Molecular mechanisms of TWIST1-regulated transcription in EMT and cancer metastasis.

TWIST1 induces epithelial-to-mesenchymal transition (EMT) to drive cancer metastasis. It is yet unclear what determines TWIST1 functions to activate or repress transcription. We found that the TWIST1 N-terminus antagonizes TWIST1-regulated gene expression, cancer growth and metastasis. TWIST1 interacts with both the NuRD complex and the NuA4/TIP60 complex (TIP60-Com) via its N-terminus. Non-acetylated TWIST1-K73/76 selectively interacts with and recruits NuRD to repress epithelial target gene transcription. Diacetylated TWIST1-acK73/76 binds BRD8, a component of TIP60-Com that also binds histone H4-acK5/8, to recruit TIP60-Com to activate mesenchymal target genes and MYC. Knockdown of BRD8 abolishes TWIST1 and TIP60-Com interaction and TIP60-Com recruitment to TWIST1-activated genes, resulting in decreasing TWIST1-activated target gene expression and cancer metastasis. Both TWIST1/NuRD and TWIST1/TIP60-Com complexes are required for TWIST1 to promote EMT, proliferation, and metastasis at full capacity. Therefore, the diacetylation status of TWIST1-K73/76 dictates whether TWIST1 interacts either with NuRD to repress epithelial genes, or with TIP60-Com to activate mesenchymal genes and MYC. Since BRD8 is essential for TWIST1-acK73/76 and TIP60-Com interaction, targeting BRD8 could be a means to inhibit TWIST1-activated gene expression.

High-throughput profiling of histone post-translational modifications and chromatin modifying proteins by reverse phase protein array.

Epigenetic variation plays a significant role in normal development and human diseases including cancer, in part through post-translational modifications (PTMs) of histones. Identification and profiling of changes in histone PTMs, and in proteins regulating PTMs, are crucial to understanding diseases, and for discovery of epigenetic therapeutic agents. In this study, we have adapted and validated an antibody-based reverse phase protein array (RPPA) platform for profiling 20 histone PTMs and expression of 40 proteins that modify histones and other epigenomic regulators. The specificity of the RPPA assay for histone PTMs was validated with synthetic peptides corresponding to histone PTMs and by detection of histone PTM changes in response to inhibitors of histone modifier proteins in cell cultures. The useful application of the RPPA platform was demonstrated with two models: induction of pluripotent stem cells and a mouse mammary tumor progression model. Described here is a robust platform that includes a rapid microscale method for histone isolation and partially automated workflows for analysis of histone PTMs and histone modifiers that can be performed in a high-throughput manner with hundreds of samples. This RPPA platform has potential for translational applications through the discovery and validation of epigenetic states as therapeutic targets and biomarkers. SIGNIFICANCE: Our study has established an antibody-based reverse phase protein array platform for global profiling of a wide range of post-translational modifications of histones and histone modifier proteins. The high-throughput platform provides comprehensive analyses of epigenetics for biological research and disease studies and may serve as screening assay for diagnostic purpose or therapy development.

Conformational Changes of RORγ During Response Element Recognition and Coregulator Engagement.

The retinoic acid receptor-related orphan receptor γ (RORγ) is a ligand-dependent transcription factor of the nuclear receptor super family that underpins metabolic activity, immune function, and cancer progression. Despite being a valuable drug target in health and disease, our understanding of the ligand-dependent activities of RORγ is far from complete. Like most nuclear receptors, RORγ must recruit coregulatory protein to enact the RORγ target gene program. To date, a majority of structural studies have been focused exclusively on the RORγ ligand-binding domain and the ligand-dependent recruitment of small peptide segments of coregulators. Herein, we examine the ligand-dependent assembly of full length RORγ:coregulator complexes on cognate DNA response elements using structural proteomics and small angle x-ray scattering. The results from our studies suggest that RORγ becomes elongated upon DNA recognition, preventing long range interdomain crosstalk. We also determined that the DNA binding domain adopts a sequence-specific conformation, and that coregulatory protein may be able to 'sense' the ligand- and DNA-bound status of RORγ. We propose a model where ligand-dependent coregulator recruitment may be influenced by the sequence of the DNA to which RORγ is bound. Overall, the efforts described herein will illuminate important aspects of full length RORγ and monomeric orphan nuclear receptor target gene regulation through DNA-dependent conformational changes.

The Prognostic Impact of Intratumoral Aryl Hydrocarbon Receptor in Primary Breast Cancer Depends on the Type of Endocrine Therapy: A Population-Based Cohort Study.

The aryl hydrocarbon receptor (AhR) is a master regulator of multiple pathways involved in breast cancer, and influences the estrogen receptor alpha (ER) and aromatase/CYP19A1. The purpose of this study was to elucidate the interplay between intratumoral levels of AhR and aromatase, patient characteristics (including AhR and CYP19A1 genotypes), clinicopathological features, and prognosis in breast cancer patients receiving adjuvant treatments. A prospective cohort of 1116 patients with primary breast cancer in Sweden, included 2002-2012, was followed until June 30th 2019 (median 8.7 years). Tumor-specific AhR (n=920) and aromatase levels (n=816) were evaluated on tissue microarrays using immunohistochemistry. Associations between cytoplasmatic (AhRcyt) and nuclear (AhRnuc) AhR levels, intratumoral aromatase, clinicopathological features, and prognosis in different treatment groups were analyzed. Low AhRcyt levels (n=183) and positive intratumoral aromatase (n=69) were associated with estrogen receptor (ER)- status and more aggressive tumors. Genotypes were not associated with their respective protein levels. The functional AhR Arg554Lys GG genotype was associated with recurrence-free survival in switch-therapy (sequential tamoxifen/aromatase inhibitors (AI) or AI/tamoxifen) treated patients (HRadj 0.42; 95% CI 0.22-0.83). High AhRcyt levels were associated with longer recurrence-free survival during the first 10 years of follow-up among tamoxifen-only treated patients (HRadj 0.40; 95% CI 0.23-0.71) compared to low AhRcyt levels, whereas an almost inverse association was seen in patients with switch-therapy (P interaction=0.023). Intratumoral aromatase had little prognostic impact. These findings warrant confirmation in an independent cohort, preferably in a randomized clinical trial comparing different endocrine regimens. They might also guide the selection of breast cancer patients for clinical trials with selective AhR modulators.

Reverse-Phase Protein Array: Technology, Application, Data Processing, and Integration.

Reverse-phase protein array (RPPA) is a high-throughput antibody-based targeted proteomics platform that can quantify hundreds of proteins in thousands of samples derived from tissue or cell lysates, serum, plasma, or other body fluids. Protein samples are robotically arrayed as microspots on nitrocellulose-coated glass slides. Each slide is probed with a specific antibody that can detect levels of total protein expression or post-translational modifications, such as phosphorylation as a measure of protein activity. Here we describe workflow protocols and software tools that we have developed and optimized for RPPA in a core facility setting that includes sample preparation, microarray mapping and printing of protein samples, antibody labeling, slide scanning, image analysis, data normalization and quality control, data reporting, statistical analysis, and management of data. Our RPPA platform currently analyzes ∼240 validated antibodies that primarily detect proteins in signaling pathways and cellular processes that are important in cancer biology. This is a robust technology that has proven to be of value for both validation and discovery proteomic research and integration with other omics data sets.

Reverse-Phase Protein Array: Technology, Application, Data Processing, and Integration.

Reverse-phase protein array (RPPA) is a high-throughput antibody-based targeted proteomics platform that can quantify hundreds of proteins in thousands of samples derived from tissue or cell lysates, serum, plasma, or other body fluids. Protein samples are robotically arrayed as microspots on nitrocellulose-coated glass slides. Each slide is probed with a specific antibody that can detect levels of total protein expression or post-translational modifications, such as phosphorylation as a measure of protein activity. Here we describe workflow protocols and software tools that we have developed and optimized for RPPA in a core facility setting that includes sample preparation, microarray mapping and printing of protein samples, antibody labeling, slide scanning, image analysis, data normalization and quality control, data reporting, statistical analysis, and management of data. Our RPPA platform currently analyzes ∼240 validated antibodies that primarily detect proteins in signaling pathways and cellular processes that are important in cancer biology. This is a robust technology that has proven to be of value for both validation and discovery proteomic research and integration with other omics data sets.

Proteomic Resistance Biomarkers for PI3K Inhibitor in Triple Negative Breast Cancer Patient-Derived Xenograft Models.

PI3K pathway activation is frequently observed in triple negative breast cancer (TNBC). However, single agent PI3K inhibitors have shown limited anti-tumor activity. To investigate biomarkers of response and resistance mechanisms, we tested 17 TNBC patient-derived xenograft (PDX) models representing diverse genomic backgrounds and varying degrees of PI3K pathway signaling activities for their tumor growth response to the pan-PI3K inhibitor, BKM120. Baseline and post-treatment PDX tumors were subjected to reverse phase protein array (RPPA) to identify protein markers associated with tumor growth response. While BKM120 consistently reduced PI3K pathway activity, as demonstrated by reduced levels of phosphorylated AKT, percentage tumor growth inhibition (%TGI) ranged from 35% in the least sensitive to 84% in the most sensitive model. Several biomarkers showed significant association with resistance, including elevated baseline levels of growth factor receptors (EGFR, pHER3 Y1197), PI3Kp85 regulatory subunit, anti-apoptotic protein BclXL, EMT (Vimentin, MMP9, IntegrinaV), NFKB pathway (IkappaB, RANKL), and intracellular signaling molecules including Caveolin, CBP, and KLF4, as well as treatment-induced increases in the levels of phosphorylated forms of Aurora kinases. Interestingly, increased AKT phosphorylation or PTEN loss at baseline were not significantly correlated to %TGI. These results provide important insights into biomarker development for PI3K inhibitors in TNBC.

BCL9/STAT3 regulation of transcriptional enhancer networks promote DCIS progression.

The molecular processes by which some human ductal carcinoma in situ (DCIS) lesions advance to the more aggressive form, while others remain indolent, are largely unknown. Experiments utilizing a patient-derived (PDX) DCIS Mouse INtraDuctal (MIND) animal model combined with ChIP-exo and RNA sequencing revealed that the formation of protein complexes between B Cell Lymphoma-9 (BCL9), phosphoserine 727 STAT3 (PS-727-STAT3) and non-STAT3 transcription factors on chromatin enhancers lead to subsequent transcription of key drivers of DCIS malignancy. Downregulation of two such targets, integrin ß3 and its associated metalloproteinase, MMP16, resulted in a significant inhibition of DCIS invasive progression. Finally, in vivo targeting of BCL9, using rosemary extract, resulted in significant inhibition of DCIS malignancy in both cell line and PDX DCIS MIND animal models. As such, our studies provide compelling evidence for future testing of rosemary extract as a chemopreventive agent in breast cancer.

Systemic distribution of progesterone receptor subtypes in human tissues.

Progesterone receptor (PR) is expressed in a wide variety of human tissues, including both reproductive and non-reproductive tissues. Upon binding to the PR, progesterone can display several non-reproductive functions, including neurosteroid activity in the central nervous system, inhibition of smooth muscle contractile activity in the gastrointestinal tract, and regulating the development and maturation of the lung. PR exists as two major isoforms, PRA and PRB. Differential expression of these PR isoforms reportedly contributes to different biological activities of the hormone. However, the distribution of the PR isoforms in human tissues has remained virtually unexplored. In this study, we immunolocalized PR expression in various human tissues using PR (1294) specific antibody, which is capable of detecting both PRA and PRB, and PRB (250H11) specific antibody. Tissues from the uterus, ovary, breast, placenta, prostate, testis, cerebrum, cerebellum, pituitary, spinal cord, esophagus, stomach, small intestine, colon, pancreas, liver, kidney, urinary bladder, lung, heart, aorta, thymus, adrenal gland, thyroid, spleen, skin, and bone were examined in four different age groups (fetal, pediatric, young, and old) in male and female subjects. PR and PRB were detected in the nuclei of cells in the female reproductive system, in both the nuclei and cytoplasm of pituitary gland and pancreatic acinar cells, and only in the cytoplasm of cells in the testis, stomach, small intestine, colon, liver, kidney, urinary bladder, lung, adrenal gland, and skin. Of particular interest, total PRB expression overlapped with that of total PR expression in most tissues but was negative in the female fetal reproductive system. The findings indicate that progesterone could affect diverse human organs differently than from reproductive organs. These findings provide new insights into the novel biological roles of progesterone in non-reproductive organs.

Progesterone receptor isoform B expression in pulmonary neuroendocrine cells decreases cell proliferation.

The progesterone receptor (PR) has been reported to play important roles in lung development and function, such as alveolarization, alveolar fluid clearance (AFC) and upper airway dilator muscle activity. In the lung, pulmonary neuroendocrine cells (PNECs) are important in the etiology and progression of lung neuroendocrine tumors (NETs). Women with lung NETs had significantly better survival rates than men, suggesting that sex steroids and their receptors, such as the PR, could be involved in the progression of lung NETs. The PR exists as two major isoforms, PRA and PRB. How the expression of different PR isoforms affects proliferation and the development of lung NETs is not well understood. To determine the role of the PR isoforms in PNECs, we constructed H727 lung NET cell models expressing PRB, PRA, Green Fluorescence Protein (GFP) (control). The expression of PRB significantly inhibited H727 cell proliferation better than that of PRA in the absence of progestin. The expression of the unrelated protein, GFP, had little to no effect on H727 cell proliferation. To better understand the role of the PR isoform in PNECs, we examined PR isoform expression in PNECs in lung tissues. A monoclonal antibody specific to the N-terminus of PRB (250H11 mAb) was developed to specifically recognize PRB, while a monoclonal antibody specific to a common N-terminus epitope present in both PRA and PRB (1294 mAb) was used to detect both PRA and PRB. Using these PR and PRB-specific antibodies, we demonstrated that PR (PRA&PRB) and PRB were expressed in the PNECs of the normal fetal and adult lung, with significantly higher PR expression in the fetal lung. Interestingly, PRB expression in the normal lung was associated with lower cell proliferation than PR expression, suggesting a distinct role of PRB in the PNECs. A better understanding of the molecular mechanism of PR and PR isoform signaling in lung NET cells may help in developing novel therapeutic strategies that will benefit lung NET patients in the future.

UCHL1 loss alters the cell-cycle in metastatic pancreatic neuroendocrine tumors.

Loss of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) expression by CpG promoter hypermethylation is associated with metastasis in gastroenteropancreatic neuroendocrine tumors; however, the mechanism of how UCHL1 loss contributes to metastatic potential remains unclear. In this study, we first confirmed that loss of UCHL1 expression on immunohistochemistry was significantly associated with metastatic tumors in a translational pancreatic neuroendocrine tumor (PNET) cohort, with a sensitivity and specificity of 78% and 89%, respectively. To study the mechanism driving this aggressive phenotype, BON and QGP-1 metastatic PNET cell lines, which do not produce UCHL1, were stably transfected to re-express UCHL1. In vitro assays, RNA-sequencing, and reverse-phase protein array (RPPA) analyses were performed comparing empty-vector negative controls and UCHL1-expressing cell lines. UCHL1 re-expression is associated with lower anchorage-independent colony growth in BON cells, lower colony formation in QGP cells, and a higher percentage of cells in the G0/G1 cell-cycle phase in BON and QGP cells. On RPPA proteomic analysis, there was an upregulation of cell-cycle regulatory proteins CHK2 (1.2 fold change, p=0.004) and P21 (1.2 fold change, p=0.023) in BON cells expressing UCHL1; western blot confirmed upregulation of phosphorylated CHK2 and P21. There were no transcriptomic differences detected on RNA-Sequencing between empty-vector negative controls and UCHL1-expressing cell lines. In conclusion, UCHL1 loss correlates with metastatic potential in PNETs and its re-expression induces a less aggressive phenotype in vitro, in part by inducing cell-cycle arrest through post-translational regulation of phosphorylated CHK2. UCHL1 re-expression should be considered as a functional biomarker in detecting PNETs capable of metastasis.

Correction to: The Emerging Roles of Steroid Hormone Receptors in Ductal Carcinoma in Situ (DCIS) of the Breast.

The original version of this article unfortunately contained mistakes.

Mammary Precancerous Stem and Non-Stem Cells Evolve into Cancers of Distinct Subtypes.

There are distinct cell subpopulations in normal epithelial tissue, including stem cells, progenitor cells, and more differentiated cells, all of which have been extensively studied for their susceptibility to tumorigenesis. However, normal cells usually have to progress through a precancerous lesion state before becoming a full-blown tumor. Precancerous early lesions are heterogeneous, and the cell subset that is the primary source of the eventual tumor remains largely unknown. By using mouse models that are tailored to address this question, we identified a keratin 6a-expressing precancerous stem cell (PcSC) subset and a more differentiated whey acidic protein-positive (WAP+) cell subset in mammary precancerous lesions initiated by the Wnt1 oncogene. Both cell subsets rapidly progressed to cancer upon introduction of constitutively active versions of either HRAS or BRAF. However, the resulting tumors were dramatically different in protein profiles and histopathology: keratin 6a+ precancerous cells gave rise to adenocarcinoma, whereas WAP+ cells yielded metaplastic carcinoma with severe squamous differentiation and more robust activation of MEK/ERK signaling. Therefore, both stem and non-stem cells in mammary precancerous lesions can contribute to the eventual cancers, but their differentiation status determines the resulting cancer phenotype. This work identifies a previously unknown player in cancer heterogeneity and suggests that cancer prevention should target precancerous cells broadly and not be limited to PcSC. SIGNIFICANCE: This work uses a novel mouse mammary gland cancer model to show that tumors initiated from different precancerous mammary epithelial cells are distinct.

Consequences of early postnatal lipopolysaccharide exposure on developing lungs in mice.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of infants that is characterized by interrupted lung development. Postnatal sepsis causes BPD, yet the contributory mechanisms are unclear. To address this gap, studies have used lipopolysaccharide (LPS) during the alveolar phase of lung development. However, the lungs of infants who develop BPD are still in the saccular phase of development, and the effects of LPS during this phase are poorly characterized. We hypothesized that chronic LPS exposure during the saccular phase disrupts lung development by mechanisms that promote inflammation and prevent optimal lung development and repair. Wild-type C57BL6J mice were intraperitoneally administered 3, 6, or 10 mg/kg of LPS or a vehicle once daily on postnatal days (PNDs) 3-5. The lungs were collected for proteomic and genomic analyses and flow cytometric detection on PND6. The impact of LPS on lung development, cell proliferation, and apoptosis was determined on PND7. Finally, we determined differences in the LPS effects between the saccular and alveolar lungs. LPS decreased the survival and growth rate and lung development in a dose-dependent manner. These effects were associated with a decreased expression of proteins regulating cell proliferation and differentiation and increased expression of those mediating inflammation. While the lung macrophage population of LPS-treated mice increased, the T-regulatory cell population decreased. Furthermore, LPS-induced inflammatory and apoptotic response and interruption of cell proliferation and alveolarization was greater in alveolar than in saccular lungs. Collectively, the data support our hypothesis and reveal several potential therapeutic targets for sepsis-mediated BPD in infants.

The Emerging Roles of Steroid Hormone Receptors in Ductal Carcinoma in Situ (DCIS) of the Breast.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor to most types of invasive breast cancer (IBC). Although it is estimated only one third of untreated patients with DCIS will progress to IBC, standard of care for treatment is surgery and radiation. This therapeutic approach combined with a lack of reliable biomarker panels to predict DCIS progression is a major clinical problem. DCIS shares the same molecular subtypes as IBC including estrogen receptor (ER) and progesterone receptor (PR) positive luminal subtypes, which encompass the majority (60-70%) of DCIS. Compared to the established roles of ER and PR in luminal IBC, much less is known about the roles and mechanism of action of estrogen (E2) and progesterone (P4) and their cognate receptors in the development and progression of DCIS. This is an underexplored area of research due in part to a paucity of suitable experimental models of ER+/PR + DCIS. This review summarizes information from clinical and observational studies on steroid hormones as breast cancer risk factors and ER and PR as biomarkers in DCIS. Lastly, we discuss emerging experimental models of ER+/PR+ DCIS.

TGF-ß1 programmed myeloid-derived suppressor cells (MDSC) acquire immune-stimulating and tumor killing activity capable of rejecting established tumors in combination with radiotherapy.

Cancer-induced myeloid-derived suppressor cells (MDSC) play an important role in tumor immune evasion. MDSC programming or polarization has been proposed as a strategy for leveraging the developmental plasticity of myeloid cells to reverse MDSC immune suppressive functions, or cause them to acquire anti-tumor activity. While MDSC derived ex vivo from murine bone marrow precursor cells with tumor-conditioned medium efficiently suppressed T cell proliferation, MDSC derived from conditioned medium in presence of TGF-ß1 (TGFß-MDSC) acquired a novel immune-stimulatory phenotype, losing the ability to inhibit T cell proliferation and acquiring enhanced antigen-presenting capability. Altered immune function was associated with SMAD-2 dependent upregulation of maturation and costimulatory molecules, and downregulation of inducible nitric oxide synthase (iNOS), an effector mechanism of immunosuppression. TGFß-MDSC also upregulated FAS-ligand expression, leading to FAS-dependent killing of murine human papillomavirus (HPV)-associated head and neck cancer cells and tumor spheroids in vitro and anti-tumor activity in vivo. Radiation upregulated FAS expression on tumor cells, and the combination of radiotherapy and intratumoral injection of TGFß-MDSC strongly enhanced class I expression on tumor cells and induction of HPV E7 tetramer-positive CD8 + T cells, leading to clearance of established tumors and long-term survival. TGFß-MDSC derived from human PBMC with tumor conditioned medium also lost immunosuppressive function and acquired tumor-killing activity. Thus, TGFß1 mediated programming of nascent MDSC leads to a potent anti-tumor phenotype potentially suitable for adoptive immunotherapy.

A mouse model engineered to conditionally express the progesterone receptor-B isoform.

Using a Rosa26 gene targeting strategy in mouse embryonic stem cells, we have generated a new transgenic mouse (Pgr-B LSL ), which is designed to conditionally express the epitope-tagged mouse progesterone receptor-B (PGR-B) isoform when crossed with a specific cre driver mouse. To functionally validate this transgenic mouse, we crossed the Pgr-B LSL mouse with the MMTV-CREA transgenic mouse to create the MMTV-CREA/Pgr-B LSL bigenic (termed PR-B:OE to denote PGR-B overexpressor). As expected, transgene-derived PGR-B protein was specifically targeted to the virgin mammary gland epithelium. At a functional level, the PR-B:OE bigenic exhibited abnormal mammary morphogenesis-dilated epithelial ducts, precocious alveologenesis and lateral side-branching, along with a prominent proliferative signature-that resulted in pregnant PR-B:OE mice unable to exhibit mammary gland terminal differentiation at parturition. Because of this developmental failure, the PR-B:OE mammary gland was incapable of producing milk resulting in early neonatal death of otherwise healthy litters. This first line of analysis demonstrates the utility of the Pgr-B LSL mouse to examine the role of the PGR-B isoform in different physiologic and pathophysiologic systems that are responsive to progesterone.

Metformin inhibits stromal aromatase expression and tumor progression in a rodent model of postmenopausal breast cancer.

BACKGROUND: Obesity and type II diabetes are linked to increased breast cancer risk in postmenopausal women. Patients treated with the antidiabetic drug metformin for diabetes or metabolic syndrome have reduced breast cancer risk, a greater pathologic complete response to neoadjuvant therapy, and improved breast cancer survival. We hypothesized that metformin may be especially effective when targeted to the menopausal transition, as this is a lifecycle window when weight gain and metabolic syndrome increase, and is also when the risk for obesity-related breast cancer increases. METHODS: Here, we used an 1-methyl-1-nitrosourea (MNU)-induced mammary tumor rat model of estrogen receptor (ER)-positive postmenopausal breast cancer to evaluate the long-term effects of metformin administration on metabolic and tumor endpoints. In this model, ovariectomy (OVX) induces rapid weight gain, and an impaired whole-body response to excess calories contributes to increased tumor glucose uptake and increased tumor proliferation. Metformin treatment was initiated in tumor-bearing animals immediately prior to OVX and maintained for the duration of the study. RESULTS: Metformin decreased the size of existing mammary tumors and inhibited new tumor formation without changing body weight or adiposity. Decreased lipid accumulation in the livers of metformin-treated animals supports the ability of metformin to improve overall metabolic health. We also found a decrease in the number of aromatase-positive, CD68-positive macrophages within the tumor microenvironment, suggesting that metformin targets the immune microenvironment in addition to improving whole-body metabolism. CONCLUSIONS: These findings suggest that peri-menopause/menopause represents a unique window of time during which metformin may be highly effective in women with established, or at high risk for developing, breast cancer.

TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis.

Receptor tyrosine kinases (RTKs) are important drivers of cancers. In addition to genomic alterations, aberrant activation of WT RTKs plays an important role in driving cancer progression. However, the mechanisms underlying how RTKs drive prostate cancer remain incompletely characterized. Here we show that non-proteolytic ubiquitination of RTK regulates its kinase activity and contributes to RTK-mediated prostate cancer metastasis. TRAF4, an E3 ubiquitin ligase, is highly expressed in metastatic prostate cancer. We demonstrated here that it is a key player in regulating RTK-mediated prostate cancer metastasis. We further identified TrkA, a neurotrophin RTK, as a TRAF4-targeted ubiquitination substrate that promotes cancer cell invasion and found that inhibition of TrkA activity abolished TRAF4-dependent cell invasion. TRAF4 promoted K27- and K29-linked ubiquitination at the TrkA kinase domain and increased its kinase activity. Mutation of TRAF4-targeted ubiquitination sites abolished TrkA tyrosine autophosphorylation and its interaction with downstream proteins. TRAF4 knockdown also suppressed nerve growth factor (NGF) stimulated TrkA downstream p38 MAPK activation and invasion-associated gene expression. Furthermore, elevated TRAF4 levels significantly correlated with increased NGF-stimulated invasion-associated gene expression in prostate cancer patients, indicating that this signaling axis is significantly activated during oncogenesis. Our results revealed a posttranslational modification mechanism contributing to aberrant non-mutated RTK activation in cancer cells.

Mass Spectrometry-Based Proteomics Reveals Potential Roles of NEK9 and MAP2K4 in Resistance to PI3K Inhibition in Triple-Negative Breast Cancers.

Activation of PI3K signaling is frequently observed in triple-negative breast cancer (TNBC), yet PI3K inhibitors have shown limited clinical activity. To investigate intrinsic and adaptive mechanisms of resistance, we analyzed a panel of patient-derived xenograft models of TNBC with varying responsiveness to buparlisib, a pan-PI3K inhibitor. In a subset of patient-derived xenografts, resistance was associated with incomplete inhibition of PI3K signaling and upregulated MAPK/MEK signaling in response to buparlisib. Outlier phosphoproteome and kinome analyses identified novel candidates functionally important to buparlisib resistance, including NEK9 and MAP2K4. Knockdown of NEK9 or MAP2K4 reduced both baseline and feedback MAPK/MEK signaling and showed synthetic lethality with buparlisib in vitro A complex in/del frameshift in PIK3CA decreased sensitivity to buparlisib via NEK9/MAP2K4-dependent mechanisms. In summary, our study supports a role for NEK9 and MAP2K4 in mediating buparlisib resistance and demonstrates the value of unbiased omic analyses in uncovering resistance mechanisms to targeted therapy.Significance: Integrative phosphoproteogenomic analysis is used to determine intrinsic resistance mechanisms of triple-negative breast tumors to PI3K inhibition. Cancer Res; 78(10); 2732-46. ©2018 AACR.