Repurpose Spironolactone for Enhancing Chemotherapy Response in Bladder Cancer. Molecular cancer therapeutics\| PubMed ID: 35247903	2022
Retinoblastoma Protein Paralogs and Tumor Suppression. Frontiers in genetics\| PubMed ID: 35368709	2022

David W. Goodrich

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