By Hugo Francisco de SouzaReviewed by Susha Cheriyedath, M.Sc.Oct 23 2024
New study reveals that specialized ribosomes, driven by cytokine signals, help tumors evade the immune system, offering a breakthrough pathway for enhancing cancer immunotherapies.
Study: P-stalk ribosomes act as master regulators of cytokine-mediated processes. Image Credit: ALIOUI MA / Shutterstock
In a recent study published in the journal Cell, researchers conducted extensive translational experiments on both in vivo murine model systems and in vitro human melanoma cell lines to enhance our understanding of the processes involved in cytokine-induced cellular rewiring, especially following ribosomal changes.
PSR Role in T-Cell Recognition: The study reveals that P-stalk ribosome (PSR) knockdown dramatically reduces CD8+ T cell recognition of tumor antigens, underscoring the central role of PSRs in enabling immune detection of tumors.
Study findings reveal that P-stalk ribosomes (PSRs) are essential mediators of cellular signal convergence, influencing pro- and anti-inflammatory cytokine regulation. These processes progress via previously unverified translation mechanisms, particularly phosphorylation. PSRs specifically drive the translation of mRNAs critical for cytokine responses, including those involved in antigen presentation and immune surveillance. Notably, this mechanistic interaction was found to be cell- and cancer-type agnostic, providing a generalized mechanism of tumor immune evasion. These findings are, hence, invaluable in helping prevent or treat multiple cancers in the future.
Background
One of the hallmarks allowing cancers to remain a leading cause of human mortality is their ability to effectively and consistently evade their host's immune mechanisms, preventing their isolation and removal by T cells. This property is exploited in relatively novel cancer interventions collectively known as immune checkpoint blockade (ICB). ICB comprises a class of drugs that helps improve cancer outcomes by preventing the action of 'checkpoint proteins' that mask cancers from T cells, thereby enhancing patients' intrinsic cancer-fighting capabilities.
Unfortunately, many patients demonstrate unresponsiveness to ICB interventions, prompting a reassessment of the mechanisms underpinning their modes of action. Recent research suggests that T-cell infiltration into the tumor microenvironment (TME), a cornerstone of ICB mediation, produces several cytokines (regulatory proteins). While some cytokines serve beneficial roles as biomarkers of ICB responses, others, particularly inhibitory cytokines like TGF-β, may rewire cancer- and immune-cell behavior, compromising tumor antigen presentation and accelerating immune evasion.
"However, despite this major rewiring and the significant phenotypic consequence of it, our understanding of how cytokines modify tumor behavior at the post-transcriptional level is still limited," the authors note. Analysis of cytokine responses has tended to focus on transcriptional regulation, and while some examples of post-transcriptional regulation of cytokine responses exist, there is still much to learn in this context.
Understanding the mechanisms underpinning PSR-driven regulation of cytokines and the subsequent influence of these cytokines on cellular rewiring would allow clinicians and researchers to develop novel translational or post-translational modalities that may arrest cancer metastasis and enhance its visibility to the host immune system, thereby increasing the efficacy of ICBs.
About the study
In the present study, researchers hypothesize the existence of a specific post-translational mechanism triggered by cytokines, resulting in ribosomal rewiring via P-stalk incorporation. They test this hypothesis in both in vitro human melanoma cell lines (Mel624 and M026) and in vivo murine models (female 6-8-week-old mice).
To validate the association between cytokine exposure and identify specific ribosomal proteins (RPs) involved in regulating tumor cell responses to cytokine exposure, a combination of flow cytometry (to detect ribosomal activation via the increased expression of human leukocyte antigens [HLA]) and proteomics (to evaluate expression levels of specific immunoproteasome subunits) was used. Sucrose density gradient centrifugation identified and isolated actively expressing ribosome populations, and liquid chromatography-mass spectrometry (LC-MS) computed the relative expression levels of different cell types.
The above analysis revealed that translational ribosomes across both melanoma cell lines significantly upregulated P1 protein levels as part of the P-stalk complex. Measurements of total-, polysome-, and sub-polysome P1 levels confirmed these findings. Experiment replication across various cancer types (ovarian, colorectal, and retinal) demonstrated that this mechanism is consistent across diverse tissues and tumor types, further supporting the generalizability of the PSR function.
Short hairpin RNA (shRNA) was used to knock down (KD) P1 expression, and the functional relevance (immune cell visibility) of PSRs was subsequently evaluated. The Cancer Genome Atlas Project (TCGA) database was leveraged to elucidate if observed findings were limited to the under-investigation population or could be generalized to several cancer types. Study findings revealed that these findings were generalizable, highlighting that PSRs selectively translate mRNAs involved in immunosurveillance, a key process in immune evasion.
"If antigens are correctly presented on tumor cells, they will be recognized by T cells, leading to their activation (measured by IFNγ and TNF-α production and CD107a cell surface expression)," the study explains. While KD of eL28 did not affect T-cell recognition, KD of P1 resulted in a strong and significant decrease in recognition by both MART-1 and NY-ESO-1-specific CD8+ T cells. A live imaging killing assay confirmed this finding, with shP1-expressing cells being far less efficiently killed by T cells compared with either shL28 or a scrambled control.
The main cytokines observed to mediate PSR action were interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). While the former was observed to activate PSR function, the latter was observed to inhibit PSR action. These contrasting occurrences (and their relative impacts) were evaluated by coculturing melanoma cell lines with T-cells and modulating concentrations of under-investigation cytokines.
Study findings
Prolonged PSR Activation: The study found that PSR activation by inflammatory cytokines (like IFN-γ and TNF-α) is delayed, occurring 48 hours after exposure, suggesting that these ribosomes are part of a long-term immune alert state rather than an immediate response.
The present study highlights four novel findings: Firstly, cytokines mediate the formation and activity regulation of the PSR. These can either be pro-inflammatory cytokines (which initiate PSR formation and activate the complex) or anti-inflammatory cytokines (which also trigger PSR formation but have the opposite effect on PSR activity). PSRs act as a critical regulatory hub where these opposing cytokine signals converge.
Secondly, the study reveals that PSR selectively translates key mRNAs that regulate antigen presentation and immune responses, particularly during cytokine exposure. Thirdly, these regulatory mechanisms are achieved via the phosphorylation of PSR proteins. Fourth, and most importantly, these results and observations are generalizable to several (if not all) cancer types and are independent of species (identical mechanisms were identified in murine and human cells).
"Our discovery also raises several additional questions. We have shown that P-stalk incorporation into the ribosome is regulated by both inflammatory (IFNγ/TNF-α, IFNɑ/IFNβ, IL-1β, IL-6, and IL17-A) and inhibitory (TGF-β) cytokines," the researchers explain. It will also be interesting to know how widespread this role of the PSR is. Our analysis of both mouse and human primary cells, as well as the TCGA datasets, suggests that cytokine response is regulated in this manner in a large number of tissues.
Conclusions
The present study identifies and validates PSR as a master regulator of cytokine responses and their associated cell rewiring, which plays a critical role in tumor immune evasion. It highlights post-translational mechanisms as the mediators of gene expression and underscores the generalizability of these findings across several cancer types and host species.
Together, these findings provide novel insights into the process of tumor T-cell escape, elucidate surprisingly low success rates of ICB interventions in some patients, and provide the groundwork for future therapeutic approaches targeting PSRs to improve immune-based cancer therapies.
Journal reference: Dopler, A., Alkan, F., Malka, Y., van der Kammen, R., Hoefakker, K., Taranto, D., Kocabay, N., Mimpen, I., Ramirez, C., Malzer, E., Isaeva, O. I., Kerkhoff, M., Gangaev, A., Silva, J., Ramalho, S., Hoekman, L., Altelaar, M., Beijersbergen, R., Akkari, L., ... Faller, W. J. (2024). P-stalk ribosomes act as master regulators of cytokine-mediated processes. In Cell. Elsevier BV, DOI - 10.1016/j.cell.2024.09.039, https://www.cell.com/cell/fulltext/S0092-8674(24)01139-5