| Peer-reviewed | Experimental study | Mice/cells |
YAP, a protein known to help cancer cells survive treatment, behaves very differently in cell culture models depending on how they are grown in the lab, highlighting the importance of mimicking tumour architecture to guide better drug strategies.
The study, published today as a Reviewed Preprint in eLife, is described as important by the editors. They say the authors provide compelling evidence supporting the role of YAP in tumour treatment resistance and its differential activation in lab experiments based on whether it is grown in a flat monolayer (2D), or a three-dimensional (3D) spheroid culture. The work has the potential to define more biologically relevant cell culture model systems for drug resistance and targetable pathways to overcome drug resistance.
Non-small cell lung cancer (NSCLC) is the most common form of lung cancer in the US, accounting for approximately 85% of all cases*. Two frequent genetic ‘drivers’ of NSCLC progression are mutations in the KRAS gene – which normally helps control cell growth but, when mutated, leaves growth signals permanently switched on – and the EGFR gene – which normally responds to growth signals but can become overactive when mutated. Drugs that block EGFR activity have revolutionised treatment for EGFR-mutant NSCLC, but tumours often recur within a year. Likewise, new drugs that inhibit KRAS activity have shown promise in early clinical trials, but patients typically only remain progression-free for about six months. The acquired genetic mechanisms that allow NSCLC cells to survive treatment are well studied and include additional mutations of EGFR or amplification of mutant KRAS. However, less is known about how these cells become treatment-resistant through non-genetic means – that is, without any new mutations.
NSCLC cells sense mechanical forces from their surroundings through a dense, fibrous network of collagen called the extracellular matrix. The increased environmental rigidity can force YAP into the nucleus, the control center of a cell, which in turn activates genes that help the cancer cells grow and persist. A small group of drug-tolerant persister cells (DTPs) can survive even when most of the tumour dies after treatment, and YAP activity in DTPs has been identified as a mechanism underlying treatment resistance using cell culture models.
“These findings have often been reached in two-dimensional, monolayer cultures that do not reflect the real three-dimensional environment of a tumour,” says lead author Rachel Nakagawa, a PhD student in the Department of Cell & Tissue Biology, University of California, San Francisco, US. “To address this, scientists have developed three-dimensional spheroid cultures that better replicate cell-cell interactions, cell-extracellular matrix interactions, and nutrient gradients – which are crucial for understanding tumour tissue behaviour and drug responses.”
Nakagawa and colleagues set out to confirm YAP’s role in helping cancer cells resist treatment and compare its activity in tumours grown in monolayers versus spheroids.
They began by implanting human EGFR-mutant NSCLC cells in mice and treating them for five days with the EGFR inhibitor afatinib. After treatment, the tumours had shrunk by about 90%. They then used immunofluorescence microscopy to track YAP’s position within any remaining DTP cells. In the absence of treatment, YAP is typically located within the cytoplasm – the gelatinous liquid that fills cells – suggesting that this pathway is not active prior to drug administration. However, the team found that roughly 60% of the surviving DTP cells had YAP inside their nucleus, where it can activate survival genes, compared to only 10% in untreated NSCLC cells. This indicates that drug administration can select for cells in which relocation and activation of YAP in NSCLC tumour cells has occurred.
Next, the researchers grew the same EGFR-mutant NSCLC cell lines in the lab in both monolayers on rigid plastic, and spheroids on ultra-low adhesion plates. They treated both cultures with afatinib and assessed the proportion of surviving cells. NSCLC cells grown in a monolayer largely survived treatment, but stopped dividing, whereas those grown in spheroids died en masse, mirroring the results in mice. The team’s analysis showed that the cells in monolayers retained active EGFR signalling and kept YAP localised in the nucleus. Conversely, tumour cells grown in spheroids lost EGFR signalling activity and sequestered YAP in the cytoplasm, making them far more sensitive to drug-induced cell death.
Based on this insight, the team expanded their tests across other cancer mutations and drugs. They treated a second EGFR-mutant line with a mutant EGFR inhibitor, osimertinib, two KRAS-mutant lines with the targeted KRASG12C inhibitor, ARS-1620, and a melanoma-causing BRAF-mutant line with BRAFV600E inhibitor, vemurafenib. In 5 out of 6 cases, three-dimensional spheroid cultures were significantly more drug-sensitive than their monolayer counterparts. Additionally in these lines, YAP localised to the nucleus when cells were grown in monolayer and to the cytoplasm in spheroids. Interestingly, the remaining line, a KRAS mutant cell line, that did not show differential sensitivity to ARS-1620 in spheroids versus monolayers was found to have YAP predominantly localised to the cytoplasm in both contexts. This further suggested YAP localisation may be critical for differential drug sensitivity.
Finally, to prove YAP’s causal role in tumour treatment resistance, they engineered cells grown in spheroids to overexpress a mutated form of YAP that has increased localisation to the nucleus. These cells survived afatinib treatment at a far greater level than those in the normal spheroids, confirming that nuclear YAP alone can drive non-genetic resistance to short-term drug treatment.
“Our findings confirm YAP’s role as a non-genetic driver of treatment resistance in NSCLC cells, and that the choice between a two- and three-dimensional culture when modelling cancers in the lab can alter its activity,” says senior author Andrei Goga, Professor in the Department of Cell & Tissue Biology, and co-leader of the Breast Oncology Program, UCSF Helen Diller Comprehensive Cancer Center, University of California, San Francisco. “Standard monolayers model the small population of DTP cells seen in patients, while spheroids better reflect the real tumour environment and initial, dramatic shrinkage after treatment. This work underscores the importance of considering diverse cell culture models beyond conventional monolayers to mitigate unforeseen YAP-related impacts on drug sensitivity and resistance.”
“These data, along with exciting work from other labs, also suggest that a combinational therapy of EGFR inhibitors followed by YAP inhibitors may provide synergistic benefits to eliminate DTP cells after initial treatment,” adds Nakagawa. “Further work is necessary to determine the optimal timing for administering YAP inhibitors, as our results suggest that YAP inhibition may be less effective during initial EGFR inhibitor treatment when YAP activity is low.”
* According to Chevallier, M., Borgeaud, M., Addeo, A. & Friedlaender, A. Oncogenic driver mutations in non-small cell lung cancer: Past, present and future. World J. Clin. Oncol. 12, 217–237 (2021)
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