Transformation Of Mammary Cells Research Articles

Caspase-3 promotes oncogene-induced malignant transformation via EndoG-dependent Src-STAT3 phosphorylation

Accumulating evidence suggests that caspase-3 plays critical roles beyond apoptosis, serving pro-survival functions in malignant transformation and tumorigenesis. However, the mechanism of non-apoptotic action of caspase-3 in oncogenic transformation remains unclear. In the present study, we show that caspase-3 is consistently activated in malignant transformation induced by exogenous expression of oncogenic cocktail (c-Myc, p53DD, Oct-4, and H-Ras) in vitro as well as in the mouse mammary tumor virus-polyomavirus middle T antigen (MMTV-PyMT) mouse model of breast cancer. Genetic ablation of caspase-3 significantly attenuated oncogene-induced transformation of mammalian cells and delayed breast cancer progression in MMTV-PyMT transgenic mice. Mechanistically, active caspase-3 triggers the translocation of endonuclease G (EndoG) from mitochondria, which migrates to the nucleus, thereby induces phosphorylation of Src-STAT3 signaling pathway to facilitate oncogenic transformation. Taken together, our data suggest that caspase-3 plays pivotal role in facilitating rather than suppressing oncogene-induced malignant transformation of mammalian cells.

The human adenovirus PI3K-Akt activator E4orf1 is targeted by the tumor suppressor p53.

Human adenoviruses (HAdV) are classified as DNA tumor viruses due to their potential to mediate oncogenic transformation in non-permissive mammalian cells and certain human stem cells. To achieve transformation, the viral early proteins of the E1 and E4 regions must block apoptosis and activate proliferation: the former predominantly through modulating the cellular tumor suppressor p53 and the latter by activating cellular pro-survival and pro-metabolism protein cascades, such as the phosphoinositide 3-kinase (PI3K-Akt) pathway, which is activated by HAdV E4orf1. Focusing on HAdV-C5, we show that E4orf1 is necessary and sufficient to stimulate Akt activation through phosphorylation in H1299 cells, which is not only hindered but repressed during HAdV-C5 infection with a loss of E4orf1 function in p53-positive A549 cells. Contrary to other research, E4orf1 localized not only in the common, cytoplasmic PI3K-Akt-containing compartment, but also in distinct nuclear aggregates. We identified a novel inhibitory mechanism, where p53 selectively targeted E4orf1 to destabilize it, also stalling E4orf1-dependent Akt phosphorylation. Co-IP and immunofluorescence studies showed that p53 and E4orf1 interact, and since p53 is bound by the HAdV-C5 E3 ubiquitin ligase complex, we also identified E4orf1 as a novel factor interacting with E1B-55K and E4orf6 during infection; overexpression of E4orf1 led to less-efficient E3 ubiquitin ligase-mediated proteasomal degradation of p53. We hypothesize that p53 specifically subverts the pro-survival function of E4orf1-mediated PI3K-Akt activation to protect the cell from metabolic hyper-activation or even transformation.IMPORTANCEHuman adenoviruses (HAdV) are nearly ubiquitous pathogens comprising numerous subtypes that infect various tissues and organs. Among many encoded proteins that facilitate viral replication and subversion of host cellular processes, the viral E4orf1 protein has emerged as an intriguing yet under-investigated player in the complex interplay between the virus and its host. Nonetheless, E4orf1 has gained attention as a metabolism activator and oncogenic agent, while recent research is showing that E4orf1 may play a more important role in modulating the cellular pathways such as phosphoinositide 3-kinase-Akt-mTOR. Our study reveals a novel and general impact of E4orf1 on host mechanisms, providing a novel basis for innovative antiviral strategies in future therapeutic settings. Ongoing investigations of the cellular pathways modulated by HAdV are of great interest, particularly since adenovirus-based vectors actually serve as vaccine or gene vectors. HAdV constitute an ideal model system to analyze the underlying molecular principles of virus-induced tumorigenesis.

SAT635 Role Of The Human Prolactin Receptor I-tail In The Pathogenesis Of Breast Cancer

Abstract Disclosure: C.V. Clevenger: None. S. Shen: None. Evidence from epidemiological, cellular, and genetic analyses indicate that the hormone prolactin (PRL) and its receptor (hPRLr) in humans are significantly involved in breast cancer pathogenesis. The intermediate form of hPRLr (hPRLrI) induces significant increases in proliferation and anchorage-independent growth of normal mammary epithelia in vitro when co-expressed with the long form hPRLr (hPRLrL). However, the exact mechanism of how the hPRLrI transforms mammary epithelium is not well understood. The hPRLrI is produced by alternative splicing that induces a frameshift, resulting in a premature stop codon and a novel 13 amino acid tail ("I-Tail") gain. The ubiquitin-like protein neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8) was found to be associated with the I-tail by yeast two-hybrid analysis. Treatment with a selective NEDD8 inhibitor resulted in the altered stability of the hPRLrL and the death of breast cancer cells. Given this, we hypothesized that the hPRLrI I-tail contributes to mammary transformation. hPRLrL, hPRLrI, hPRLrIΔ13 (I-tail removed mutant due to loss of 13 C-terminal amino acid resides) were delivered to MCF10AT cells by lentivirus transduction and subsequently analyzed. Anchorage-independent cell growth was determined by soft agar assay, cell proliferation was determined with a Incucyte analysis, and cell motility was examined by wound healing assay. In addition, signaling pathway and differential gene expression alterations mediated by the I-tail were determined by RNA-seq and signaling analysis. Successful expression of the hPRLrL+hPRLrIΔ13 in the MCF10AT transfectant resulted in significant reductions or alterations of anchorage-independent growth, cell proliferation, ERK1/2 activation, and PRL-induced different gene expression. These findings demonstrate that the hPRLrI I-tail contributes to the transformation of normal mammary epithelia cells. Presentation: Saturday, June 17, 2023

Global Down-regulation of Gene Expression Induced by Mouse Mammary Tumor Virus (MMTV) in Normal Mammary Epithelial Cells.

Mouse mammary tumor virus (MMTV) is a betaretrovirus that causes breast cancer in mice. The mouse mammary epithelial cells are the most permissive cells for MMTV, expressing the highest levels of virus upon infection and being the ones later transformed by the virus due to repeated rounds of infection/superinfection and integration, leading eventually to mammary tumors. The aim of this study was to identify genes and molecular pathways dysregulated by MMTV expression in mammary epithelial cells. Towards this end, mRNAseq was performed on normal mouse mammary epithelial cells stably expressing MMTV, and expression of host genes was analyzed compared with cells in its absence. The identified differentially expressed genes (DEGs) were grouped on the basis of gene ontology and relevant molecular pathways. Bioinformatics analysis identified 12 hub genes, of which 4 were up-regulated (Angp2, Ccl2, Icam, and Myc) and 8 were down-regulated (Acta2, Cd34, Col1a1, Col1a2, Cxcl12, Eln, Igf1, and Itgam) upon MMTV expression. Further screening of these DEGs showed their involvement in many diseases, especially in breast cancer progression when compared with available data. Gene Set Enrichment Analysis (GSEA) identified 31 molecular pathways dysregulated upon MMTV expression, amongst which the PI3-AKT-mTOR was observed to be the central pathway down-regulated by MMTV. Many of the DEGs and 6 of the 12 hub genes identified in this study showed expression profile similar to that observed in the PyMT mouse model of breast cancer, especially during tumor progression. Interestingly, a global down-regulation of gene expression was observed, where nearly 74% of the DEGs in HC11 cells were repressed by MMTV expression, an observation similar to what was observed in the PyMT mouse model during tumor progression, from hyperplasia to adenoma to early and late carcinomas. Comparison of our results with the Wnt1 mouse model revealed further insights into how MMTV expression could lead to activation of the Wnt1 pathway independent of insertional mutagenesis. Thus, the key pathways, DEGs, and hub genes identified in this study can provide important clues to elucidate the molecular mechanisms involved in MMTV replication, escape from cellular anti-viral response, and potential to cause cell transformation. These data also validate the use of the MMTV-infected HC11 cells as an important model to study early transcriptional changes that could lead to mammary cell transformation.

Abstract SY04-01: Understanding and targeting RNA splicing in breast cancer

Abstract Upregulation of SR proteins, a family of 14 essential splicing factors (SFs), often occurs in solid tumors, including in breast, and promotes mammary cell transformation and metastasis in breast cancer models. SR protein alterations often occur without genomic copy number changes, suggesting misregulation at the transcriptional or post-transcriptional levels; however the underlying molecular mechanisms are incompletely understood. Here we investigate the transcriptional and post-transcriptional regulatory mechanisms that control SF-levels in normal mammary cells and their misregulation in cancer cells. We demonstrate the MYC oncogene directly regulates a network of splicing factors that are co-expressed in tumors and act together to regulate their downstream targets and promote cell invasion. In parallel, our work reveals the role of non-coding ‘poison exon’ in regulating SR proteins levels during cell differentiation and in tumors. We uncover an extensive cross-regulatory network used by the SR protein family to control their expression. Finally, we develop splice-switching antisense oligonucleotides to reverse the increased skipping of TRA2β poison exon detected in tumors, leading to selective anti-proliferative effects across a variety of cancer models. In summary, our work provides new insights into the regulatory mechanisms of SR proteins and identifies novel oncogenic spliced isoforms that represent potential biomarkers and targets for therapeutics development. Citation Format: Olga A. Anczukow. Understanding and targeting RNA splicing in breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr SY04-01.

Leucyl-tRNA synthetase is a tumour suppressor in breast cancer and regulates codon-dependent translation dynamics.

Tumourigenesis and cancer progression require enhanced global protein translation1–3. Such enhanced translation is caused by oncogenic and tumour suppressive events that drive the synthesis and activity of translational machinery4,5. Here we report the surprising observation that leucyl-tRNA synthetase (LARS) becomes repressed during mammary cell transformation and in human breast cancer. Monoallelic genetic deletion of LARS in mouse mammary glands enhanced breast cancer tumour formation and proliferation. LARS repression reduced the abundance of select leucine tRNA isoacceptors, leading to impaired leucine codon-dependent translation of growth suppressive genes including epithelial membrane protein 3 (EMP3) and gamma-glutamyltransferase 5 (GGT5). Our findings uncover a tumour suppressive tRNA synthetase and reveal that dynamic repression of a specific tRNA synthetase—along with its downstream cognate tRNAs—elicits a downstream codon-biased translational gene network response that enhances breast tumour formation and growth.

De novo and cell line models of human mammary cell transformation reveal an essential role for Yb-1 in multiple stages of human breast cancer

Breast cancer heterogeneity has made it challenging to identify mechanisms critical to the initial stages of their genesis in vivo. Here, we sought to interrogate the role of YB-1 in newly arising human breast cancers as well as in established cell lines. In a first series of experiments, we found that short-hairpin RNA-mediated knockdown of YB-1 in MDA-MB-231 cells blocked both their local tumour-forming and lung-colonising activity in immunodeficient mice. Conversely, upregulated expression of YB-1 enhanced the poor in vivo tumorigenicity of T47D cells. We then found that YB-1 knockdown also inhibits the initial generation in mice of invasive ductal carcinomas and ductal carcinomas in situ from freshly isolated human mammary cells transduced, respectively, with KRASG12D or myristoylated-AKT1. Interestingly, increased expression of HIF1α and G3BP1, two YB-1 translational targets and elements of a stress-adaptive programme, mirrored the levels of YB-1 in both transformed primary and established MDA-MB-231 breast cancer cells.

Polymer-Assisted Metallization of Mammalian Cells.

Developing biotemplating techniques to translate microorganisms and cultured mammalian cells into metallic biocomposites is of great interest for biosensors, electronics, and energy. The metallization of viruses and microbial cells is successfully demonstrated via a genetic engineering strategy or electroless deposition. However, it is difficult to transform mammalian cells into metallic biocomposites because of the complicated genes and the delicate morphological features. Herein, "polymer-assisted cell metallization" (PACM) is reported as a general method for the transformation of mammalian cells into metallic biocomposites. PACM includes a first step of in situ polymerization of functional polymer on the surface and in the interior of the mammalian cells, and a subsequent electroless deposition of metal to convert the polymer-functionalized cells into metallic biocomposites, which retain the micro- and nanostructures of the mammalian cells. This new biotemplating method is compatible with different cell types and metals to yield a wide variety of metallic biocomposites with controlled structures and properties.

Abstract LB226: Maternal microbiome protects host against clonal de novo transformation, early onset systemic metastasis, and sudden death

Abstract The role of maternal microbiome transmitted at birth in cancer control is poorly understood. We have developed the first germfree B6 mouse model of breast cancer to investigate the role of the maternal microbiome in controlling oncogenic/metastatic frequencies of pro-oncogenic mammary cells. In this model, a DNA barcoded, primitive normal mouse mammary epithelial cell encoding MMTV-PyMT oncogene, was transplanted in large numbers into conventional or germfree B6 mice. Next-gen sequencing analysis of the DNA barcodes in tissues enabled us to clonally track millions of cells and measure the frequency and growth dynamics of clones at the primary site and their systemic distribution in circulation and all vital organs, generating an unprecedented high-definition map of cancer progression. Our results show that in conventional B6 mice with maternal microbial transmission at-birth, a small fraction (~0.01%) of transplanted cells transform de novo and produce slow growing, late-onset benign tumors (mouse median survival of > 1 year). In contrast, in germfree B6 mice, a >10-fold higher frequency of cells transform de novo and generate early-onset, highly aggressive metastatic clones, and are frequently associated with features leading to early euthanasia or endpoint (i.e. sudden death, intracardiac metastasis, paralysis, swollen abdomen, early multiorgan aggressive metastasis) (median survival of ~4 months; p<0.005 vs conventional mice). However, postnatal intervention with healthy fecal implants reconstituted gut microbiota in germfree B6 mice and significantly restored protective effects against cancer progression and sudden death, as well as improved quality/duration of life (median survival of >1 year; p<0.005 vs germfree mice; p=ns vs conventional mice). We found maternal microbial transmission fully protected against systemic metastasis. GC-MS analysis identified significantly distinct primary tumor metabolome associated with conventional (non-metastatic) mice vs germfree (metastatic) mice. Metagenomic analysis of fecal samples from a subset of tumor bearing conventional (non-metastatic) mice vs fecal implanted (late-onset, less aggressive metastatic) germfree mice identified significant association between metastasis and altered microbial communities. These findings using novel germfree mouse model of metastasis and cellular DNA barcoding technology provide first direct evidence that diverse microbial communities transmitted at birth play a critical role in protecting the host from clonal de novo mammary cell transformation and progression to systemic disease and thereby improving quality and duration of life. Citation Format: Nagarajan Kannan, Syed Mohammed Musheer Aalam, Xiaojia Tang, Krishna Kalari, Purna Kashyap, Jun Chen, Stephen Johnson, Anguraj Sadanandam, Mark Sherman. Maternal microbiome protects host against clonal de novo transformation, early onset systemic metastasis, and sudden death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB226.

Abstract PD7-03: A living biobank of normal mammary organoids derived from patients at low and increased risk of developing breast cancer

Abstract Background: Mutations in BRCA1 and BRCA2 are associated with a high risk of developing breast cancer, but the earliest molecular changes that lead to the transformation of mammary cells in the setting of BRCA1/2 heterozygosity remain unknown. We previously demonstrated that breast organoid cultures derived from histologically normal tissues can preserve all of the major mammary epithelial lineages for further study in vitro1. In addition, organoid culture medium is fully defined, enabling modulation of the medium to enhance the growth of distinct mammary subpopulations. Here, we undertook the development of a large biobank of breast organoids derived from patients with and without increased breast cancer risk, including patients with inherited mutations in BRCA1 and BRCA2. Methods: Breast samples with normal histology (taken from reduction mammoplasties or prophylactic mastectomies), were digested for ~2 hours using collagenase, embedded in basement membrane extract, and grown in a fully-defined organoid medium1. CyTOF profiling was performed using our previously published mammary-specific heavy metal-tagged antibody panel1. Lentiviral transduction was performed to knock-down BRCA1 and/or overexpress mutant p53. Murine engraftment of normal breast organoids was performed by intra-ductal injection of ~1,000 organoids into NCG mice using a modification of protocols for breast cancer cell lines2, with removal of mammary glands after 3 months for detailed immunohistochemistry analysis. Results: A living biobank of > 100 normal breast organoids derived from patients with and without inherited mutations in breast cancer predisposition genes, including BRCA1 and BRCA2, was established. Cultures were generated with high efficiency (>95%) and could be serially passaged with the longest cultures > 16 months. CyTOF profiling of organoids revealed the maintenance of multiple epithelial cell subtypes, with at least one subtype of luminal cells enriched in tissues and organoids from BRCA1/2 mutation carriers. Protein and RNA expression patterns of breast organoids were found to correlate with patient tissues analyzed using a combination of single-cell analyses (CyTOF, single-cell RNA sequencing, and immunohistochemistry). The impact of factors present in the organoid medium on distinct mammary epithelial cell subtypes was assessed by CyTOF, enabling identification of conditions that promote expansion of cell populations that are enriched in tissues from BRCA1/2 mutation carriers. Furthermore, normal breast cell types could be engrafted into the murine mammary gland, and could be modified by lentiviral transduction for gene transfer, enabling future studies of the tumorigenic potential of distinct normal and premalignant epithelial cell subtypes. Conclusion: We have shown that organoid cultures can be used to propagate normal breast tissues with high efficiency, preserve normal as well as potential premalignant breast epithelial cell types, and model methods to inhibit precancerous cells in vitro. Thus, organoids are a useful complement to murine and other models of breast cancer development, and can ultimately be used to identify potential cancer interception strategies for patients at high risk of developing breast cancer.

Key Epigenetic Events Involved in the Maintenance of Breast Cancer Stem Cells.

Cancer stem cells (CSCs) are a small subpopulation of cells within tumors and play significant roles in tumorigenesis, metastasis, resistance to treatment, and relapse. They are defined by self-renewal and multi-lineage differentiation, and aggressiveness. Epigenetic modifications, including DNA methylation and acetylation, histone modifications, and non-coding. RNAs (ncRNAs), are partly responsible for CSC potentials and are involved in the modification of key components of crucial pathways such as Notch and Wnt signaling in breast cancer. In this review, we present an overview of the pathways and epigenetic events that lead to the transformation of mammary gland stem cells to breast CSCs (BCSCs). Based on the data presented here, important pathways such as TGF-β/SMAD2 and Wnt/β-catenin and epigenetic modifications, including histone modifications, DNA methylations, and microRNAs, play important roles in BCSC formation and maintenance. Epigenetic events can alter the expression of genes and functional RNAs, resulting in tumor initiation and progression. Thus, a better understanding of epigenetic modifications involved in BCSC maintenance signaling pathways may help to eliminate or suppress BCSCs and overcome cancer by generating more effective and efficient therapeutic agents.

ATM Paradoxically Promotes Oncogenic Transformation via Transcriptional Reprogramming

The role of the ataxia-telangiectasia-mutated (ATM) gene in human malignancies, especially in solid tumors, remains poorly understood. In the present study, we explored the involvement of ATM in transforming primary human cells into cancer stem cells. We show that ATM plays an unexpected role in facilitating oncogene-induced malignant transformation through transcriptional reprogramming. Exogenous expression of an oncogene cocktail induced a significant amount of DNA double-strand breaks in human fibroblasts that caused persistent activation of ATM, which in turn enabled global transcriptional reprogramming through chromatin relaxation, allowing oncogenic transcription factors to access chromatin. Consistently, deficiencies in ATM significantly attenuated oncogene-induced transformation of human cells. In addition, ATM inhibition significantly reduced tumorigenesis in a mouse model of mammary cancer. ATM and cellular DNA damage response therefore play a previously unknown role in facilitating rather than suppressing oncogene-induced malignant transformation of mammalian cells. SIGNIFICANCE: These findings uncover a novel pro-oncogenic role for ATM and show that contrary to established theory, ATM does not always function as a tumor suppressor; its function is however dependent on cell type.

The DPF Domain As a Unique Structural Unit Participating in Transcriptional Activation, Cell Differentiation, and Malignant Transformation.

The DPF (double PHD finger) domain consists of two PHD fingers organized in tandem. The two PHD-finger domains within a DPF form a single structure that interacts with the modification of the N-terminal histone fragment in a way different from that for single PHD fingers. Several histone modifications interacting with the DPF domain have already been identified. They include acetylation of H3K14 and H3K9, as well as crotonylation of H3K14. These modifications are found predominantly in transcriptionally active chromatin. Proteins containing DPF belong to two classes of protein complexes, which are the transcriptional coactivators involved in the regulation of the chromatin structure. These are the histone acetyltransferase complex belonging to the MYST family and the SWI/SNF chromatin-remodeling complex. The DPF domain is responsible for the specificity of the interactions between these complexes and chromatin. Proteins containing DPF play a crucial role in the activation of the transcription of a number of genes expressed during the development of an organism. These genes are important in the differentiation and malignant transformation of mammalian cells.

MEAN VALUE OF LET FOR ONCOGENIC EFFECTS OF RADON AND ITS PROGENY

The topic of the article is to define the average value of linear energy transfer (LET) for carcinogenic effects of radon progeny. The microdosimetric model of boundary specific energy is used. It follows that the effect at high LET should decrease approximately with the third power of LET. This is verified by the analysis of the relationship between radiation effects ratio and LET in published experiments with oncogenic transformation of mammalian cells irradiated with the monoenergetic alpha particles. If these cells are exposed with the radon irradiator, our analysis leads us to conclude that the oncogenic effect of radon progeny is comparable to that of alpha particles with a LET of 75keV/μm. It is about a quarter lower than the LET value, where the effect of the monoenergetic alpha particles reaches its maximum level. Some implications for lung cancer due to radon inhalation may also be carefully examined.

Initiation of human mammary cell tumorigenesis by mutant KRAS requires YAP inactivation

High YAP activity is associated with poor prognosis human breast cancers, but its role during the initial stage of mammary cell transformation is unknown. To address this question, we designed experiments that exploit the ability of KRASG12D-transduced subsets of freshly isolated normal human mammary cells to form invasive tumors rapidly and efficiently when transplanted into immunodeficient mice. Initial examination of the newly developing tumors thus generated revealed a consistent marked loss of nuclear YAP, independent of the initial primary human mammary cell type transduced. Conversely, co-transduction of the same subsets of primary human mammary cells with KRASG12D plus the constitutively active YAPS127A prevented tumor formation. These findings contrast with the enhanced display of transformed properties obtained when the immortalized, but non-tumorigenic MCF10A cells are transduced just with YAPS127A. In addition, we show that YAPS127A-transduction of the human MDA-MB-231 breast cancer cell line (that carry a similar KRAS mutation) enhances their metastatic activity in vivo. We also discover that the KRASG12D-induced early loss of YAP in primary human mammary cells is associated with their induced secretion of amphiregulin. Collectively, these findings suggest that YAP can differentially affect the acquisition of malignant properties by human mammary cells at different stages of their transformation.

Early Steps of Mammary Stem Cell Transformation by Exogenous Signals; Effects of Bisphenol Endocrine Disrupting Chemicals and Bone Morphogenetic Proteins.

Estrogens are major regulators of the mammary gland development, notably during puberty, via estrogen receptor (ER) activation, leading to the proliferation and differentiation of mammary cells. In addition to estrogens, the bone morphogenetic proteins (BMPs) family is involved in breast stem cell/progenitor commitment. However, these two pathways that synergistically contribute to the biology of the normal mammary gland have also been described to initiate and/or promote breast cancer development. In addition to intrinsic events, lifestyle habits and exposure to environmental cues are key risk factors for cancer in general, and especially for breast cancer. In the latter case, bisphenol A (BPA), an estrogen-mimetic compound, is a critical pollutant both in terms of the quantities released in our environment and of its known and speculated effects on mammary gland biology. In this review, we summarize the current knowledge on the actions of BMPs and estrogens in both normal mammary gland development and breast cancer initiation, dissemination, and resistance to treatment, focusing on the dysregulations of these processes by BPA but also by other bisphenols, including BPS and BPF, initially considered as safer alternatives to BPA.

E1B-55K-Mediated Regulation of RNF4 SUMO-Targeted Ubiquitin Ligase Promotes Human Adenovirus Gene Expression.

Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB-associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4- and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention.

PI3K Positively Regulates YAP and TAZ in Mammary Tumorigenesis Through Multiple Signaling Pathways.

Breast cancer is a leading cause of death in women worldwide. Active mutations of PI3K catalytic subunit PIK3CA (e.g., H1047R) and amplification of its homolog PIK3CB are observed in a large number of breast cancers. In recent years, aberrant activation of Transcriptional coactivator with PDZ binding motif (TAZ) and its paralog Yes-associated protein (YAP) have also been found to be important for breast cancer development and progression. However, whether PI3K interacts with YAP/TAZ during mammary tumorigenesis is unknown. Through a systematic gain-of-function screen for kinases involved in mammary tumorigenesis, we identified PIK3CB as a transformation-inducing kinase in breast cells. We further determined that PIK3CB positively regulates YAP and TAZ to promote transformation and inhibit mammary cell death in vitro PIK3CB coexpression with TAZ, rather than PIK3CB or TAZ alone, in human MCF10A nontumorigenic mammary cells is sufficient for tumor formation in mice in vivo Interestingly, we also determined that PIK3CA-H1047R enhances YAP and TAZ activity in mammary tumorigenesis in vitro Mechanistically, the regulation of YAP/TAZ by both PIK3CA and PIK3CB occurs through multiple signaling pathways including LATS-dependent and LATS-independent pathways. Therefore, in this study, we determine that PI3K and YAP/TAZ interact to promote breast cancer cell transformation.Implications: This study provides the first evidence that the Hippo pathway effectors TAZ and YAP are critical mediators of PI3K-induced mammary tumorigenesis and synergistically function together with PI3K in transformation of mammary cells. These findings may provide a novel rationale for targeting YAP/TAZ alone or in combination with PI3K inhibitors for breast cancer therapy in the future. Mol Cancer Res; 16(6); 1046-58. ©2018 AACR.

Essential roles of Caspase-3 in facilitating Myc-induced genetic instability and carcinogenesis.

The mechanism for Myc-induced genetic instability is not well understood. Here we show that sublethal activation of Caspase-3 plays an essential, facilitative role in Myc-induced genomic instability and oncogenic transformation. Overexpression of Myc resulted in increased numbers of chromosome aberrations and γH2AX foci in non-transformed MCF10A human mammary epithelial cells. However, such increases were almost completely eliminated in isogenic cells with CASP3 gene ablation. Furthermore, we show that endonuclease G, an apoptotic nuclease downstream of Caspase-3, is directly responsible for Myc-induced genetic instability. Genetic ablation of either CASP3 or ENDOG prevented Myc-induced oncogenic transformation of MCF10A cells. Taken together, we believe that Caspase-3 plays a critical, unexpected role in mediating Myc-induced genetic instability and transformation in mammalian cells.

Abstract 914: Age-associated neoplastic transformation of mammary stem cells

Abstract Incidence rate of breast cancer increases significantly with age. However, the mechanism of age-associated mammary tumorigenesis is unknown. Although mammary stem/progenitor cells have been proposed as the cells of origin for breast cancer, the mechanism remains largely elusive. Previously we discovered that in the old (28 to 30 month-old) mouse mammary gland, the luminal cells in the hyperplastic lesions elevated expression of CD49f, which is a basal cell marker. This led to an increased percentage of mammary stem cells (MaSCs) marked by CD49fhi in the old in comparison to the young (3 to 6 month-old). Similarly, old (>60 yr) human mammary ducts in the adjacent normal tissues from patients with breast cancer showed more hyperplastic lesions and CD49fhi luminal cells than young (<40 yr) tissues. Regenerated mammary glands by the old CD49fhi MaSCs through in vivo transplantation in cleared mammary fat pads showed more hyperplastic and dysplastic lesions than those regenerated by the young CD49fhi MaSCs, suggesting the old CD49fhi MaSCs contain transformed MaSCs. Moreover, gene set enrichment analysis of whole genome transcriptome data showed that they also have elevated expression of luminal cell specific genes and reduced expression of basal cell specific genes. Through large scale screening with flow cytometry guided by bioinformatics analysis, we found that Lin- CD24med CD49fhi CD13+ CD73- mammary epithelial cells had increased number of MaSCs. By comparing young (3 to 6 months old) and old Balb/C mice, we found that the frequency of this specific MaSCs was significantly increased by four folds in old mammary epithelial cells (young: 4%, old: 16%). In contrast, relative percentage of Lin- CD24med CD49fhi CD13- CD73+ cells significantly decreased (young: 46%, old: 16%) during aging while the CD13 and CD73 double positive or double negative subpopulations remained unchanged. CD13+ CD73- cells generated significantly many more mammospheres (around 40 spheres per 1,000 cells) in suspension culture than other three types of cells (around 2 spheres per 1,000 cells). However, there was no significant difference between the young and old in terms of sphere formation efficiency for any of the four subpopulations marked by CD13 and CD73. We are studying whether anyone of these four subpopulation from CD49fhi old cells will generate more hyperplastic and dysplastic lesions in regenerated glands than the other subpopulations as well as than the corresponding young subpopulation. Our study show that CD13 and CD73 may be the markers for the identification of tumor-initiating MaSCs. Citation Format: Xiang Gu, Hakim Bouamar, Kyle Pressley, LuZhe Sun. Age-associated neoplastic transformation of mammary stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 914. doi:10.1158/1538-7445.AM2017-914