Chimeric RNAs reveal putative neoantigen peptides for developing tumor vaccines for breast cancer.

Introduction: We present here a strategy to identify immunogenic neoantigen candidates from unique amino acid sequences at the junctions of fusion proteins which can serve as targets in the development of tumor vaccines for the treatment of breastcancer. Method: We mined the sequence reads of breast tumor tissue that are usually discarded as discordant paired-end reads and discovered cancer specific fusion transcripts using tissue from cancer free controls as reference. Binding affinity predictions of novel peptide sequences crossing the fusion junction were analyzed by the MHC Class I binding predictor, MHCnuggets. CD8+ T cell responses against the 15 peptides were assessed through in vitro Enzyme Linked Immunospot (ELISpot). Results: We uncovered 20 novel fusion transcripts from 75 breast tumors of 3 subtypes: TNBC, HER2+, and HR+. Of these, the NSFP1-LRRC37A2 fusion transcript was selected for further study. The 3833 bp chimeric RNA predicted by the consensus fusion junction sequence is consistent with a read-through transcription of the 5'-gene NSFP1-Pseudo gene NSFP1 (NSFtruncation at exon 12/13) followed by trans-splicing to connect withLRRC37A2 located immediately 3' through exon 1/2. A total of 15 different 8-mer neoantigen peptides discovered from the NSFP1 and LRRC37A2 truncations were predicted to bind to a total of 35 unique MHC class I alleles with a binding affinity of IC50<500nM.); 1 of which elicited a robust immune response. Conclusion: Our data provides a framework to identify immunogenic neoantigen candidates from fusion transcripts and suggests a potential vaccine strategy to target the immunogenic neopeptides in patients with tumors carrying the NSFP1-LRRC37A2 fusion.

The molecular heterogeneity of the precancerous breast affects drug efficacy.

In the therapeutic domain, targeted therapies have been shown to be generally more effective when given to patients with tumors that harbor the targeted aberration. This principle has not been tested in cancer prevention despite evidence that molecular heterogeneity accompanies the multi-step progression to invasive disease. We hypothesized that efficacy of agents targeting the precancerous state varies based on timing of the treatment relative to the underlying molecular changes. MCF10A cell line-based model of the multi-step progression to TNBC was used. Global proteomic patterns were obtained and growth-inhibitory effects of selected agents were correlated with the underlying molecular stage of progression. These analyses revealed that most protein alterations were acquired in the normal-to-atypia (preneoplasia) transition, with only handful aberrations acquired hereafter. The efficacy of small molecule inhibitors of the AKT/MEK pathway was associated with the underlying pathway levels. Similarly, fluvastatin was more effective in inhibiting cell proliferation earlier in the progression model. However, the nonspecific inhibitors, aspirin and metformin, were equally ineffective in inhibiting proliferation across the progression model. Our data provides proof-of-principle that in the prevention domain, treatment with agents developed to target specific pathways, will need to consider the molecular heterogeneity of the precancerous breast in order to achieve maximum efficacy.

Gene signature associated with resistance to fluvastatin chemoprevention for breast cancer.

BACKGROUND: Although targeting of the cholesterol pathway by statins prevents breast cancer development in mouse models, efficacy is not absolute. Therefore, the goal of this study is to investigate if the upregulation in the cholesterol biosynthesis pathway genes associates with response to statin chemoprevention and may potentially be used as response biomarkers. METHODS: Expression of cholesterol biosynthesis pathway genes was initially derived from the RNA sequencing of MCF10A cell line- based breast cancer progression model system and subsequently validated by quantitative PCR assay. Response to fluvastatin was assessed in vitro using the MCF10A cell line model system, including a statin resistant cell line that was generated (MCF10.AT1-R), and measured using colony forming assays. In vivo efficacy of statin for chemoprevention was assessed in the SV40C3 TAg mouse model. Mammary tumors were identified by histologic analysis of the mammary glands. Mammary glands without histologic evidence of high-grade lesions (in situ and/or invasive carcinoma) were considered responsive to statin treatment. RESULTS: We found more than 70% of a published multi-gene fluvastatin resistance signature to be significantly upregulated during breast cancer progression and inversely correlated with statin inhibition of cellular growth and proliferation. This inherent statin resistance gene signature was also largely shared with the signature of acquired resistance to fluvastatin in MCF10.AT1-R cell line model of acquired statin resistance. These inherent resistance genes and genes exclusive to acquired statin resistance map to steroid-, and terpenoid backbone- biosynthesis pathway. We found upregulation of ~ 80% of cholesterol biosynthesis pathway genes in the tumor bearing mammary glands of SV40 C3TAg transgenic mouse model of TNBC, suggesting the involvement of cholesterol biosynthesis pathway in resistance to statin chemoprevention in vivo. A panel of 13-genes from the pathway significantly associated with response to statin treatment, as did the expression level of HMGCR alone in a mouse model of breast cancer suggesting their utility to predict the efficacy of statin chemoprevention. CONCLUSIONS: High basal level, or restorative upregulation, in the cholesterol biosynthesis pathway genes appear to be strongly associated with resistance to statin chemoprevention for breast cancer and may serve as a biomarker to tailor statin treatment to individuals who are most likely to benefit.

Concordant Androgen-Regulated Expression of Divergent Rhox5 Promoters in Sertoli Cells.

Concordant transcriptional regulation can generate multiple gene products that collaborate to achieve a common goal. Here we report a case of concordant transcriptional regulation that instead drives a single protein to be produced in the same cell type from divergent promoters. This gene product-the RHOX5 homeobox transcription factor-is translated from 2 different mRNAs with different 5' untranslated regions (UTRs) transcribed from alternative promoters. Despite the fact that these 2 promoters-the proximal promoter (Pp) and the distal promoter (Pd)-exhibit different patterns of tissue-specific activity, share no obvious sequence identity, and depend on distinct transcription factors for expression, they exhibit a remarkably similar expression pattern in the testes. In particular, both depend on androgen signaling for expression in the testes, where they are specifically expressed in Sertoli cells and have a similar stage-specific expression pattern during the seminiferous epithelial cycle. We report evidence for 3 mechanisms that collaborate to drive concordant Pp/Pd expression. First, both promoters have an intrinsic ability to respond to androgen receptor and androgen. Second, the Pp acts as an enhancer to promote androgen-dependent transcription from the Pd. Third, Pd transcription is positively autoregulated by the RHOX5 protein, which is first produced developmentally from the Pp. Together, our data support a model in which the Rhox5 homeobox gene evolved multiple mechanisms to activate both of its promoters in Sertoli cells to produce Rhox5 in an androgen-dependent manner during different phases of spermatogenesis.

Efficacy of fluvastatin and aspirin for prevention of hormonally insensitive breast cancer.

PURPOSE: Primary prevention of hormonally insensitive breast cancers remains an important clinical need and repurposing existing low-toxicity drugs represents a low-cost, efficient strategy for meeting this goal. This study targeted the cholesterol pathway using fluvastatin, a cholesterol-lowering drug, and aspirin, an AMPK activator that acts as a brake in the cholesterol pathway, in a transgenic mouse model of triple-negative breast cancer (TNBC). METHODS: Using SV40C3 TAg mice, the efficacy and mechanism of fluvastatin, aspirin, or both in combination were compared with vehicle alone. RESULTS: Sixteen-weeks of fluvastatin treatment resulted in significant delay in onset of tumors (20 weeks vs. 16.8 weeks in vehicle treatment, p = 0.01) and inhibited tumor incidence and tumor multiplicity by 50% relative to the vehicle control. In animals that developed tumors, fluvastatin treatment inhibited tumor weight by 75% relative to vehicle control. Aspirin alone did not significantly affect tumor latency, tumor incidence or tumor burden compared to vehicle control. Fluvastatin and aspirin in combination delayed the onset of tumors but failed to inhibit tumor incidence and tumor multiplicity. The growth-inhibitory effects of fluvastatin were mediated through increased FAS/FASL mediated apoptotic cell death that was characterized by increased cleaved PARP and driven in part by depletion of an isoprenoid, geranyl geranyl pyrophosphate (GGPP). CONCLUSIONS: In line with NCI's emphasis to repurpose low-toxicity drugs for prevention of cancer, fluvastatin was effective for prevention of TNBC and warrants further clinical testing. Aspirin did not provide chemopreventive benefit.

Erratum: Ju, Z., et al. Integrative Analyses of Multilevel Omics Reveal Preneoplastic Breast to Possess a Molecular Landscape That Is Globally Shared with Invasive Basal-Like Breast Cancer (Running Title: Molecular Landscape of Basal-Like Breast Cancer Progression). Cancers 2020, 12, 722.

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Integrative Analyses of Multilevel Omics Reveal Preneoplastic Breast to Possess a Molecular Landscape That is Globally Shared with Invasive Basal-Like Breast Cancer (Running Title: Molecular Landscape of Basal-Like Breast Cancer Progression).

To characterize molecular changes accompanying the stepwise progression to breast cancer and to identify functional target pathways, we performed miRNA and RNA sequencing using MCF10A cell lines based model system that replicates the multi-step progression involving normal, preneoplastic, ductal carcinoma in situ, and invasive carcinoma cells, where the carcinoma most resemble the basal-like subgroup of human breast cancers. These analyses suggest that 70% of miRNA alterations occurred during the initial progression from normal to a preneoplastic stage. Most of these early changes reflected a global upregulation of miRNAs. This was consistent with a global increase in the miRNA-processing enzyme DICER, which was upregulated as a direct result of loss of miRNA let-7b-5p. Several oncogenic and tumor suppressor pathways were also found to change early, prior to histologic stigmata of cancer. Our finding that most genomic changes in the progression to basal-like breast cancer occurred in the earliest stages of histologic progression has implications for breast cancer prevention and selection of appropriate control tissues in molecular studies. Furthermore, in support of a functional significance of let-7b-5p loss, we found its low levels to predict poor disease-free survival and overall survival in breast cancer patients.

The isomiR-140-3p-regulated mevalonic acid pathway as a potential target for prevention of triple negative breast cancer.

BACKGROUND: Prevention of triple-negative breast cancer (TNBC) is hampered by lack of knowledge about the drivers of tumorigenesis. METHODS: To identify molecular markers and their downstream networks that can potentially be targeted for TNBC prevention, we analyzed small RNA and RNA sequencing of a cell line model that represent early stages of TNBC development. We have identified direct gene targets of isomiRNA-140-3p and by using cell-based and in vivo model systems we have demonstrated the utility of targeting downstream pathways for prevention of TNBC. RESULTS: These analyses showed that 5'isomiRNA of miR-140-3p (miR-140-3p-1) and its novel direct gene targets, HMG-CoA reductase (HMGCR) and HMG-CoA synthase 1(HMGCS1), key enzymes in the cholesterol biosynthesis pathway, were deregulated in the normal-to-preneoplastic transition. Upregulation in the cholesterol pathway creates metabolic vulnerability that can be targeted. Consistent with this hypothesis, we found direct targeting of miR-140-3p-1 and its downstream pathway by fluvastatin to inhibit growth of these preneoplastic MCF10.AT1 cells. However, although, fluvastatin inhibited the growth of MCF10.AT1-derived xenografts, histological progression remained unchanged. The cholesterol pathway is highly regulated, and HMGCR enzymatic activity inhibition is known to trigger a feedback response leading to restoration of the pathway. Indeed, we found fluvastatin-induced HMGCR transcript levels to be directly correlated with the degree of histological progression of lesions, indicating that the extent of cholesterol pathway suppression directly correlates with abrogation of the tumorigenic process. To block the HMGCR feedback response to statins, we treated resistant preneoplastic cells with an activator of AMP-activated protein kinase (AMPK), a brake in the cholesterol feedback pathway. AMPK activation by aspirin and metformin effectively abrogated the statin-induced aberrant upregulation of HMGCR and sensitized these resistant cells to fluvastatin. CONCLUSIONS: These results suggest the potential use of combined treatment with statin and aspirin for prevention of TNBC.

Regulation of miRNA-29c and its downstream pathways in preneoplastic progression of triple-negative breast cancer.

Little is understood about the early molecular drivers of triple-negative breast cancer (TNBC), making the identification of women at risk and development of targeted therapy for prevention significant challenges. By sequencing a TNBC cell line-based breast cancer progression model we have found that miRNA-29c is progressively lost during TNBC tumorigenesis. In support of the tumor suppressive role of miRNA 29c, we found that low levels predict poor overall patient survival and, conversely, that ectopic expression of miRNA-29c in preneoplastic cell models inhibits growth. miRNA-29c exerts its growth inhibitory effects through direct binding and regulation of TGFB-induced factor homeobox 2 (TGIF2), CAMP-responsive element binding protein 5 (CREB5), and V-Akt murine thymoma viral oncogene homolog 3 (AKT3). miRNA-29c regulation of these gene targets seems to be functionally relevant, as TGIF2, CREB5, and AKT3 were able to rescue the inhibition of cell proliferation and colony formation caused by ectopic expression of miRNA-29c in preneoplastic cells. AKT3 is an oncogene of known relevance in breast cancer, and as a proof of principle we show that inhibition of phosphoinositide 3-kinase (PI3K) activity, a protein upstream of AKT3, suppressed proliferation in TNBC preneoplastic cells. We explored additional opportunities for prevention of TNBC by studying the regulation of miRNA-29c and identified DNA methylation to have a role in the inhibition of miRNA-29c during TNBC tumorigenesis. Consistent with these observations, we found 5 aza-cytadine to relieve the suppression of miRNA-29c. Together, these results demonstrate that miRNA-29c loss plays a key role in the early development of TNBC.

Annexin A1 Preferentially Predicts Poor Prognosis of Basal-Like Breast Cancer Patients by Activating mTOR-S6 Signaling.

INTRODUCTION: Annexin A1 (ANXA1) is an anti-inflammatory protein reported to play a role in cell proliferation and apoptosis, and to be deregulated in breast cancer. The exact role of annexin A1 in the biology of breast cancer remains unclear. We hypothesized that the annexin A1 plays an oncogenic role in basal subtype of breast cancer by modulating key growth pathway(s). METHODS: By mining the Cancer Genome Atlas (TCGA)-Breast Cancer dataset and manipulating annexin A1 levels in breast cancer cell lines, we studied the role of annexin A1 in breast cancer and underlying signaling pathways. RESULTS: Our in-silico analysis of TCGA-breast cancer dataset demonstrated that annexin A1 mRNA expression is higher in basal subtype compared to luminal and HER2 subtypes. Within the basal subtype, patients show significantly poorer overall survival associated with higher expression of annexin A1. In both TCGA patient samples and cell lines, annexin A1 levels were significantly higher in basal-like breast cancer than luminal and Her2/neu-positive breast cancer. Stable annexin A1 knockdown in TNBC cell lines suppressed the mTOR-S6 pathway likely through activation of AMPK but had no impact on the MAPK, c-Met, and EGFR pathways. In a cell migration assay, annexin A1-depleted TNBC cells showed delayed migration as compared to wild-type cells, which could be responsible for poor patient prognosis in basal like breast cancers that are known to express higher annexin A1. CONCLUSIONS: Our data suggest that annexin A1 is prognostic only in patients with basal like breast cancer. This appears to be in part due to the role of annexin A1 in activating mTOR-pS6 pathway.

Hormone-induced and DNA demethylation-induced relief of a tissue-specific and developmentally regulated block in transcriptional elongation.

Genome-wide studies have revealed that genes commonly have a high density of RNA polymerase II just downstream of the transcription start site. This has raised the possibility that genes are commonly regulated by transcriptional elongation, but this remains largely untested in vivo, particularly in vertebrates. Here, we show that the proximal promoter from the Rhox5 homeobox gene recruits polymerase II and begins elongating in all tissues and cell lines that we tested, but it only completes elongation in a tissue-specific and developmentally regulated manner. Relief of the elongation block is associated with recruitment of the elongation factor P-TEFb, the co-activator GRIP1, the chromatin remodeling factor BRG1, and specific histone modifications. We provide evidence that two mechanisms relieve the elongation block at the proximal promoter: demethylation and recruitment of androgen receptor. Together, our findings support a model in which promoter proximal pausing helps confer tissue-specific and developmental gene expression through a mechanism regulated by DNA demethylation-dependent nuclear hormone receptor recruitment.

Suppression of Akt-mTOR pathway-a novel component of oncogene induced DNA damage response barrier in breast tumorigenesis.

DNA damage has been thought to be directly associated with the neoplastic progression by enabling mutations in tumor suppressor genes and activating/and amplifying oncogenes ultimately resulting in genomic instability. DNA damage causes activation of the DNA damage response (DDR) that is an important cellular mechanism for maintaining genomic integrity in the face of genotoxic stress. While the cellular response to genotoxic stress has been extensively studied in cancer models, less is known about the cellular response to oncogenic stress in the premalignant context. In the present study, by using breast tissues samples from women at different risk levels for invasive breast cancer (normal, proliferative breast disease and ductal carcinoma in situ) we found that DNA damage is inversely correlated with risk of invasive breast cancer. Similarly, in MCF10A based in vitro model system where we recapitulated high DNA damage conditions as seen in patient samples by stably cloning in cyclin E, we found that high levels of oncogene induced DNA damage, by triggering inhibition of a major proliferative pathway (AKT), inhibits cell growth and causes cells to die through autophagy. These data suggest that AKT-mTOR pathway is a novel component of oncogene induced DNA damage response in immortalized 'normal-like' breast cells and its suppression may contribute to growth arrest and arrest of the breast tumorigenesis.

DNA demethylation-dependent AR recruitment and GATA factors drive Rhox5 homeobox gene transcription in the epididymis.

Mammalian male fertility depends on the epididymis, a highly segmented organ that promotes sperm maturation and protects sperm from oxidative damage. Remarkably little is known about how gene expression is controlled in the epididymis. A candidate to regulate genes crucial for epididymal function is reproductive homeobox gene on X chromosome (RHOX)5, a homeobox transcription factor essential for optimal sperm motility that is expressed in the caput region of the epididymis. Here, we report the identification of factors that control Rhox5 gene expression in epididymal cells in a developmentally regulated and region-specific fashion. First, we identify GATA transcription factor-binding sites in the Rhox5 proximal promoter (Pp) necessary for Rhox5 expression in epididymal cells in vitro and in vivo. Adjacent to the GATA sites are androgen-response elements, which bind to the nuclear hormone receptor androgen receptor (AR), and are responsible for the AR-dependent expression of Rhox5 in epididymal cells. We provide evidence that AR is recruited to the Pp in a region-specific and developmentally regulated manner in the epididymis that is dictated not only by differential AR availability but differential methylation of the Pp. Site-specific methylation of the Pp cytosine and guanine separated by one phosphate, most of which overlap with androgen-response elements, inhibited both AR occupancy at the Pp and Pp-dependent transcription in caput epididymal cells. Together, our data support a model in which DNA methylation, AR, and GATA factors collaborate to dictate the unique developmental and region-specific expression pattern of the RHOX5 homeobox transcription factor in the caput epididymis, which in turn controls the expression of genes critical for promoting sperm motility and function.

Identification of a microRNA that activates gene expression by repressing nonsense-mediated RNA decay.

Nonsense-mediated decay (NMD) degrades both normal and aberrant transcripts harboring stop codons in particular contexts. Mutations that perturb NMD cause neurological disorders in humans, suggesting that NMD has roles in the brain. Here, we identify a brain-specific microRNA-miR-128-that represses NMD and thereby controls batteries of transcripts in neural cells. miR-128 represses NMD by targeting the RNA helicase UPF1 and the exon-junction complex core component MLN51. The ability of miR-128 to regulate NMD is a conserved response occurring in frogs, chickens, and mammals. miR-128 levels are dramatically increased in differentiating neuronal cells and during brain development, leading to repressed NMD and upregulation of mRNAs normally targeted for decay by NMD; overrepresented are those encoding proteins controlling neuron development and function. Together, these results suggest the existence of a conserved RNA circuit linking the microRNA and NMD pathways that induces cell type-specific transcripts during development.

The rhox homeobox gene cluster is imprinted and selectively targeted for regulation by histone h1 and DNA methylation.

Histone H1 is an abundant and essential component of chromatin whose precise role in regulating gene expression is poorly understood. Here, we report that a major target of H1-mediated regulation in embryonic stem (ES) cells is the X-linked Rhox homeobox gene cluster. To address the underlying mechanism, we examined the founding member of the Rhox gene cluster-Rhox5-and found that its distal promoter (Pd) loses H1, undergoes demethylation, and is transcriptionally activated in response to loss of H1 genes in ES cells. Demethylation of the Pd is required for its transcriptional induction and we identified a single cytosine in the Pd that, when methylated, is sufficient to inhibit Pd transcription. Methylation of this single cytosine prevents the Pd from binding GA-binding protein (GABP), a transcription factor essential for Pd transcription. Thus, H1 silences Rhox5 transcription by promoting methylation of one of its promoters, a mechanism likely to extend to other H1-regulated Rhox genes, based on analysis of ES cells lacking DNA methyltransferases. The Rhox cluster genes targeted for H1-mediated transcriptional repression are also subject to another DNA methylation-regulated process: Xp imprinting. Remarkably, we found that only H1-regulated Rhox genes are imprinted, not those immune to H1-mediated repression. Together, our results indicate that the Rhox gene cluster is a major target of H1-mediated transcriptional repression in ES cells and that H1 is a candidate to have a role in Xp imprinting.

GATA factors and androgen receptor collaborate to transcriptionally activate the Rhox5 homeobox gene in Sertoli cells.

How Sertoli-specific expression is initiated is poorly understood. Here, we address this issue using the proximal promoter (Pp) from the Rhox5 homeobox gene. Its Sertoli cell-specific expression is achieved, in part, through a negative regulatory element that inhibits Pp transcription in non-Sertoli cell lines. Complementing this negative regulation is positive regulation conferred by four androgen-response elements (AREs) that interact with the androgen receptor (AR), a nuclear hormone receptor expressed at high levels in Sertoli cells. A third control mechanism is provided by a consensus GATA-binding site that is crucial for Pp transcription both in vitro and in vivo. Several lines of evidence suggested that GATA factors and AR act cooperatively to activate Pp transcription: (i) the GATA-binding site crucial for Pp transcription is in close proximity to two of the AREs, (ii) GATA and AR form a complex with the Pp in vitro, (iii) overexpression of GATA factors rescued expression from mutant Pp constructs harboring defective AREs, and (iv) incubation of a Sertoli cell line with testosterone triggered corecruitment of AR and GATA4 to the Pp. Collectively, our results suggest that the Rhox5 gene achieves Sertoli cell-specific transcription using a combinatorial strategy involving negative and cooperative positive regulation.

Resveratrol inhibits proliferation, induces apoptosis, and overcomes chemoresistance through down-regulation of STAT3 and nuclear factor-kappaB-regulated antiapoptotic and cell survival gene products in human multiple myeloma cells.

Whether resveratrol, a component of red grapes, berries, and peanuts, could suppress the proliferation of multiple myeloma (MM) cells by interfering with NF-kappaB and STAT3 pathways, was investigated. Resveratrol inhibited the proliferation of human multiple myeloma cell lines regardless of whether they were sensitive or resistant to the conventional chemotherapy agents. This stilbene also potentiated the apoptotic effects of bortezomib and thalidomide. Resveratrol induced apoptosis as indicated by accumulation of sub-G(1) population, increase in Bax release, and activation of caspase-3. This correlated with down-regulation of various proliferative and antiapoptotic gene products, including cyclin D1, cIAP-2, XIAP, survivin, Bcl-2, Bcl-xL, Bfl-1/A1, and TRAF2. In addition, resveratrol down-regulated the constitutive activation of AKT. These effects of resveratrol are mediated through suppression of constitutively active NF-kappaB through inhibition of IkappaBalpha kinase and the phosphorylation of IkappaBalpha and of p65. Resveratrol inhibited both the constitutive and the interleukin 6-induced activation of STAT3. When we examined CD138(+) plasma cells from patients with MM, resveratrol inhibited constitutive activation of both NF-kappaB and STAT3, leading to down-regulation of cell proliferation and potentiation of apoptosis induced by bortezomib and thalidomide. These mechanistic findings suggest that resveratrol may have a potential in the treatment of multiple myeloma.

Regulation and function of the Rhox5 homeobox gene.

Recently, a large cluster of homeobox genes was discovered on the X chromosome that is expressed in reproductive tissues after birth. It is postulated that these reproductive homeobox genes on the X chromosome (Rhox) encode transcription factors that regulate gametogenesis. In support of this, male mice lacking the founding member of this gene cluster, Rhox5, are subfertile, exhibiting increased germ-cell apoptosis and a defect in sperm motility. To identify RHOX5 targets, microarray analyses were used to identify genes differently expressed in postnatal testes from Rhox5-null and control littermates. Highly overrepresented were genes that encode proteins involved in cellular metabolism. Several lines of evidence indicated that one of these, insulin II, is a direct target of RHOX5. Microarray analysis was also used to identify genes differentially expressed in response to physiological levels of Rhox5 in a Sertoli-cell line. Among the few genes identified, the netrin-1 receptor UNC5c, a proapoptotic molecule that is inhibited by RHOX5, was also regulated in vivo, and is thus a candidate to be downstream of RHOX5 in a prosurvival germ-cell pathway. To understand the means by which Rhox5 expression is restricted to Sertoli nurse cells in the testis, a variety of molecular approaches were used in both Sertoli-cell lines and mice. This analysis revealed that both positive and negative cis elements collaborate to confer Sertoli cell-specific gene expression. Acting on the positive cis elements are androgen receptor and GATA transcription factors. Collectively, the results of this study provide an initial glimpse into the regulatory networks that control spermatogenesis.

A metabolic enzyme doing double duty as a transcription factor.

Many kinds of multifunctional regulatory proteins have been identified that perform distinct biochemical functions in the nucleus, the cytoplasm, or both. Here we describe the recent discovery by Hall et al. (2004) of a new type of multifunctional protein: a metabolic enzyme that doubles as a transcription factor. This enzyme, Arg5,6, functions as a catalytic enzyme in ornithine biosynthesis and also binds and regulates the promoters of nuclear and mitochondrial genes. It may also regulate precursor mRNA metabolism. We discuss how proteins that serve as both metabolic enzymes and transcription factors might have evolved.

Induction of IFN-regulated factors and antitumoral surveillance by transfected placebo plasmid DNA.

Delivery of DNA encoding therapeutic genes in vivo has great potential for treating malignancy as well as genetic diseases. Delivery of placebo DNA without a transgene is used as a control in gene therapy studies. It is tacitly assumed by most investigators that the protein expressed from the transfected DNA has phenotypic consequences, but that the consequences are not from the DNA itself. Here, we demonstrate that transfection of control plasmid DNA (that does not express a gene product) into tumor cell lines induces a dramatic (>10-fold) increase in the expression of the interferon (IFN)-regulated genes IRF7, STAT1, MIG (approved gene symbol CXCL9), MHCI (MICA), and CD11a (ITGAL) in tumor cell lines. Induction of these genes inhibits tumor development and tumor growth in immunocompetent mice that are immunized with apoptotic tumor cells. The antibody depletion study indicates that the underlying mechanism by which transfection of control DNA induces IFN-regulated genes is the induction of a secreting factor(s) such as IFN-beta. Three lines of evidence indicate that DNA transfection-mediated induction of IFN-regulatory genes is independent of TLR9. The three lines of evidence are: (1) TLR9 is not expressed in either SCCVII or 4T1 cell line, (2) activation of TLR9 downstream signaling molecules is not associated with the induction of gene expression, and (3) the secretion factor(s) obtained from the conditioned medium of DNA-transfected SCCVII tumor cells induces the same type of gene expression in the 4T1 tumor cell line, which is refractory to the gene induction by DNA transfection. Our finding indicates that the 4T1 tumor cell line, which is resistant to the DNA transfection-mediated induction of IFN-regulated genes, can be used to determine the real therapeutic gene function.