Pax Family Research Articles

Genome-Wide Identification and Evolutionary and Mutational Analysis of the Bos taurus Pax Gene Family.

Bos taurus is known for its tolerance of coarse grains, adaptability, high temperature, humidity, and disease resistance. Primarily, cattle are raised for their meat and milk, and pinpointing genes associated with traits relevant to meat production can enhance their overall productivity. The aim of this study was to identify the genome, analyze the evolution, and explore the function of the Pax gene family in B. taurus to provide a new molecular target for breeding in meat-quality-trait cattle. In this study, 44 Pax genes were identified from the genome database of five species using bioinformatics technology, indicating that the genetic relationships of bovids were similar. The Pax3 and Pax7 protein sequences of the five animals were highly consistent. In general, the Pax gene of the buffalo corresponds to the domestic cattle. In summary, there are differences in affinity between the Pax family genes of buffalo and domestic cattle in the Pax1/9, Pax2/5/8, Pax3/7, and Pax4/6 subfamilies. We believe that Pax1/9 has an effect on the growth traits of buffalo and domestic cattle. The Pax3/7 gene is conserved in the evolution of buffalo and domestic animals and may be a key gene regulating the growth of B. taurus. The Pax2/5/8 subfamily affects coat color, reproductive performance, and milk production performance in cattle. The Pax4/6 subfamily had an effect on the milk fat percentage of B. taurus. The results provide a theoretical basis for understanding the evolutionary, structural, and functional characteristics of the Pax family members of B. taurus and for molecular genetics and the breeding of meat-production B. taurus species.

Abstract P50: PAX9 Activates Cell Death via Autophagic Degradation of EGFR in Oral squamous Cell Carcinoma

Abstract PAired boX 9 (PAX9) gene belongs to the PAX family and is critically essential for physiology and development. It has a significant role in maintaining squamous cell differentiation, and its downregulation is associated with tumor initiation and malignant transformation. In this study, we have analyzed the tumor suppressive potential of PAX9 by activating autophagy-dependent apoptosis in oral squamous cell carcinoma (OSCC). PAX9 overexpression inhibited cell growth and triggered apoptosis in OSCC. Moreover, PAX9 restricted orosphere formation and epithelial-mesenchymal transition in OSCC. Similarly, overexpression of PAX9 in OSCC induced autophagy activation and displayed enhanced lysosomal function in the transcriptional-dependent mechanism. Interestingly, PAX9-activated autophagy caused EGFR degradation leading to OSCC inhibition, thus establishing that PAX9 could induce lethal autophagy. In addition, PAX9 involves chemosensitizing OSCC to anticancer drugs, which exhibited profound growth inhibitory potential in OSCC. To check the status of PAX9 expression in OSCC in patient tissue, we performed IHC, and surprisingly, the expression of PAX9 was found to decreased in a higher grade of cancer tissues compared to the normal tissues. Further, we established a DMBA-induced oral carcinoma in hamster model, and it was observed that the expression of PAX9 was decreased in the 12th and 16th weeks of DMBA-exposed tissue compared to the normal tissue. On a mechanistic view, PAX9 expression was reduced with enhanced expression of DNMT1 in exposure to carcinogen in OSCC in vitro, indicating a potential association of methylation in controlling the expression of PAX9 during carcinogenesis. Citation Format: Chandra Sekhar Bhol, Gautam Sethi, Sujit Kumar Bhutia. PAX9 Activates Cell Death via Autophagic Degradation of EGFR in Oral squamous Cell Carcinoma [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P50.

Prognostic value of PAX8 in small cell lung cancer

ObjectivesSmall cell lung cancer (SCLC) shows poor prognosis since it metastasizes widely at early stage. Paired box gene (PAX) 8 is a transcriptional factor of PAX family, of which the expression in lung cancer is a controversial issue, and its prognostic value of PAX8 in SCLC is still unclear. Materials and methodsOverall, 184 subjects who were pathologically diagnosed with SCLC were enrolled in the study. Immunohistochemical analysis of PAX8 and Ki-67 were performed. The correlations between PAX8 expression and clinical features or Ki-67 index were further analyzed. Subsequently, an analysis of the association between PAX8, stage, Ki-67 status, and overall survival (OS) were performed in 169 subjects with follow-up information. ResultsPAX8 was positive in 53.8% (99/184) SCLC specimens. The positive rate is significantly higher in extensive-stage specimens (61.0%) than in limited-stage specimens (45.24%). PAX8 expression is positively correlated with Ki-67 index (P = 0.001) while negatively correlated with OS (HR = 3.725, 95% CI 1.943–7.139, P＜0.001). In combination groups, the PAX8 negative and limited stage group had the most promising OS. ConclusionPAX8 expression rate in SCLC specimens is not low. It has prognostic value in small cell lung cancer.

Pax-8: Molecular biology, pathophysiology, and potential pathogenesis.

Transcription factors, as the convergence points of multiple signaling pathways in eukaryotic cells, are closely involved in disease development. Pax-8, an important transcription factor belonging to the Pax family, exerts a crucial influence on the regulation of gene expression required for both physiological conditions and pathological processes. Pax-8 contributes to the pathogenesis of many human diseases, ranging from cardiovascular disease to many cancers, and therefore, it can be imagined that Pax-8 holds great therapeutic potential. In this review, we summarize the structure, distribution, function, and regulatory mechanisms of Pax-8 to provide a new research direction for Pax-8.

Translation of paired box 6 (PAX6) mRNA is IRES-mediated and inhibited by cymarin in breast cancer cells.

Paired box 6 (PAX6) is a member of the PAX family and plays an essential role in cancer cell cycle progression, colony formation, proliferation and invasion. Its expression is upregulated in many cancers including breast cancer, but the process of PAX6 mRNA translation has rarely been studied. We found that PAX6 translation level increased in MCF-7 breast cancer cells treated with the chemotherapeutic drug adriamycin (ADM), which might be attributable to internal ribosome entry site (IRES)-mediated translation. By modifying a bicistronic luciferase plasmid that is widely used to examine IRES activity, we found that the 469-base 5'-UTR of PAX6 mRNA contains an IRES element and that core IRES activity is located between nucleotides 159 and 333. Moreover, PAX6 IRES activity was induced during ADM treatment, which may be the main reason for the elevated level of PAX6 protein. We also found that cymarin, a cardiac glycoside, acts as an inhibitor of PAX6 protein expression by impairing its IRES-mediated translation. Furthermore, MCF-7 cell proliferation was suppressed during treatment with cymarin. These results provide novel insights into the translation mechanism of PAX6 in breast cancer cells and suggest that cymarin is a promising candidate for the treatment of breast cancer via targeting the expression of PAX6.

In Vivo Evaluation of Analgesic Activities of Dioscorea dumetorum (Kunth) Pax and Tragia benthamii Bak

An analgesic is any member of the group of drugs used to achieve analgesia, relief from pain without loss of consciousness. Pain is one of the devastating health problems commonly treated with medicinal plants. Dioscorea dumetorum (Kunth) Pax (Family Dioscoreaceae) is one of the popular species of yam, usually found in Africa and mostly consumed in West Africa. Tragia benthamii Bak. is an herbaceous twining or trailing plant which belongs to the Euphobiaceae family. The plant is indigenous to Ivory Coast extending wide spread across Africa including Nigeria. This study investigated the analgesic activities of Dioscorea dumetorum and Tragia benthamii leaves’ methanol extract on mice. Acetic acid-induced writhing method was engaged. Thirty-two (32) healthy and energetic mice were grouped into eight (8) of four (4) mice. Indomethacin (25 mg/kg) was administered to the positive control group, negative control group received distilled water (10 ml/kg) while each extract treatment groups received 50, 100, 200 mg/kg respectively. Analgesic activities of the plants’ extract were assessed by counting the number of writhes for 30 minutes after a latency period of 5 minutes. Reduction in the number of writhes as compared to the control groups was considered as evidence for analgesic potential of the plants. In this study, the positive control (Indomethacin), D. dumetorum and T. benthamii significantly decreased the mean number of abdominal constrictions or writhes in a dose dependent manner following the order of 200>100>50 mg/kg. The result showed that the potency of the two plants’ extract was superior to that of Indomethacin but T. benthamii (200 mg/kg) exhibited higher analgesic activity. Values of reduction time in writhing were significantly different (P<0.05) among the treatments. This research revealed effective potential analgesic effects of D. dumetorum and T. benthamii which could be attributed to the presence of phytochemicals in them. Therefore, further studies to explain their mechanisms of action should be conducted to aid the discovery of new therapeutic agents for the treatment of pains.

Long-Term Treatment with Bortezomib Induces Specific Methylation Changes in Differentiated Neuronal Cells.

Simple SummaryWe exposed LUHMES cells, differentiated into mature neurons, to bortezomib (BTZ) in two treatment cycles and analyzed the methylomes of these cells after each cycle, controlling the analysis for the methylation changes potentially induced by the long-term culture. Our results show that BTZ induces methylation changes that may affect cell morphogenesis, neurogenesis, and neurotransmission. These changes are specifically enriched within transcription factor binding sites of EBF, PAX, DLX, LHX, and HNF family members, which have been shown to regulate neurogenesis and neuronal differentiation. We further show that the observed methylation changes are not present in the SH-SY5Y cells that we used to study mechanisms of development of BTZ resistance. Altogether, our results show that BTZ treatment induces very specific changes in the methylomes of neuronal cells.Bortezomib (BTZ) is proteasome inhibitor, effectively used in the treatment of multiple myeloma, but frequently discontinued due to peripheral neuropathy, which develops in patients after consecutive treatment cycles. The molecular mechanisms affected by BTZ in neuronal cells, which result in neuropathy, remain unknown. However, BTZ is unlikely to lead to permanent morphological nerve damage, because neuropathy reverses after discontinuation of treatment, and nerve cells have very limited renewal capacity. We have previously shown that BTZ induces methylation changes in SH-SY5Y cells, which take part in the development of treatment resistance. Here, we hypothesized that BTZ affects the methylomes of mature neurons, and these changes are associated with BTZ neurotoxicity. Thus, we studied methylomes of neuronal cells, differentiated from the LUHMES cell line, after cycles of treatment with BTZ. Our results show that BTZ induces specific methylation changes in mature neurons, which are not present in SH-SY5Y cells after BTZ treatment. These changes appear to affect genes involved in morphogenesis, neurogenesis, and neurotransmission. Furthermore, identified methylation changes are significantly enriched within binding sites of transcription factors previously linked to neuron physiology, including EBF, PAX, DLX, LHX, and HNF family members. Altogether, our results indicate that methylation changes are likely to be involved in BTZ neurotoxicity.

PAX9 in Cancer Development.

Paired box 9 (PAX9) is a transcription factor of the PAX family functioning as both a transcriptional activator and repressor. Its functional roles in the embryonic development of various tissues and organs have been well studied. However, its roles and molecular mechanisms in cancer development are largely unknown. Here, we review the current understanding of PAX9 expression, upstream regulation of PAX9, and PAX9 downstream events in cancer development. Promoter hypermethylation, promoter SNP, microRNA, and inhibition of upstream pathways (e.g., NOTCH) result in PAX9 silencing or downregulation, whereas gene amplification and an epigenetic axis upregulate PAX9 expression. PAX9 may contribute to carcinogenesis through dysregulation of its transcriptional targets and related molecular pathways. In summary, extensive studies on PAX9 in its cellular and tissue contexts are warranted in various cancers, in particular, HNSCC, ESCC, lung cancer, and cervical SCC.

Differential Regulation of Retinoic Acid Signaling in Fanconi Anemia

Fanconi anemia (FA) is a rare genetic disease characterized by heterogeneous congenital defects, and increased risk for early‐onset bone marrow failure and cancer. Biallelic mutations in any one of 23 genes leads to FA. In vitro experiments have established a coordinated role for the FA gene network in the repair of DNA interstrand crosslinks (ICLs). However, the endogenous source(s) of DNA damage and chromosome instability in FA patients remain largely unknown. Recent studies have suggested that the FA proteins may play an important role in the repair of DNA damage caused by genotoxic reactive aldehydes produced during cellular metabolism, e.g., acetaldehyde and formaldehyde.To discover novel metabolic pathways linked to FA, we recently performed RNA‐seq analysis on telomerase‐immortalized mutant and functionally complemented FA‐D2 (FANCD2‐/‐) patient cells. Gene ontology enrichment analysis uncovered dysregulation of retinoid metabolism and retinoic acid signaling in mutant FA‐D2 (FANCD2‐/‐) cells. For example, we observed increased expression of the retinoid metabolism genes RDH10, ALDH1A1, and CYP26B1, in the absence of FANCD2. RDH10 converts retinol to retinaldehyde; ALDH1A1 converts retinaldehyde to retinoic acid; CYP26B1 degrades excess retinoic acid. In addition, we observed altered expression of multiple retinoic acid regulated genes including RARA, EGR1, and TGM2, and several members of the HOX, PAX, and SOX gene families. Using immunoblotting and qPCR, we have validated differential expression of many of these genes. We have also observed altered retinoid metabolism in Fancd2‐/‐ mouse cells. Furthermore, in preliminary flow cytometry experiments, we have determined that FA‐D2 (FANCD2‐/‐) patient cells exhibit increased aldehyde dehydrogenase activity.These findings are potentially highly significant and suggest that dysregulated retinoid metabolism and retinoic acid signaling ‐ as well as retinaldehyde‐associated genotoxicity ‐ might be key molecular features of FA. Further characterization of this newly discovered link could pave the way for the discovery of new therapeutic targets for FA patients.

Genetic Regulation of Vertebrate Forebrain Development by Homeobox Genes.

Forebrain development in vertebrates is regulated by transcription factors encoded by homeobox, bHLH and forkhead gene families throughout the progressive and overlapping stages of neural induction and patterning, regional specification and generation of neurons and glia from central nervous system (CNS) progenitor cells. Moreover, cell fate decisions, differentiation and migration of these committed CNS progenitors are controlled by the gene regulatory networks that are regulated by various homeodomain-containing transcription factors, including but not limited to those of the Pax (paired), Nkx, Otx (orthodenticle), Gsx/Gsh (genetic screened), and Dlx (distal-less) homeobox gene families. This comprehensive review outlines the integral role of key homeobox transcription factors and their target genes on forebrain development, focused primarily on the telencephalon. Furthermore, links of these transcription factors to human diseases, such as neurodevelopmental disorders and brain tumors are provided.

Paired box 8 in organogenesis and oncogenesis – a review

Introduction and aim. Paired box 8 (PAX-8) is a specific transcription factor known as a protein product gene that plays an essential role in organogenesis and oncogenesis. The aim of this paper was to discuss structure and function of PAX-8. The aim of this study is to determine the utility of PAX-8 in cytology effusions with metastatic tumor. Material and methods. This article is a review done in regards to discuss the role knowledge on PAX-8 especially in oncogenesis and organogenesis. Analysis of the literature. Current information about PAX-8 is presented. Conclusion. The PAX family of genes plays an important role in the formation of tissues and organs during embryonic development and in maintaining the proper functioning of certain cells after birth.

Identification of Pax protein inhibitors that suppress target gene expression and cancer cell proliferation

The Pax family of developmental control genes are frequently deregulated in human disease. In the kidney, Pax2 is expressed in developing nephrons but not in adult proximal and distal tubules, whereas polycystic kidney epithelia or renal cell carcinoma continues to express high levels. Pax2 reduction in mice or cell culture can slow proliferation of cystic epithelial cells or renal cancer cells. Thus, inhibition of Pax activity may be a viable, cell-type-specific therapy. We designed an unbiased, cell-based, high-throughput screen that identified triazolo pyrimidine derivatives that attenuate Pax transactivation ability. We show that BG-1 inhibits Pax2-positive cancer cell growth and target gene expression but has little effect on Pax2-negative cells. Chromatin immunoprecipitation suggests that these inhibitors prevent Pax protein interactions with the histone H3K4 methylation complex at Pax target genes in renal cells. Thus, these compounds may provide structural scaffolds for kidney-specific inhibitors with therapeutic potential.

EHMT2 (G9a) activation in mantle cell lymphoma and its associated DNA methylation and gene expression.

Objective:The function of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) has been studied in several cancers; however, little is known about its role in mantle cell lymphoma (MCL). Thus, this study aimed to characterize the significance and function of EHMT2 in MCL.Methods:EHMT2 expression in MCL and reactive hyperplasia (RH) were investigated by immunohistochemistry. Genome-wide analysis of DNA methylation was performed on EHMT2 + MCL samples. The function of EHMT2 was determined by CCK8, flow cytometry, and western blot assays. Gene expression profile analysis was performed before and after EHMT2 knockdown to search for EHMT2-regulated genes. Co-immunoprecipitation (Co-IP) experiments were conducted to identify the proteins interacting with EHMT2.Results:EHMT2 was expressed in 68.57% (24/35) of MCLs but not in any RHs. Genome-wide analysis of DNA methylation on EHMT2 + MCLs revealed that multiple members of the HOX, FOX, PAX, SOX, and CDX families were hypermethylated or hypomethylated in EHMT2 + MCLs. BIX01294, a EHMT2 inhibitor, inhibited MCL cell growth and stalled cells in the G1 phase. Additionally, BIX01294 downregulated the expressions of cell cycle proteins, cyclin D1, CDK4, and P21, but upregulated the expressions of apoptosis-related proteins, Bax and caspase-3. Co-IP experiments revealed that EHMT2 interacted with UHRF1, HDAC1, and HDAC2 but not with HDCA3. After EHMT2 knockdown, multiple genes were regulated, including CD5 and CCND1, mostly enriched in the Tec kinase signaling pathway. In addition, several genes (e.g., MARCH1, CCDC50, HIP1, and WNT3) were aberrantly methylated in EHMT2 + MCLs.Conclusions:For the first time, we determined the significance of EHMT2 in MCL and identified potential EHMT2-regulated genes.

Ocular disease-associated mutations diminish the mitotic chromosome retention ability of PAX6

Transcription factors play a key role in maintaining cell identity. One mechanism of such cell memory after multiple rounds of cell division cycles is through persistent mitotic chromosome binding, although how individual transcription factors achieve mitotic chromosome retention is not completely understood. Here we show that PAX6, a lineage-determining transcription factor, coats mitotic chromosomes. Using deletion and point mutants associated with human ocular diseases in live-cell imaging analysis, we identified two regions, MCR-D1 and MCR-D2, that were responsible for mitotic chromosome retention of PAX6. We also identified three nuclear localization signals (NLSs) that contributed to mitotic chromosome retention independent of their nuclear import functions. Full mitotic chromosome retention required the presence of DNA-binding domains as well as NLSs within MCR-Ds. Furthermore, disease-associated mutations and NLS mutations changed the distribution of intrinsically disordered regions (IDRs) in PAX6. Our findings not only identify PAX6 as a novel mitotic chromosome retention factor but also demonstrate that the mechanism of mitotic chromosome retention involves sequence-specific DNA binding, NLSs, and molecular conformation determined by IDRs. These findings link mitotic chromosome retention with PAX6-related pathogenesis and imply similar mechanisms for other lineage-determining factors in the PAX family.

Unraveling the Mysteries of PAX8 in Reproductive Tract Cancers.

Paired Box 8 (PAX8) is a lineage-specific transcription factor that has essential roles during embryogenesis and tumorigenesis. The importance of PAX8 in the development of the reproductive system is highlighted by abnormalities observed upon the loss or mutation of this PAX family member. In cancer, PAX8 expression is deregulated in a key set of neoplasms, including those arising from the Müllerian ducts. The roles of PAX8 in oncogenesis are diverse and include epigenetic remodeling, stimulation of proliferation, inhibition of apoptosis, and regulation of angiogenesis. PAX8 can interact with different protein partners during cancer progression and may exhibit significant function-altering alternative splicing. Moreover, expression of PAX8 in cancer can also serve as a biomarker for diagnostic and prognostic purposes. In this review, we focus on the roles of PAX8 in cancers of the reproductive system. Understanding the diverse mechanisms of action of PAX8 in development and oncogenesis may identify new vulnerabilities in malignancies that currently lack effective therapies.

Upregulation of the PPAR signaling pathway and accumulation of lipids are related to the morphological and structural transformation of the dragon-eye goldfish eye

Goldfish comprise around 300 different strains with drastically altered and aesthetical morphologies making them suitable models for evolutionary developmental biology. The dragon-eye strain is characterized by protruding eyes (analogous to those of Chinese dragons). Although the strain has been selected for about 400 years, the mechanism of its eye development remains unclear. In this study, a stable dragon-eye goldfish strain with a clear genetic background was rapidly established and studied. We found that upregulation of the PPAR signaling pathway accompanied by an increase in lipid accumulation might trigger the morphological and structural transformation of the eye in dragon-eye goldfish. At the developmental stage of proptosis (eye protrusion), downregulation of the phototransduction pathway was consistent with the structural defects and myopia of the dragon-eye strain. With the impairment of retinal development, cytokine-induced inflammation was activated, especially after proptosis, similar to the pathologic symptoms of many human ocular diseases. In addition, differentially expressed transcription factors were significantly enriched in the PAX and homeobox families, two well-known transcription factor families involved in eye development. Therefore, our findings reveal the dynamic changes in key pathways during eye development in dragon-eye goldfish, and provide insights into the molecular mechanisms underlying drastically altered eyes in goldfish and human ocular disease.

Pax9 is essential for granulopoiesis but dispensable for erythropoiesis in zebrafish

Paired Box (Pax) gene family, a group of transcription regulators have been implicated in diverse physiological processes. However, their role during hematopoiesis which generate a plethora of blood cells remains largely unknown. Using a previously reported single cell transcriptomics data, we analyzed the expression of individual Pax family members in hematopoietic cells in zebrafish. We have identified that Pax9, which is an essential regulator for odontogenesis and palatogenesis, is selectively localized within a single cluster of the hematopoietic lineage. To further analyze the function of Pax9 in hematopoiesis, we generated two independent pax9 knock-out mutants using the CRISPR-Cas9 technique. We found that Pax9 appears to be an essential regulator for granulopoiesis but dispensable for erythropoiesis during development, as lack of pax9 selectively decreased the number of neutrophils with a concomitant decrease in the expression level of neutrophil markers. In addition, embryos, where pax9 was functionally disrupted by injecting morpholinos, failed to increase the number of neutrophils in response to pathogenic bacteria, suggesting that Pax9 is not only essential for developmental granulopoiesis but also emergency granulopoiesis. Due to the inability to initiate emergency granulopoiesis, innate immune responses were severely compromised in pax9 morpholino-mediated embryos, increasing their susceptibility and mortality. Taken together, our data indicate that Pax9 is essential for granulopoiesis and promotes innate immunity in zebrafish larvae.

Molecular Modelling of Islet β-Cell Adaptation to Inflammation in Pregnancy and Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM), a metabolic disease that develops with the increase in insulin resistance during late pregnancy, is currently one of the most common complications affecting pregnancy. The polygenic nature of GDM, together with the interplay between different genetic variants with nutritional and environmental factors has hindered the full understanding of the etiology of this disease. However, an important genetic overlap has been found with type 2 diabetes mellitus (T2DM) and, as in the case of T2DM, most of the identified loci are associated with β-cell function. Early detection of GDM and adequate interventions to control the maternal glycemia are necessary to avoid the adverse outcomes for both the mother and the offspring. The in utero exposure to the diabetic milieu predispose these children for future diseases, among them T2DM, originating a vicious circle implicated in the increased prevalence of both GDM and T2DM. The involvement of inflammatory processes in the development of GDM highlights the importance of pancreatic β-cell factors able to favor the adaptation processes required during gestation, concomitantly with the protection of the islets from an inflammatory milieu. In this regard, two members of the Pax family of transcription factors, PAX4 and PAX8, together with the chromatin remodeler factor HMG20A, have gained great relevance due to their involvement in β-cell mass adaptation together with their anti-inflammatory properties. Mutations in these factors have been associated with GDM, highlighting these as novel candidates for genetic screening analysis in the identification of women at risk of developing GDM.

Systematic alanine scanning of PAX8 paired domain reveals functional importance of the N-subdomain.

Thyroid-specific transcription factor PAX8 has an indispensable role in the thyroid gland development, which is evidenced by the facts that PAX8/Pax8 mutations cause congenital hypothyroidism in humans and mice. More than 90% of known PAX8 mutations were located in the paired domain, suggesting the central role of the domain in exerting the molecular function. Structure-function relationships of PAX8, as well as other PAX family transcription factors, have never been investigated in a systematic manner. Here, we conducted the first alanine scanning mutagenesis study, in which 132 alanine variants located in the paired domain of PAX8 were created and systematically evaluated in vitro. We found that 76 alanine variants (55%) were loss of function (LOF) variants (defined by <30% activity as compared with wild type PAX8). Importantly, the distribution of LOF variants were skewed, with more frequently observed in the N-subdomain (65% of the alanine variants in the N-subdomain) than in the C-subdomain (45%). Twelve out of 13 alanine variants in residues that have been affected in patients with congenital hypothyroidism were actually LOF, suggesting that the alanine scanning data can be used to evaluate the functional importance of mutated residues. Using our in vitro data, we tested the accuracy of seven computational algorithms for pathogenicity prediction, showing that they are sensitive but not specific to evaluate on the paired domain alanine variants. Collectively, our experiment-based data would help better understand the structure-function relationships of the paired domain, and would provide a unique resource for pathogenicity prediction of future PAX8 variants.

Abstract P2-06-09: Identification of oncogenic gene fusions in triple-negative breast cancer metastatic to the brain

Abstract BACKGROUND: As many as 24% of breast cancer (BC) patients develop metastasis to the brain and 27% of these occur in patients with triple negative (TN) BC. The brain (#2) and lung (#1) are the most common sites of TNBC metastasis. Half of women with metastatic (m) TNBC will die within one year. Novel therapies for women with mTNBC, especially to the brain, are desperately needed. TNBCs often exhibit genomic instability, resulting in gene fusions and other chromosomal rearrangements. Such alterations are long recognized as oncogenic drivers and effective drug targets in hematologic and other solid malignancies. We aimed to identify these alterations in mTNBC with Segmental Transcript Analysis (STA), a novel population-based algorithm ranking genes based on likelihood of harboring a gene rearrangement by identifying abrupt changes in transcript expression. RESULTS: We performed RNAseq on 25 mTNBC, 4 matched primary BC, 2 normal cerebellum and 2 normal cerebral cortex samples. TNBC subtype analysis showed an enrichment of basal-like (BL) 2 and mesenchymal (M) subtypes among mTNBC to the brain compared to primary (p) BC in the TCGA. A principal component analysis demonstrated mTNBC and their matched primaries clustered together, indicating the pairs were more closely related to each other genetically than to other mTNBCs. When the gene expression patterns of pBC were compared with their brain metastases following adjustment for contamination with normal brain tissue, an upregulation of mesenchymal genes (p63/p73 axis) and primitive/embryonic genes (NOTCH and PAX family) were noted in the brain mTNBC. We confirmed a modest to marked upregulation of p63 expression by immunohistochemistry across all TNBC subtypes, most notably in BL1 and BL2. STA found three gene rearrangements: EHF-WT1, NF1-VWDE and JAG1-Chr20. Two of these are potential driver gene rearrangements. The first, in a tumor of M subtype, results from fusion of the 5'UTR of EHF to WT1. The resulting fusion transcript is predicted to encode a truncated WT1 protein; loss of the N-terminal regulatory region of WT1 would likely cause constitutive activation. We also identified a truncating rearrangement in the Notch ligand JAG1 in a tumor of LAR subtype. JAG1 is a membrane bound ligand for the Notch receptor and the breakpoint occurs just distal to the DSL domain essential for Notch interaction. This rearrangement occurs within an intergenic chromosomal region, introduces an abrupt stop codon and generates a truncated protein lacking a transmembrane domain. An intact signal peptide should localize the protein to the endoplasmic reticulum; however, loss of the transmembrane domain would likely lead to secretion rather than membrane insertion. This alteration is predicted to constitutively stimulate Notch activity in a paracrine fashion. If the rearrangement functions in this manner, neutralizing antibodies to the N-terminal portion of JAG1 should have therapeutic efficacy. DISCUSION: EHF-WT1 and JAG1-Chr20 are potential driver rearrangements novel to brain mTNBC. The JAG1 rearrangement predicts constitutive production of Notch transcription factors and is potentially targetable. Continued interrogation of mTNBC for discovery of additional oncogenic fusions driving BC biology is warranted. Citation Format: Gonzalez-Ericsson P, Lehmann B, Mobley B, Chen Y-Y, Pietenpol J, Sanders ME. Identification of oncogenic gene fusions in triple-negative breast cancer metastatic to the brain [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-09.