3D culture reveals a signaling network

The behavior of a cell is significantly influenced by its context. Epithelial cells derived from glandular organs such as the breast recreate their glandular organization when grown under 3D culture conditions. While traditional monolayer cultures are powerful tools to understand how cells proliferate, grow and respond to stress, they do not recreate the 3D property observed in vivo. Multiple studies demonstrate that 3D organization can reveal novel and unexpected insights into the mechanisms by which normal and tumorderived epithelial cells function. In the present article we comment on a study that reports identification of a RasV12-induced IL-6 signaling network in mammary epithelial cells in 3D cultures

Dysregulation of Cell Polarity Proteins Synergize with Oncogenes or the Microenvironment to Induce Invasive Behavior in Epithelial Cells

Changes in expression and localization of proteins that regulate cell and tissue polarity are frequently observed in carcinoma. However, the mechanisms by which changes in cell polarity proteins regulate carcinoma progression are not well understood. Here, we report that loss of polarity protein expression in epithelial cells primes them for cooperation with oncogenes or changes in tissue microenvironment to promote invasive behavior. Activation of ErbB2 in cells lacking the polarity regulators Scribble, Dlg1 or AF-6, induced invasive properties. This cooperation required the ability of ErbB2 to regulate the Par6/aPKC polarity complex. Inhibition of the ErbB2-Par6 pathway was sufficient to block ErbB2-induced invasion suggesting that two polarity hits may be needed for ErbB2 to promote invasion. Interestingly, in the absence of ErbB2 activation, either a combined loss of two polarity proteins, or exposure of cells lacking one polarity protein to cytokines IL-6 or TNFα induced invasive behavior in epithelial cells. We observed the invasive behavior only when cells were plated on a stiff matrix (Matrigel/Collagen-1) and not when plated on a soft matrix (Matrigel alone). Cells lacking two polarity proteins upregulated expression of EGFR and activated Akt. Inhibition of Akt activity blocked the invasive behavior identifying a mechanism by which loss of polarity promotes invasion of epithelial cells. Thus, we demonstrate that loss of polarity proteins confers phenotypic plasticity to epithelial cells such that they display normal behavior under normal culture conditions but display aggressive behavior in response to activation of oncogenes or exposure to cytokines

Prostate cancer-associated SPOP mutations confer resistance to BET inhibitors through stabilization of BRD4

The bromodomain and extra-terminal (BET) family of proteins, comprised of four members including BRD2, BRD3, BRD4 and the testis-specific isoform BRDT, largely function as transcriptional co-activators 1–3 and play critical roles in various cellular processes, including cell cycle, apoptosis, migration and invasion 4,5. As such, BET proteins enhance the oncogenic functions of major cancer drivers by either elevating their expression such as c-Myc in leukemia 6,7 or by promoting transcriptional activities of oncogenic factors such as AR and ERG in the prostate cancer setting 8. Pathologically, BET proteins are frequently overexpressed and clinically linked to various types of human cancers 5,9,10, therefore pursued as attractive therapeutic targets for selective inhibition in patients. To this end, a number of bromodomain inhibitors, including JQ1 and I-BET, have been developed 11,12 and shown promising outcomes in early clinical trials. Despite resistance to BET inhibitor has been documented in pre-clinical models 13–15 the molecular mechanisms underlying acquired resistance are largely unknown. Here, we report that Cullin 3SPOP earmarks BET proteins including BRD2, BRD3 and BRD4 for ubiquitination-mediated degradation. Pathologically, prostate cancer-associated SPOP mutants fail to interact with and promote the destruction of BET proteins, leading to their elevated abundance in SPOP-deficient prostate cancer. As a result, prostate cancer cells and prostate cancer patient-derived organoids harboring SPOP mutations are more resistant to BET inhibitor-induced cell growth arrest and apoptosis. Therefore, our results elucidate the tumor suppressor role of SPOP in prostate cancer by negatively controlling BET protein stability, and also provide a molecular mechanism for BET inhibitor resistance in prostate cancer patients bearing SPOP mutations

Identification of PTPN23 as a novel regulator of cell invasion in mammary epithelial cells from a loss-of-function screen of the ‘PTP-ome'

We used an RNAi-mediated loss-of-function screen to study systematically the role of the protein tyrosine phosphatase (PTP) superfamily of enzymes in mammary epithelial cell motility in the absence or presence of the oncoprotein tyrosine kinase ERBB2. We report that although shRNAs directed against most of the PTP family were without effect, suppression of three PTPs—PRPN23, PTPRG, and PTPRR—enhanced cell motility. Furthermore, we found that suppression of PTPN23, but not PTPRG or PTPRR, induced cell invasion. Suppression of PTPN23 increased E-cadherin internalization, impaired early endosome trafficking of E-cadherin, induced the expression of mesenchymal proteins, and caused cell scattering. The activity of SRC and β-catenin was elevated when PTPN23 was suppressed. Moreover, we identified SRC, E-cadherin, and β-catenin as direct substrates of PTPN23. Inhibition of SRC with the small molecular inhibitor SU6656 blocked the effects of PTPN23 depletion. These findings suggest that loss of PTPN23 may increase the activity of SRC and the phosphorylation status of the E-cadherin/β-catenin signaling complex to promote tumor growth and invasive behavior in breast cancer. In addition, our studies highlight functional specificity among PTPs and reveal new roles for PTPs in mammary epithelial cell biology