Mechanical stress shapes the cancer cell response to neddylation inhibition.

BACKGROUND: The inhibition of neddylation by the preclinical drug MLN4924 represents a new strategy to combat cancer. However, despite being effective against hematologic malignancies, its success in solid tumors, where cell-cell and cell-ECM interactions play essential roles, remains elusive. METHODS: Here, we studied the effects of MLN4924 on cell growth, migration and invasion in cultured prostate cancer cells and in disease-relevant prostate tumoroids. Using focused protein profiling, drug and RNAi screening, we analyzed cellular pathways activated by neddylation inhibition. RESULTS: We show that mechanical stress induced by MLN4924 in prostate cancer cells significantly affects the therapeutic outcome. The latter depends on the cell type and involves distinct Rho isoforms. In LNCaP and VCaP cells, the stimulation of RhoA and RhoB by MLN4924 markedly upregulates the level of tight junction proteins at cell-cell contacts, which augments the mechanical strain induced by Rho signaling. This "tight junction stress response" (TJSR) causes the collapse of cell monolayers and a characteristic rupture of cancer spheroids. Notably, TJSR is a major cause of drug-induced apoptosis in these cells. On the other hand, in PC3 cells that underwent partial epithelial-to-mesenchymal transition (EMT), the stimulation of RhoC induces an adverse effect by promoting amoeboid cell scattering and invasion. We identified complementary targets and drugs that allow for the induction of TJSR without stimulating RhoC. CONCLUSIONS: Our finding that MLN4924 acts as a mechanotherapeutic opens new ways to improve the efficacy of neddylation inhibition as an anticancer approach.

Direct transfection of clonal organoids in Matrigel microbeads: a promising approach toward organoid-based genetic screens.

Organoid cultures in 3D matrices are relevant models to mimic the complex in vivo environment that supports cell physiological and pathological behaviors. For instance, 3D epithelial organoids recapitulate numerous features of glandular tissues including the development of fully differentiated acini that maintain apico-basal polarity with hollow lumen. Effective genetic engineering in organoids would bring new insights in organogenesis and carcinogenesis. However, direct 3D transfection on already formed organoids remains challenging. One limitation is that organoids are embedded in extracellular matrix and grow into compact structures that hinder transfection using traditional techniques. To address this issue, we developed an innovative approach for transgene expression in 3D organoids by combining single-cell encapsulation in Matrigel microbeads using a microfluidic device and electroporation. We demonstrate that direct electroporation of encapsulated organoids reaches up to 80% of transfection efficiency. Using this technique and a morphological read-out that recapitulate the different stages of tumor development, we further validate the role of p63 and PTEN as key genes in acinar development in breast and prostate tissues. We believe that the combination of controlled organoid generation and efficient 3D transfection developed here opens new perspectives for flow-based high-throughput genetic screening and functional genomic applications.

Distinct outcomes of CRL-Nedd8 pathway inhibition reveal cancer cell plasticity.

Inhibition of protein degradation by blocking Cullin-RING E3 ligases (CRLs) is a new approach in cancer therapy though of unknown risk because CRL inhibition may stabilize both oncoproteins and tumor suppressors. Probing CRLs in prostate cancer cells revealed a remarkable plasticity of cells with TMPRSS2-ERG translocation. CRL suppression by chemical inhibition or knockdown of RING component RBX1 led to reversible G0/G1 cell cycle arrest that prevented cell apoptosis. Conversely, complete blocking of CRLs at a higher inhibitor dose-induced cytotoxicity that was amplified by knockdown of CRL regulator Cand1. We analyzed cell signaling to understand how varying degrees of CRL inhibition translated to distinct cell fates. Both tumor suppressor and oncogenic cell signaling pathways and transcriptional activities were affected, with pro-metastatic Wnt/ß-catenin as the most upregulated. Suppression of the NF-κB pathway contributed to anti-apoptotic effect, and androgen receptor (AR) and ERG played decisive, though opposite, roles: AR was involved in protective quiescence, whereas ERG promoted apoptosis. These data define AR-ERG interaction as a key plasticity and survival determinant in prostate cancer and suggest supplementary treatments that may overcome drug resistance mechanisms regulated by AR-ERG interaction.

Facile bench-top fabrication of enclosed circular microchannels provides 3D confined structure for growth of prostate epithelial cells.

We present a simple bench-top method to fabricate enclosed circular channels for biological experiments. Fabricating the channels takes less than 2 hours by using glass capillaries of various diameters (from 100 µm up to 400 µm) as a mould in PDMS. The inner surface of microchannels prepared in this way was coated with a thin membrane of either Matrigel or a layer-by-layer polyelectrolyte to control cellular adhesion. The microchannels were then used as scaffolds for 3D-confined epithelial cell culture. To show that our device can be used with several epithelial cell types from exocrine glandular tissues, we performed our biological studies on adherent epithelial prostate cells (non-malignant RWPE-1 and invasive PC3) and also on breast (non-malignant MCF10A) cells We observed that in static conditions cells adhere and proliferate to form a confluent layer in channels of 150 µm in diameter and larger, whereas cellular viability decreases with decreasing diameter of the channel. Matrigel and PSS (poly (sodium 4-styrenesulphonate)) promote cell adhesion, whereas the cell proliferation rate was reduced on the PAH (poly (allylamine hydrochloride))-terminated surface. Moreover infusing channels with a continuous flow did not induce any cellular detachment. Our system is designed to simply grow cells in a microchannel structure and could be easily fabricated in any biological laboratory. It offers opportunities to grow epithelial cells that support the formation of a light. This system could be eventually used, for example, to collect cellular secretions, or study cell responses to graduated hypoxia conditions, to chemicals (drugs, siRNA, …) and/or physiological shear stress.

The modulation of attachment, growth and morphology of cancerous prostate cells by polyelectrolyte nanofilms.

The behaviour of cancerous epithelial prostatic cells (PC3) growing on polyelectrolytes (PE) coatings was compared to the behaviour of immortalized normal prostatic cells (PNT-2). The cell behaviour was evaluated and quantified in terms of initial cell attachment, growth, metabolic activity, morphometry, adhesion, apoptosis and stress related gene expression. Both the anionic PSS (poly(sodium 4-styrenesulphonate))-terminated surface and cationic PAH (poly(allylamine hydrochloride))-terminated surfaces were not cytotoxic. The initial attachment of cells was better on the PAH-terminated surface compared to fibronectin. However, the proliferation rate of PC3 cells was reduced on the PAH-terminated surface and slightly increased on the PSS coatings. Only PAH prevented the clustering phenotype of PC3 and reduced the number of focal adhesion points as compared to fibronectin or PSS coatings. In contrast, none of the PE surfaces significantly affected the biological responses of PNT-2 cells. PAH-terminating films provide a tool to preferentially modulate the growth of some cancerous phenotypes, in this case as a micro-environment that reduces the growth of metastatic PC3 cells.

Label-free analysis of prostate acini-like 3D structures by lensfree imaging.

We present a lensfree imaging method to analyze polarity in RWPE1 prostate epithelial cells that form polarized acini with lumen under standard tridimensional (3D) culture conditions. The first event in epithelial carcinogenesis is loss of polarity, followed by uncontrolled proliferation leading to metastasis. We demonstrate that it is possible to use optical signatures to discriminate 3D objects with distinct polarities in a large field of view. The three metrics we present here are designed as image processing tools to discriminate acini from spheroids without any 3D reconstruction. To demonstrate that our lensfree imaging platform may be used to study the 3D organization of epithelial cells, we analyzed and quantified the modulation of dynamic processes, e.g., the polarity of acini and the merging of polarized structures, upon transforming growth factor beta-1 (TGF beta-1) addition to the culture media. Hence, coupling lensfree microscopy with 3D cell culture provides an innovative tool to study epithelial tissue morphogenesis in a large field of view and to elucidate the regulation of growth, morphogenesis and differentiation in normal and cancerous human prostate cells. Moreover, such biosensor would be a powerful tool to follow cancer progression and to evaluate anti-cancer drugs.