Twist alters the breast tumor microenvironment via choline kinase to facilitate an aggressive phenotype.

Twist (TWIST1) is a gene required for cell fate specification in embryos and its expression in mammary epithelium can initiate tumorigenesis through the epithelial-mesenchymal transition. To identify downstream target genes of Twist in breast cancer, we performed microarray analysis on the transgenic breast cancer cell line, MCF-7/Twist. One of the targets identified was choline kinase whose upregulation resulted in increased cellular phosphocholine and total choline containing compounds-a characteristic observed in highly aggressive metastatic cancers. To study the interactions between Twist, choline kinase, and their effect on the microenvironment, we used 1H magnetic resonance spectroscopy and found significantly higher phosphocholine and total choline, as well as increased phosphocholine/glycerophosphocholine ratio in MCF-7/Twist cells. We also observed significant increases in extracellular glucose, lactate, and [H +] ion concentrations in the MCF-7/Twist cells. Magnetic resonance imaging of MCF-7/Twist orthotopic breast tumors showed a significant increase in vascular volume and permeability surface area product compared to control tumors. In addition, by reverse transcription-quantitative polymerase chain reaction, we discovered that Twist upregulated choline kinase expression in estrogen receptor negative breast cancer cell lines through FOXA1 downregulation. Moreover, using The Cancer Genome Atlas database, we observed a significant inverse relationship between FOXA1 and choline kinase expression and propose that it could act as a modulator of the Twist/choline kinase axis. The data presented indicate that Twist is a driver of choline kinase expression in breast cancer cells via FOXA1 resulting in the generation of an aggressive breast cancer phenotype.

Twist activates miR-22 to suppress estrogen receptor alpha in breast cancer.

TWIST1 (Twist) is a basic helix-loop-helix transcription factor that is overexpressed in many cancers and promotes tumor cell invasion, metastasis, and recurrence. In this study, we demonstrate that Twist upregulates expression of microRNA 22 (miR-22) which, in turn, downregulates estrogen receptor alpha (ER) expression in breast cancer. Initial analysis of miR-22 and Twist expression in a panel of breast cancer cell lines showed a direct correlation between Twist and miR-22 levels with miR-22 being highly expressed in ER negative cell lines. Overexpressing Twist caused increased miR-22 levels while downregulating it led to decreased miR-22 expression. To characterize the upstream promoter region of miR-22, we utilized rapid amplification of cDNA ends and identified the transcription start site and the putative promoter region of miR-22. Mechanistically, we determined that Twist, in combination with HDAC1 and DNMT3B, transcriptionally upregulates miR-22 expression by binding to E-boxes in the proximal miR-22 promoter. We also established that miR-22 causes an increase in growth in 3D but not 2D cultures. Importantly, we observed a direct correlation between increased breast cancer grade and Twist and miR-22 expression. We also identified two potential miR-22 binding sites in the 3'-UTR region of ER and confirmed by promoter assays that miR-22 regulates ER expression by binding to both target sites. These results reveal a novel pathway of ER suppression by Twist through miR-22 activation that could potentially promote the ER negative phenotype in breast cancers.

Genomic pathways modulated by Twist in breast cancer.

BACKGROUND: The basic helix-loop-helix transcription factor TWIST1 (Twist) is involved in embryonic cell lineage determination and mesodermal differentiation. There is evidence to indicate that Twist expression plays a role in breast tumor formation and metastasis, but the role of Twist in dysregulating pathways that drive the metastatic cascade is unclear. Moreover, many of the genes and pathways dysregulated by Twist in cell lines and mouse models have not been validated against data obtained from larger, independant datasets of breast cancer patients. METHODS: We over-expressed the human Twist gene in non-metastatic MCF-7 breast cancer cells to generate the estrogen-independent metastatic breast cancer cell line MCF-7/Twist. These cells were inoculated in the mammary fat pad of female severe compromised immunodeficient mice, which subsequently formed xenograft tumors that metastasized to the lungs. Microarray data was collected from both in vitro (MCF-7 and MCF-7/Twist cell lines) and in vivo (primary tumors and lung metastases) models of Twist expression. Our data was compared to several gene datasets of various subtypes, classes, and grades of human breast cancers. RESULTS: Our data establishes a Twist over-expressing mouse model of breast cancer, which metastasizes to the lung and replicates some of the ontogeny of human breast cancer progression. Gene profiling data, following Twist expression, exhibited novel metastasis driver genes as well as cellular maintenance genes that were synonymous with the metastatic process. We demonstrated that the genes and pathways altered in the transgenic cell line and metastatic animal models parallel many of the dysregulated gene pathways observed in human breast cancers. CONCLUSIONS: Analogous gene expression patterns were observed in both in vitro and in vivo Twist preclinical models of breast cancer metastasis and breast cancer patient datasets supporting the functional role of Twist in promoting breast cancer metastasis. The data suggests that genetic dysregulation of Twist at the cellular level drives alterations in gene pathways in the Twist metastatic mouse model which are comparable to changes seen in human breast cancers. Lastly, we have identified novel genes and pathways that could be further investigated as targets for drugs to treat metastatic breast cancer.

Functional roles of FAP-α in metabolism, migration and invasion of human cancer cells.

Fibroblast activation protein-α (FAP-α) is a transmembrane serine protease that is attracting significant interest as it is expressed by a subgroup of cancer-associated fibroblasts that play a role in immune suppression and cancer metastasis. FAP-α is also expressed by some cancer cells, such as melanoma, colorectal and breast cancer cells. Triple negative breast cancer (TNBC) is an aggressive cancer that urgently requires identification of novel targets for therapy. To expand our understanding of the functional roles of FAP-α in TNBC we engineered a human TNBC cell line, MDA-MB-231, to stably overexpress FAP-α and characterized changes in metabolism by 1H magnetic resonance spectroscopy, cell proliferation, migration characterized by wound healing, and invasion. FAP-α overexpression resulted in significant alterations in myoinositol, choline metabolites, creatine, and taurine, as well as a significant increase of migration and invasion, although proliferation remained unaltered. The increase of migration and invasion are consistent with the known activities of FAP-α as an exopeptidase and endopeptidase/gelatinase/collagenase in tissue remodeling and repair, and in cell migration. We additionally determined the effects of FAP-α overexpression on the human fibrosarcoma HT1080 cell line that showed increased migration, accompanied by limited changes in metabolism that identified the dependency of the metabolic changes on cell type. These metabolic data identify a previously unknown role of FAP-α in modifying cancer cell metabolism in the TNBC cell line studied here that may provide new insights into its functional roles in cancer progression.

Glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5) expression correlates with malignant choline phospholipid metabolite profiles in human breast cancer.

Altered choline phospholipid metabolism is a hallmark of cancer, leading to malignant choline metabolite profiles consisting of low glycerophosphocholine (GPC) and high phosphocholine (PC) in human breast cancers. Glycerophosphocholine phosphodiesterase (GPC-PDE) catalyzes the degradation of GPC to free choline and glycerol-3-phosphate. The gene(s) encoding for the GPC-PDE(s) responsible for GPC degradation in breast cancers have not yet been identified. Here, we demonstrate for the first time that the GPC-PDE encoded by glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5) is associated with breast cancer malignancy. Two human breast cancer cell lines (n = 8 and n = 10) and primary human breast tumor samples (n = 19) were studied with combined MRS and quantitative reverse transcription-polymerase chain reaction to investigate several isoforms of GDPD expression with respect to choline phospholipid metabolite levels. Of the five GDPDs tested, GDPD5 was found to be significantly overexpressed in highly malignant estrogen receptor negative (ER(-)) compared with weakly malignant estrogen receptor positive (ER(+)) human breast cancer cells (p = 0.027) and breast tumors from patients (p = 0.015). GDPD5 showed significantly positive correlations with PC (p < 0.001), total choline (tCho) (p = 0.007) and PC/GPC (p < 0.001) levels in human breast tumors. GDPD5 showed a trend towards a negative correlation with GPC levels (p = 0.130). Human breast cancers with malignant choline metabolite profiles consisting of low GPC and high PC levels highly co-expressed GDPD5, choline kinase alpha (CHKA) and phosphatidylcholine-specific phospholipase D1 (PLD1), whereas cancers containing high GPC and relatively low PC levels displayed low co-expression of GDPD5, CHKA and PLD1. GDPD5, CHKA and PLD1 were significantly overexpressed in highly malignant ER(-) tumors in our patient cohort. Our study identified GDPD5 as a GPC-PDE that probably participates in the regulation of choline phospholipid metabolism in breast cancer, which possibly occurs in cooperation with CHKA and PLD1.

RK-33, a small molecule inhibitor of host RNA helicase DDX3, suppresses multiple variants of SARS-CoV-2.

SARS-CoV-2, the virus behind the deadly COVID-19 pandemic, continues to spread globally even as vaccine strategies are proving effective in preventing hospitalizations and deaths. However, evolving variants of the virus appear to be more transmissive and vaccine efficacy towards them is waning. As a result, SARS-CoV-2 will continue to have a deadly impact on public health into the foreseeable future. One strategy to bypass the continuing problem of newer variants is to target host proteins required for viral replication. We have used this host-targeted antiviral (HTA) strategy that targets DDX3, a host DEAD-box RNA helicase that is usurped by SARS-CoV-2 for virus production. We demonstrated that targeting DDX3 with RK-33, a small molecule inhibitor, reduced the viral load in four isolates of SARS-CoV-2 (Lineage A, and Lineage B Alpha, Beta, and Delta variants) by one to three log orders in Calu-3 cells. Furthermore, proteomics and RNA-seq analyses indicated that most SARS-CoV-2 genes were downregulated by RK-33 treatment. Also, we show that the use of RK-33 decreases TMPRSS2 expression, which may be due to DDX3s ability to unwind G-quadraplex structures present in the TMPRSS2 promoter. The data presented supports the use of RK-33 as an HTA strategy to control SARS-CoV-2 infection, irrespective of its mutational status, in humans.

RK-33, a small molecule inhibitor of host RNA helicase DDX3, suppresses multiple variants of SARS-CoV-2.

SARS-CoV-2, the virus behind the deadly COVID-19 pandemic, continues to spread globally even as vaccine strategies are proving effective in preventing hospitalizations and deaths. However, evolving variants of the virus appear to be more transmissive and vaccine efficacy toward them is waning. As a result, SARS-CoV-2 will continue to have a deadly impact on public health into the foreseeable future. One strategy to bypass the continuing problem of newer variants is to target host proteins required for viral replication. We have used this host-targeted antiviral (HTA) strategy that targets DDX3X (DDX3), a host DEAD-box RNA helicase that is usurped by SARS-CoV-2 for virus production. We demonstrated that targeting DDX3 with RK-33, a small molecule inhibitor, reduced the viral load in four isolates of SARS-CoV-2 (Lineage A, and Lineage B Alpha, Beta, and Delta variants) by one to three log orders in Calu-3 cells. Furthermore, proteomics and RNA-seq analyses indicated that most SARS-CoV-2 genes were downregulated by RK-33 treatment. Also, we show that the use of RK-33 decreases TMPRSS2 expression, which may be due to DDX3s ability to unwind G-quadraplex structures present in the TMPRSS2 promoter. The data presented support the use of RK-33 as an HTA strategy to control SARS-CoV-2 infection, irrespective of its mutational status, in humans.

Targeting host DEAD-box RNA helicase DDX3X for treating viral infections.

DDX3X or DDX3, a member of the DEAD (asp, glu, ala, asp) box RNA helicase family of proteins, is a multifunctional protein, which is usurped by several viruses and is vital to their production. To date, 18 species of virus from 12 genera have been demonstrated to be dependent on DDX3 for virulence. In addition, DDX3 has been shown to function within 7 of 10 subcellular regions that are involved in the metabolism of viruses. As such, due to its direct interaction with viral components across most or all stages of viral life cycles, DDX3 can be considered an excellent host target for pan-antiviral drug therapy and has been reported to be a possible broad-spectrum antiviral target. Along these lines, it has been demonstrated that treatment of virally infected cells with small molecule inhibitors of DDX3 blunts virion productions. On the other hand, DDX3 bolsters an innate immune response and viruses have evolved capacities to sequester or block DDX3, which dampens an innate immune response. Thus, enhancing DDX3 production or co-targeting direct viral products that interfere with DDX3's modulation of innate immunity would also diminish virion production. Here we review the evidence that supports the hypothesis that modulating DDX3's agonistic and antagonistic functions during viral infections could have an important impact on safely and efficiently subduing a broad-spectrum of viral infections.

Divergent organ-specific isogenic metastatic cell lines identified using multi-omics exhibit differential drug sensitivity.

BACKGROUND: Monitoring and treating metastatic progression remains a formidable task due, in part, to an inability to monitor specific differential molecular adaptations that allow the cancer to thrive within different tissue types. Hence, to develop optimal treatment strategies for metastatic disease, an important consideration is the divergence of the metastatic cancer growing in visceral organs from the primary tumor. We had previously reported the establishment of isogenic human metastatic breast cancer cell lines that are representative of the common metastatic sites observed in breast cancer patients. METHODS: Here we have used proteomic, RNAseq, and metabolomic analyses of these isogenic cell lines to systematically identify differences and commonalities in pathway networks and examine the effect on the sensitivity to breast cancer therapeutic agents. RESULTS: Proteomic analyses indicated that dissemination of cells from the primary tumor sites to visceral organs resulted in cell lines that adapted to growth at each new site by, in part, acquiring protein pathways characteristic of the organ of growth. RNAseq and metabolomics analyses further confirmed the divergences, which resulted in differential efficacies to commonly used FDA approved chemotherapeutic drugs. This model system has provided data that indicates that organ-specific growth of malignant lesions is a selective adaptation and growth process. CONCLUSIONS: The insights provided by these analyses indicate that the rationale of targeted treatment of metastatic disease may benefit from a consideration that the biology of metastases has diverged from the primary tumor biology and using primary tumor traits as the basis for treatment may not be ideal to design treatment strategies.

Targeting RNA helicase DDX3 in stem cell maintenance and teratoma formation.

DDX3 is an RNA helicase that has antiapoptotic properties, and promotes proliferation and transformation. Besides the role of DDX3 in transformed cells, there is evidence to indicate that DDX3 expression is at its highest levels during early embryonic development and is also expressed in germ cells of adults. Even though there is a distinct pattern of DDX3 expression during embryonic development and in adults, very little is known regarding its role in embryonic stem cells and pluripotency. In this work, we examined the relationship between DDX3 and human embryonic stem cells and its differentiated lineages. DDX3 expression was analyzed by immunohistochemistry in human embryonic stem cells and embryonal carcinoma cells. From the data obtained, it was evident that DDX3 was overexpressed in undifferentiated stem cells compared to differentiated cells. Moreover, when DDX3 expression was abrogated in multiple stem cells, proliferation was decreased, but differentiation was facilitated. Importantly, this resulted in reduced potency to induce teratoma formation. Taken together, these findings indicate a distinct role for DDX3 in stem cell maintenance.

Targeting DDX3 in Medulloblastoma Using the Small Molecule Inhibitor RK-33.

Medulloblastoma is the most common malignant tumor that arises from the cerebellum of the central nervous system. Clinically, medulloblastomas are treated by surgery, radiation, and chemotherapy, all of which result in toxicity and morbidity. Recent reports have identified that DDX3, a member of the RNA helicase family, is mutated in medulloblastoma. In this study, we demonstrate the role of DDX3 in driving medulloblastoma. With the use of a small molecule inhibitor of DDX3, RK-33, we could inhibit growth and promote cell death in two medulloblastoma cell lines, DAOY and UW228, with IC50 values of 2.5 µM and 3.5 µM, respectively. Treatment of DAOY and UW228 cells with RK-33 caused a G1 arrest, resulted in reduced TCF reporter activity, and reduced mRNA expression levels of downstream target genes of the WNT pathway, such as Axin2, CCND1, MYC, and Survivin. In addition, treatment of DAOY and UW228 cells with a combination of RK-33 and radiation exhibited a synergistic effect. Importantly, the combination of RK-33 and 5 Gy radiation caused tumor regression in a mouse xenograft model of medulloblastoma. Using immunohistochemistry, we observed DDX3 expression in both pediatric (55%) and adult (66%) medulloblastoma patients. Based on these results, we conclude that RK-33 is a promising radiosensitizing agent that inhibits DDX3 activity and down-regulates WNT/ß-catenin signaling and could be used as a frontline therapeutic strategy for DDX3-expressing medulloblastomas in combination with radiation.

Twist is a transcriptional repressor of E-cadherin gene expression in breast cancer.

Twist is a basic helix loop helix protein that plays a role both in human development and in cancer biogenesis. While characterizing the effects of Twist on breast epithelial cell transformation, we identified E-cadherin as a target gene that is down-regulated by Twist. In this study, we demonstrate that Twist can transcriptionally repress E-cadherin in breast cancer cells. Using transient promoter assays, we show that Twist can down-regulate E-cadherin promoter activity by up to two folds. This is further supported by immunoblot analyses which indicates that over-expression of Twist decreases E-cadherin protein levels in breast cancer cell lines. Subsequently, chromatin immunoprecipitation performed on MCF-7/Twist and Hs578 T (high level of endogenous Twist expression) confirmed Twist binding to the E-cadherin promoter. Finally, the functional relevance of this regulation was verified by quantitative real-time PCR and immunohistochemistry on a cohort of breast cancer samples.

Global Effects of DDX3 Inhibition on Cell Cycle Regulation Identified by a Combined Phosphoproteomics and Single Cell Tracking Approach.

DDX3 is an RNA helicase with oncogenic properties. The small molecule inhibitor RK-33 is designed to fit into the ATP binding cleft of DDX3 and hereby block its activity. RK-33 has shown potent activity in preclinical cancer models. However, the mechanism behind the antineoplastic activity of RK-33 remains largely unknown. In this study we used a dual phosphoproteomic and single cell tracking approach to evaluate the effect of RK-33 on cancer cells. MDA-MB-435 cells were treated for 24 hours with RK-33 or vehicle control. Changes in phosphopeptide abundance were analyzed with quantitative mass spectrometry using isobaric mass tags (Tandem Mass Tags). At the proteome level we mainly observed changes in mitochondrial translation, cell division pathways and proteins related to cell cycle progression. Analysis of the phosphoproteome indicated decreased CDK1 activity after RK-33 treatment. To further evaluate the effect of DDX3 inhibition on cell cycle progression over time, we performed timelapse microscopy of Fluorescent Ubiquitin Cell Cycle Indicators labeled cells after RK-33 or siDDX3 exposure. Single cell tracking indicated that DDX3 inhibition resulted in a global delay in cell cycle progression in interphase and mitosis. In addition, we observed an increase in endoreduplication. Overall, we conclude that DDX3 inhibition affects cells in all phases and causes a global cell cycle progression delay.

Twist overexpression induces in vivo angiogenesis and correlates with chromosomal instability in breast cancer.

Aggressive cancer phenotypes are a manifestation of many different genetic alterations that promote rapid proliferation and metastasis. In this study, we show that stable overexpression of Twist in a breast cancer cell line, MCF-7, altered its morphology to a fibroblastic-like phenotype, which exhibited protein markers representative of a mesenchymal transformation. In addition, it was observed that MCF-7/Twist cells had increased vascular endothelial growth factor (VEGF) synthesis when compared with empty vector control cells. The functional changes induced by VEGF in vivo were analyzed by functional magnetic resonance imaging (MRI) of MCF-7/Twist-xenografted tumors. MRI showed that MCF-7/Twist tumors exhibited higher vascular volume and vascular permeability in vivo than the MCF-7/vector control xenografts. Moreover, elevated expression of Twist in breast tumor samples obtained from patients correlated strongly with high-grade invasive carcinomas and with chromosome instability, particularly gains of chromosomes 1 and 7. Taken together, these results show that Twist overexpression in breast cancer cells can induce angiogenesis, correlates with chromosomal instability, and promotes an epithelial-mesenchymal-like transition that is pivotal for the transformation into an aggressive breast cancer phenotype.

Role of DDX3 in the pathogenesis of inflammatory bowel disease.

When crypt stem cells of the gastrointestinal tract become injured, the result is increased synthesis of pro-inflammatory cytokines and matrix metalloproteinases by their progeny - the colonic epithelium. Chronic inflammation of the gastrointestinal tract is a characteristic of inflammatory bowel disease, which includes Crohn's Disease and Ulcerative Colitis. In our ongoing investigation to decipher the characteristic functions of a RNA helicase gene, DDX3, we identified high DDX3 expression by immunohistochemistry of colon biopsy samples, which included chronic/mild Morbus Crohn, active Morbus Crohn, Chronic/mild Colitis Ulcerosa and active Colitis Ulcerosa in epithelium and stromal compartments. We used a small molecule inhibitor of DDX3, RK-33, on two human colonic epithelial cell lines, HCEC1CT and HCEC2CT and found that RK-33 was able to decrease expression of MMP-1, MMP-2, MMP-3, and MMP-10. Moreover, forced differentiation of a human colonic cancer cell line, HT29, resulted in decreased DDX3 levels, indicating that DDX3 contributes to the modulation of colonic epithelium differentiation. In conclusion, our results revealed novel functions of DDX3 in inflammatory bowel disease and indicate a potential for using RK-33 as a systemic therapy to promote not only differentiation of transformed colonic epithelium but also to reduce MMP expression and thus elicit a decreased inflammatory response.

Organ-specific isogenic metastatic breast cancer cell lines exhibit distinct Raman spectral signatures and metabolomes.

Molecular characterization of organ-specific metastatic lesions, which distinguish them from the primary tumor, will provide a better understanding of tissue specific adaptations that regulate metastatic progression. Using an orthotopic xenograft model, we have isolated isogenic metastatic human breast cancer cell lines directly from organ explants that are phenotypically distinct from the primary tumor cell line. Label-free Raman spectroscopy was used and informative spectral bands were ascertained as differentiators of organ-specific metastases as opposed to the presence of a single universal marker. Decision algorithms derived from the Raman spectra unambiguously identified these isogenic cell lines as unique biological entities - a finding reinforced through metabolomic analyses that indicated tissue of origin metabolite distinctions between the cell lines. Notably, complementarity of the metabolomics and Raman datasets was found. Our findings provide evidence that metastatic spread generates tissue-specific adaptations at the molecular level within cancer cells, which can be differentiated with Raman spectroscopy.

Combination treatment using DDX3 and PARP inhibitors induces synthetic lethality in BRCA1-proficient breast cancer.

Triple-negative breast cancers have unfavorable outcomes due to their inherent aggressive behavior and lack of targeted therapies. Breast cancers occurring in BRCA1 mutation carriers are mostly triple-negative and harbor homologous recombination deficiency, sensitizing them to inhibition of a second DNA damage repair pathway by, e.g., PARP inhibitors. Unfortunately, resistance against PARP inhibitors in BRCA1-deficient cancers is common and sensitivity is limited in BRCA1-proficient breast cancers. RK-33, an inhibitor of the RNA helicase DDX3, was previously demonstrated to impede non-homologous end-joining repair of DNA breaks. Consequently, we evaluated DDX3 as a therapeutic target in BRCA pro- and deficient breast cancers and assessed whether DDX3 inhibition could sensitize cells to PARP inhibition. High DDX3 expression was identified by immunohistochemistry in breast cancer samples of 24% of BRCA1 (p = 0.337) and 21% of BRCA2 mutation carriers (p = 0.624), as compared to 30% of sporadic breast cancer samples. The sensitivity to the DDX3 inhibitor RK-33 was similar in BRCA1 pro- and deficient breast cancer cell lines, with IC50 values in the low micromolar range (2.8-6.6 µM). A synergistic interaction was observed for combination treatment with RK-33 and the PARP inhibitor olaparib in BRCA1-proficient breast cancer, with the mean combination index ranging from 0.59 to 0.62. Overall, we conclude that BRCA pro- and deficient breast cancers have a similar dependency upon DDX3. DDX3 inhibition by RK-33 synergizes with PARP inhibitor treatment, especially in breast cancers with a BRCA1-proficient background.

Nuclear DDX3 expression predicts poor outcome in colorectal and breast cancer.

PURPOSE: DEAD box protein 3 (DDX3) is an RNA helicase with oncogenic properties that shuttles between the cytoplasm and nucleus. The majority of DDX3 is found in the cytoplasm, but a subset of tumors has distinct nuclear DDX3 localization of yet unknown biological significance. This study aimed to evaluate the significance of and mechanisms behind nuclear DDX3 expression in colorectal and breast cancer. METHODS: Expression of nuclear DDX3 and the nuclear exporter chromosome region maintenance 1 (CRM1) was evaluated by immunohistochemistry in 304 colorectal and 292 breast cancer patient samples. Correlations between the subcellular localization of DDX3 and CRM1 and the difference in overall survival between patients with and without nuclear DDX3 were studied. In addition, DDX3 mutants were created for in vitro evaluation of the mechanism behind nuclear retention of DDX3. RESULTS: DDX3 was present in the nucleus of 35% of colorectal and 48% of breast cancer patient samples and was particularly strong in the nucleolus. Nuclear DDX3 correlated with worse overall survival in both colorectal (hazard ratio [HR] 2.34, P<0.001) and breast cancer (HR 2.39, P=0.004) patients. Colorectal cancers with nuclear DDX3 expression more often had cytoplasmic expression of the nuclear exporter CRM1 (relative risk 1.67, P=0.04). In vitro analysis of DDX3 deletion mutants demonstrated that CRM1-mediated export was most dependent on the N-terminal nuclear export signal. CONCLUSION: Overall, we conclude that nuclear DDX3 is partially CRM1-mediated and predicts worse survival in colorectal and breast cancer patients, putting it forward as a target for therapeutic intervention with DDX3 inhibitors under development in these cancer types.

RK-33 Radiosensitizes Prostate Cancer Cells by Blocking the RNA Helicase DDX3.

Despite advances in diagnosis and treatment, prostate cancer is the most prevalent cancer in males and the second highest cause of cancer-related mortality. We identified an RNA helicase gene, DDX3 (DDX3X), which is overexpressed in prostate cancers, and whose expression is directly correlated with high Gleason scores. Knockdown of DDX3 in the aggressive prostate cancer cell lines DU145 and 22Rv1 resulted in significantly reduced clonogenicity. To target DDX3, we rationally designed a small molecule, RK-33, which docks into the ATP-binding domain of DDX3. Functional studies indicated that RK-33 preferentially bound to DDX3 and perturbed its activity. RK-33 treatment of prostate cancer cell lines DU145, 22Rv1, and LNCaP (which have high DDX3 levels) decreased proliferation and induced a G1 phase cell-cycle arrest. Conversely, the low DDX3-expressing cell line, PC3, exhibited few changes following RK-33 treatment. Importantly, combination studies using RK-33 and radiation exhibited synergistic effects both in vitro and in a xenograft model of prostate cancer demonstrating the role of RK-33 as a radiosensitizer. Taken together, these results indicate that blocking DDX3 by RK-33 in combination with radiation treatment is a viable option for treating locally advanced prostate cancer. Cancer Res; 76(21); 6340-50. ©2016 AACR.

miRNA expression patterns in normal breast tissue and invasive breast cancers of BRCA1 and BRCA2 germ-line mutation carriers.

miRNA deregulation has been found to promote carcinogenesis. Little is known about miRNA deregulation in hereditary breast tumors as no miRNA expression profiling studies have been performed in normal breast tissue of BRCA1 and BRCA2 mutation carriers. miRNA profiles of 17 BRCA1- and 9 BRCA2-associated breast carcinomas were analyzed using microarrays. Normal breast tissues from BRCA1 and BRCA2 mutation carriers (both n = 5) and non-mutation carriers (n = 10) were also included. Candidate miRNAs were validated by qRT-PCR. Breast carcinomas showed extensive miRNA alteration compared to normal breast tissues in BRCA1 and BRCA2 mutation carriers. Moreover, normal breast tissue from BRCA1 mutation carriers already showed miRNA alterations compared to non-mutation carriers. Chromosomal distribution analysis showed several hotspots containing down- or up-regulated miRNAs. Pathway analysis yielded many similarities between the BRCA1 and BRCA2 axes with miRNAs involved in cell cycle regulation, proliferation and apoptosis. Lesser known pathways were also affected, including cellular movement and protein trafficking. This study provides a comprehensive insight into the potential role of miRNA deregulation in BRCA1/2-associated breast carcinogenesis. The observed extensive miRNA deregulation is likely the result of genome-wide effects of chromosomal instability caused by impaired BRCA1 or BRCA2 function. This study's results also suggest the existence of common pathways driving breast carcinogenesis in both BRCA1 and BRCA2 germ-line mutation carriers.