WWOX Loss Research Articles

Abstract C119: Exploring the functional role of LRIG1 and WWOX tumor suppressors in breast cancer disparities

Abstract Breast cancer is the most commonly diagnosed cancer among women in the US. Despite a lower rate of breast cancer incidence, Black women in the US suffer higher rates of mortality compared to White women. This disparity poses a major clinical issue for Black women in the US, and we are particularly interested in understanding how molecular and environmental mechanisms may be contributing to these disproportionate outcomes. Our previous study found that in a cohort of women from Baltimore, Maryland (110 Black, 75 White), higher neighborhood deprivation was associated with decreased gene body methylation and gene expression of two tumor suppressor genes, LRIG1 and WWOX. These findings were unique to the tumor, which potentially points to ancestry-specific somatic mutations that may impact gene and protein expression for LRIG1 and WWOX. The goal of this project is to determine gene and protein differences in LRIG1 and WWOX by population group in women with and without breast cancer as it relates to neighborhood-level socioeconomic deprivation. We hypothesize that LRIG1 and WWOX exhibit decreased gene and protein expression in Black women compared to White women. Using the same diverse cohort from Baltimore, Maryland, we identified and conducted DNA genotyping on clinically relevant mutations of LRIG1 and WWOX using amplification by PCR and detection by fluorescent reporters. We found that the genotype frequencies in two single nucleotide polymorphisms (SNPs) for LRIG1, rs1403626 (P=6.3e-1) and rs2306272 (P=0.005967), differ significantly between population groups in these breast tissue samples, an interesting finding that matches our previous predictions. We also identified a dose-dependent relationship between LRIG1 gene expression and rs1403626 SNP genotypes. Additional patient genotyping will be conducted to determine relationships between LRIG1 and WWOX SNPs, differences in DNA methylation, and correlations with neighborhood deprivation measures. To interrogate the impact on protein expression, we will construct LRIG1 and WWOX knockdown mutants in estrogen receptor (ER)-positive and ER-negative commercially available breast cancer cell lines derived from Black and White patients, and determine the functional consequences of LRIG1 and WWOX loss by immunofluorescence and western blot, including how this may differ by population group. These studies will give us a better understanding of how the expression of tumor suppressor genes may contribute to the higher susceptibility of Black women developing more aggressive breast tumors. Citation Format: Angel H. Pajimola, Brittany Jenkins-Lord. Exploring the functional role of LRIG1 and WWOX tumor suppressors in breast cancer disparities [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C119.

Abstract 15153: Inhibition of the Tumor Suppressor Wwox Expression Alters Mitochondrial Dynamics in PASMC

Introduction: Pulmonary arterial hypertension (PAH) contributes to the morbidity and mortality of several patients with different pulmonary and cardiac diseases. Among other factors, the pathogenesis of PAH involves exaggerated pulmonary artery smoot muscle cell (PASMC) hyperproliferation, resistance to apoptosis and energy metabolism shift associated with alterations in mitochondrial metabolism and dynamics. The WW domain-containing oxidoreductase gene ( WWOX ) is a tumor suppressor gene that has the ability to interact with a large number of proteins, exerting its activity through a wide variety of molecular actions in specific cells. WWOX loss affects cell proliferation, motility, and deregulates expression of genes involved in cell cycle regulation, metabolism, and DNA damage in a variety of cancers. Considering that a similar phenotype is observed in cancer cells and PAH, we sought to investigate whether the loss of WWOX in PASMC is associated abnormalities of mitochondrial metabolism and dynamics in PAH. Methods and Results: In PASMC, inhibition of WWOX expression by short-interfering RNA (siRNA) decreased oxygen consumption by 30% when compared with siRNA control. Dysregulation of mitochondrial bioenergetics in WWOX-silenced PASMC was attributed to modulation of the oxidative state of mitochondrial electron transport chain proteins, showing that succinate dehydrogenase (SDH)(complex II) expression decreased by 15% in relation to siRNA control. JC-1 and mitotraker red CMXROS staining demonstrated that WWOX- silenced PASMC had hyperpolarized and fragmented mitochondria that generated more reactive oxygen species than the control. Changes in mitochondrial dynamics were detected with increased phosphorylation levels of the fission protein, dynamin-related protein 1 (DRP-1) and, decreased Mitofusin 2 and 1 protein levels, positive regulators of mitochondrial fusion. Conclusion: Loss of WWOX expression causes changes in PASMC mitochondrial metabolism and dynamics stimulating these cells to use different resources as a way to reprogram their metabolism and ensure their survival. These alterations may contribute, for example, to the stimulation of vascular remodeling, which consequently promote the pathogenesis of PAH.

WWOX Loss of Function in Neurodevelopmental and Neurodegenerative Disorders.

The WWOX gene was initially discovered as a putative tumor suppressor. More recently, its association with multiple central nervous system (CNS) pathologies has been recognized. WWOX biallelic germline pathogenic variants have been implicated in spinocerebellar ataxia type 12 (SCAR12; MIM:614322) and in early infantile epileptic encephalopathy (EIEE28; MIM:616211). WWOX germline copy number variants have also been associated with autism spectrum disorder (ASD). All identified germline genomic variants lead to partial or complete loss of WWOX function. Importantly, large-scale genome-wide association studies have also identified WWOX as a risk gene for common neurodegenerative conditions such as Alzheimer’s disease (AD) and multiple sclerosis (MS). Thus, the spectrum of CNS disorders associated with WWOX is broad and heterogeneous, and there is little understanding of potential mechanisms at play. Exploration of gene expression databases indicates that WWOX expression is comparatively higher in the human cerebellar cortex than in other CNS structures. However, RNA in-situ hybridization data from the Allen Mouse Brain Atlas show that specific regions of the basolateral amygdala (BLA), the medial entorhinal cortex (EC), and deep layers of the isocortex can be singled out as brain regions with specific higher levels of Wwox expression. These observations are in close agreement with single-cell RNA-seq data which indicate that neurons from the medial entorhinal cortex, Layer 5 from the frontal cortex as well as GABAergic basket cells and granule cells from cerebellar cortex are the specific neuronal subtypes that display the highest Wwox expression levels. Importantly, the brain regions and cell types in which WWOX is most abundantly expressed, such as the EC and BLA, are intimately linked to pathologies and syndromic conditions in turn associated with this gene, such as epilepsy, intellectual disability, ASD, and AD. Higher Wwox expression in interneurons and granule cells from cerebellum points to a direct link to the described cerebellar ataxia in cases of WWOX loss of function. We now know that total or partial impairment of WWOX function results in a wide and heterogeneous variety of neurodegenerative conditions for which the specific molecular mechanisms remain to be deciphered. Nevertheless, these observations indicate an important functional role for WWOX in normal development and function of the CNS. Evidence also indicates that disruption of WWOX expression at the gene or protein level in CNS has significant deleterious consequences.

Wwox Deletion in Mouse B Cells Leads to Genomic Instability, Neoplastic Transformation, and Monoclonal Gammopathies.

WWOX (WW domain containing oxidoreductase) expression loss is common in various cancers and characteristic of poor prognosis. Deletions, translocations, and loss of expression affecting the WWOX gene are a common feature of various B cell neoplasms such as certain B cell lymphomas and multiple myeloma. However, the role of this common abnormality in B cell tumor initiation and/or progression has not been defined. In this study, we conditionally deleted Wwox early in B cell development by means of breeding Cd19-Cre transgenic mice crossed to Wwox floxed mice (Cd19 Wwox KO). We observed a significant reduced survival in Cd19 Wwox KO mice and the development of B cell neoplasms including B cell lymphomas, plasma cell neoplasias characterized by increased numbers of CD138+ populations as well as monoclonal gammopathies detected by serum protein electrophoresis. To investigate whether Wwox loss could play a role in genomic instability, we analyzed DNA repair functions during immunoglobulin class switch joining between DNA segments in antibody genes. While class switch recombination (CSR) was only slightly impaired, Wwox deficiency resulted in a dramatic shift of double strand break (DSB) repair from normal classical-NHEJ toward the microhomology-mediated alternative-NHEJ pathway, a pathway associated with chromosome translocations and genome instability. Consistent with this, Wwox deficiency resulted in a marked increase of spontaneous translocations during CSR. This work defines for the first time a role for Wwox for maintaining B cell genome stability during a process that can promote neoplastic transformation and monoclonal gammopathies.

Strategies by which WWOX-deficient metastatic cancer cells utilize to survive via dodging, compromising, and causing damage to WWOX-positive normal microenvironment

Proapoptotic tumor suppressor WWOX is upregulated in the early stage of cancer initiation, which probably provides limitation to cancer growth and progression. Later, WWOX protein is reduced to enhance cancer cell growth, migration, invasiveness and metastasis. To understand how WWOX works in controlling cancer progression, here we demonstrate that apoptotic stress mediated by ectopic WWOX stimulated cancer cells to secrete basic fibroblast growth factor (bFGF) in order to support capillary microtubule formation. This event may occur in the cancer initiation stage. Later, when WWOX loss occurs in cancer cells, hyaluronidase production is then increased in the cancer cells to facilitate metastasis. We determined that inhibition of membrane hyaluronidase Tyr216-phosphorylated Hyal-2 by antibody suppresses cancer growth in vivo. WWOX-negative (WWOX-) cells dodged WWOX+cells in the microenvironment by migrating individually backward to avoid physical contacts and yet significantly upregulating the redox activity of WWOX+parental cells or other WWOX+cell types for causing apoptosis. Upon detecting the presence of WWOX+cells from a distance, WWOX- cells exhibit activation of MIF, Hyal-2, Eph, and Wnt pathways, which converges to MEK/ERK signaling and enables WWOX- cells to evade WWOX+cells. Inhibition of each pathway by antibody or specific chemicals enables WWOX- cells to merge with WWOX+cells. In addition, exogenous TGF-β assists WWOX- cells to migrate collectively forward and merge with WWOX+cells. Metastatic WWOX- cancer cells frequently secrete high levels of TGF-β, which conceivably assists them to merge with WWOX+cells in target organs and secure a new home base in the WWOX+microenvironment. Together, loss of WWOX allows cancer cells to develop strategies to dodge, compromise and even kill WWOX-positive cells in microenvironment.

WWOX somatic ablation in skeletal muscles alters glucose metabolism

ObjectiveWWOX, a well-established tumor suppressor, is frequently lost in cancer and plays important roles in DNA damage response and cellular metabolism. MethodsWe re-analyzed several genome-wide association studies (GWAS) using the Type 2 Diabetes Knowledge Portal website to uncover WWOX's association with metabolic syndrome (MetS). Using several engineered mouse models, we studied the effect of somatic WWOX loss on glucose homeostasis. ResultsSeveral WWOX variants were found to be strongly associated with MetS disorders. In mouse models, somatic ablation of Wwox in skeletal muscle (WwoxΔSKM) results in weight gain, glucose intolerance, and insulin resistance. Furthermore, WwoxΔSKM mice display reduced amounts of slow-twitch fibers, decreased mitochondrial quantity and activity, and lower glucose oxidation levels. Mechanistically, we found that WWOX physically interacts with the cellular energy sensor AMP-activated protein kinase (AMPK) and that its loss is associated with impaired activation of AMPK, and with significant accumulation of the hypoxia inducible factor 1 alpha (HIF1α) in SKM. ConclusionsOur studies uncover an unforeseen role of the tumor suppressor WWOX in whole-body glucose homeostasis and highlight the intimate relationship between cancer progression and metabolic disorders, particularly obesity and type-2 diabetes. Subject areasGenetics, Metabolic Syndrome, Diabetes.

Wwox deletion leads to reduced GABA-ergic inhibitory interneuron numbers and activation of microglia and astrocytes in mouse hippocampus

The association of WW domain-containing oxidoreductase WWOX gene loss of function with central nervous system (CNS) related pathologies is well documented. These include spinocerebellar ataxia, epilepsy and mental retardation (SCAR12, OMIM: 614322) and early infantile epileptic encephalopathy (EIEE28, OMIM: 616211) syndromes. However, there is complete lack of understanding of the pathophysiological mechanisms at play. In this study, using a Wwox knockout (Wwox KO) mouse model (2 weeks old, both sexes) and stereological studies we observe that Wwox deletion leads to a significant reduction in the number of hippocampal GABA-ergic (γ-aminobutyric acid) interneurons. Wwox KO mice displayed significantly reduced numbers of calcium-binding protein parvalbumin (PV) and neuropeptide Y (NPY) expressing interneurons in different subfields of the hippocampus in comparison to Wwox wild-type (WT) mice. We also detected decreased levels of Glutamic Acid Decarboxylase protein isoforms GAD65/67 expression in Wwox null hippocampi suggesting lower levels of GABA synthesis. In addition, Wwox deficiency was associated with signs of neuroinflammation such as evidence of activated microglia, astrogliosis, and overexpression of inflammatory cytokines Tnf-a and Il6. We also performed comparative transcriptome-wide expression analyses of neural stem cells grown as neurospheres from hippocampi of Wwox KO and WT mice thus identifying 283 genes significantly dysregulated in their expression. Functional annotation of transcriptome profiling differences identified ‘neurological disease’ and ‘CNS development related functions’ to be significantly enriched. Several epilepsy-related genes were found differentially expressed in Wwox KO neurospheres. This study provides the first genotype-phenotype observations as well as potential mechanistic clues associated with Wwox loss of function in the brain.

Abstract 4303: Vopp1 physically interacts with Wwox and inhibits its apoptotic function in breast cancer

Abstract WWOX is a tumor suppressor gene spanning the fragile site FRA16D and targeted by loss of heterozygosity or homozygous deletion in several tumor types. Wwox protein consists of two N-terminal WW domains that mediate protein-protein interactions and a C-terminal short-chain Dehydrogenase/Reductase domain (SDR). The WW1 module acts as a versatile platform linking WWOX with numerous multiprotein complexes. Thus, Wwox suppressor activity relies on its binding capacities to various partners. Wwox has been shown to interact with known proto-oncogenes such as p73, deltaNp63, AP2γ, c-jun, Erbb4, c-Met, Runx2 and Dvl-2. At the cellular level, Wwox has been shown to be a pro-apoptotic protein that induces cell death by acting in both the nucleus and cytoplasm. More recently, emerging evidence also suggest an important role of WWOX in DNA damage response. By using a yeast two-hybrid system and co-immunoprecipitation assays, we characterized Vopp1 (Vesicular Over-expressed in cancer Pro-survival Protein 1) as a new molecular partner of the tumor suppressor Wwox. VOPP1 gene localizes at the frequently amplified 7p11.2 locus and is often co-amplified with EGFR. It is overexpressed in multiple malignancies such as glioblastoma, gastric, head and neck and lung cancers. In this study, we demonstrate that VOPP1 physically interacts with Wwox in breast cancer cells. Upon binding, Wwox is recruited to the Vopp1-containing lysosomal compartment. This recruitment inhibits Wwox-mediated apoptosis at least in part by preventing Wwox-p73 interaction. In addition, Vopp1 acts as an oncogene as shown by its capacity to potentiate cellular transformation. Moreover, in breast cancer clinical samples, VOPP1 overexpression was detected predominantly in tumors retaining Wwox expression. Vopp1 overexpression is associated to a reduced survival of breast cancer patients, especially patients with luminal B tumor types. Remarkably, the VOPP1/WWOX expression ratio showed a significantly worst prognosis outperforming either gene alone. These findings highlight the importance of Wwox compartimentation in addition to Wwox loss in human cancer pathogenesis and define Vopp1 as a novel negative regulator of Wwox whose overexpression induces breast carcinogenesis by affecting the tumor suppressive activity of Wwox. Citation Format: François Lallemand, Florian Bonin, Rosette Lidereau, Keltouma Driouch. Vopp1 physically interacts with Wwox and inhibits its apoptotic function in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4303. doi:10.1158/1538-7445.AM2017-4303

Wwox\u2013Brca1 interaction: role in DNA repair pathway choice

In this study, loss of expression of the fragile site-encoded Wwox protein was found to contribute to radiation and cisplatin resistance of cells, responses that could be associated with cancer recurrence and poor outcome. WWOX gene deletions occur in a variety of human cancer types, and reduced Wwox protein expression can be detected early during cancer development. We found that Wwox loss is followed by mild chromosome instability in genomes of mouse embryo fibroblast cells from Wwox-knockout mice. Human and mouse cells deficient for Wwox also exhibit significantly enhanced survival of ionizing radiation and bleomycin treatment, agents that induce double-strand breaks (DSBs). Cancer cells that survive radiation recur more rapidly in a xenograft model of irradiated breast cancer cells; Wwox-deficient cells exhibited significantly shorter tumor latencies vs Wwox-expressing cells. This Wwox effect has important consequences in human disease: in a cohort of cancer patients treated with radiation, Wwox deficiency significantly correlated with shorter overall survival times. In examining mechanisms underlying Wwox-dependent survival differences, we found that Wwox-deficient cells exhibit enhanced homology directed repair (HDR) and decreased non-homologous end-joining (NHEJ) repair, suggesting that Wwox contributes to DNA DSB repair pathway choice. Upon silencing of Rad51, a protein critical for HDR, Wwox-deficient cells were resensitized to radiation. We also demonstrated interaction of Wwox with Brca1, a driver of HDR, and show via immunofluorescent detection of repair proteins at ionizing radiation-induced DNA damage foci that Wwox expression suppresses DSB repair at the end-resection step of HDR. We propose a genome caretaker function for WWOX, in which Brca1–Wwox interaction supports NHEJ as the dominant DSB repair pathway in Wwox-sufficient cells. Taken together, the experimental results suggest that reduced Wwox expression, a common occurrence in cancers, dysregulates DSB repair, enhancing efficiency of likely mutagenic repair, and enabling radiation and cisplatin treatment resistance.

Fhit and Wwox loss-associated genome instability: A genome caretaker one-two punch.

Expression of Fhit and Wwox protein is frequently lost or reduced in many human cancers. In this report, we provide data that further characterizes the molecular consequences of Fhit loss in the initiation of DNA double-strand breaks (DSBs), and of Wwox loss in altered repair of DSBs. We show that loss of Fhit initiates mild genome instability in early passage mouse kidney cells, confirming that DNA damage associated with Fhit-deficiency is not limited to cancer cells. We also demonstrate that the cause of Fhit-deficient DSBs: thymidine deficiency-induced replication stress, can be resolved with thymidine supplementation in early passage mouse kidney cells before extensive genome instability occurs. As for consequences of Wwox loss in cancer, we show in a small panel of breast cancer cells and mouse embryonic fibroblasts that Wwox expression predicts response to radiation and mitomycin C, all agents that cause DSBs. In addition, loss of Wwox significantly reduced progression free survival in a cohort of ovarian cancer patients treated with platin-based chemotherapies. Finally, stratification of a cohort of squamous lung cancers by Fhit expression reveals that Wwox expression is significantly reduced in the low Fhit-expressing group, suggesting that loss of Fhit is quickly succeeded by loss of Wwox. We propose that Fhit and Wwox loss work synergistically in cancer progression and that DNA damage caused by Fhit could be targeted early in cancer initiation for prevention, while DNA damage caused by Wwox loss could be targeted later in cancer progression, particularly in cancers that develop resistance to genotoxic therapies.

WWOX: a fragile tumor suppressor.

WWOX, the WW domain-containing oxidoreductase gene at chromosome region 16q23.3-q24.1, spanning chromosomal fragile site FRA16D, encodes the 46 kDa Wwox protein, a tumor suppressor that is lost or reduced in expression in a wide variety of cancers, including breast, prostate, ovarian, and lung. The function of Wwox as a tumor suppressor implies that it serves a function in the prevention of carcinogenesis. Indeed, in vitro studies show that Wwox protein interacts with many binding partners to regulate cellular apoptosis, proliferation, and/or maturation. It has been reported that newborn Wwox knockout mice exhibit nascent osteosarcomas while Wwox(+/-) mice exhibit increased incidence of spontaneous and induced tumors. Furthermore, absence or reduction of Wwox expression in mouse xenograft models results in increased tumorigenesis, which can be rescued by Wwox re-expression, though there is not universal agreement among investigators regarding the role of Wwox loss in these experimental models. Despite this proposed tumor suppressor function, the overlap of the human WWOX locus with FRA16D sensitizes the gene to protein-inactivating deletions caused by replication stress. The high frequency of deletions within the WWOX locus in cancers of various types, without the hallmark protein inactivation-associated mutations of "classical" tumor suppressors, has led to the proposal that WWOX deletions in cancers are passenger events that occur in early cancer progenitor cells due to fragility of the genetic locus, rather than driver events which provide the cancer cell a selective advantage. Recently, a proposed epigenetic cause of chromosomal fragility has suggested a novel mechanism for early fragile site instability and has implications regarding the involvement of tumor suppressor genes at chromosomal fragile sites in cancer. In this review, we provide an overview of the evidence for WWOX as a tumor suppressor gene and put this into the context of fragility associated with the FRA16D locus.

Alternating expression levels of WWOX tumor suppressor and cancer-related genes in patients with bladder cancer.

The aim of the present study was to determine the roles of the WWOX tumor suppressor and cancer-related genes in bladder tumor carcinogenesis. Reverse transcription-quantitative polymerase chain reaction was used to analyze the status of WWOX promoter methylation (using MethylScreen™ technology) and loss of heterozygosity (LOH) in papillary urothelial cancer tissues. The associations between the expression levels of the following tumorigenesis-related genes were also assessed: The WWOX tumor suppressor gene, the MKI67 proliferation gene, the BAX, BCL2 and BIRC5 apoptotic genes, the EGFR signal transduction gene, the VEGF vascular endothelial growth factor gene, and the CCND1 and CCNE1 cell cycle genes. The results reveal a high frequency of LOH in intron 1 in the WWOX gene, as well as an association between reduced WWOX expression levels and increased promoter methylation. In addition, the present study demonstrates that in bladder tumors, apoptosis is inhibited by increased expression levels of the BCL2 gene. A correlation between the proliferation indices of the MKI67 and the BIRC5 genes was also revealed. Furthermore, the expression levels of VEGF were identified to be positively associated with those of the EGFR gene.

The relationship between EGFR gain and VHL loss in lung adenocarcinoma and poor patient survival

The prognosis of lung cancer remains poor and clinically applicable prognostic markers have not yet been satisfactory identified. Several chromosomal copy number alterations (CNAs) have been associated with metastasis, relapse, and survival of patients with lung cancer; however, no study has focused exclusively on identifying CNAs at a gene level. The aim of this study was to identify genes whose CNAs are associated with survival of patients with lung adenocarcinoma. The CNA status of a panel of 48 genes was detected by high-resolution array comparative genomic hybridization in 56 lung adenocarcinoma samples. The follow-up time of these patients was 8.5-65.7 months. The gene CNAs were analyzed for their association with patient survival. Cox univariate regression analysis revealed that EGFR gain (hazard ratio (HR) 3.84, 95% confidence interval (CI) 1.62-9.10), VHL loss (HR 4.56, 95% CI 1.85-11.27) and WWOX loss (HR 4.14, 95% CI 1.60-10.69) were each associated with poor survival. Multivariate analyses including EGFR gain, VHL loss and WWOX loss, as well as the clinicopathological variables such as age, sex, tumor size, tumor differentiation and TNM stage showed that EGFR gain (HR 4.63, 95% CI 1.69-12.7) and VHL loss (HR 4.82, 95% CI 1.41-16.43) were independent prognostic factors for poor survival, whereas WWOX loss lost statistical significance. These findings suggest that EGFR gain and VHL loss are associated with poor overall survival for lung adenocarcinoma patients and may be used as prognostic markers.