Androgen deprivation triggers a cytokine signaling switch to induce immune suppression and prostate cancer recurrence

Reviewer #1 (Public Review):

Summary:
Sha K et al aimed at identifying the mechanism of response and resistance to castration in the Pten knockout GEM model. They found elevated levels of TNF overexpressed in castrated tumors associated with an expansion of basal-like stem cells during recurrence, which they show occurring in prostate cancer cells in culture upon enzalutamide treatment. Further, the authors carry on a timed dependent analysis of the role of TNF in regression and recurrence to show that TNF regulates both processes. Similarly, CCL2, which the authors had proposed as a chemokine secreted upon TNF induction following enzalutamide treatment, is also shown to be elevated during recurrence and associated with the remodeling of an immunosuppressive microenvironment through depletion of T cells and recruitment of TAMs.

Strengths:

The paper exploits a well-established GEM model to interrogate mechanisms of response to standard-of-care treatment. This is of utmost importance since prostate cancer recurrence after ADT or ARSi marks the onset of an incurable disease stage for which limited treatments exist. The work is relevant in the confirmation that recurrent prostate cancer is mostly an immunologically "cold" tumor with an immunosuppressive immune microenvironment

Weaknesses:

While the data is consistent and the conclusions are mostly supported and justified, the findings overall are incremental and of limited novelty. The role of TNF and NF-kB signaling in tumor progression and the role of the CCL2-CCR2 in shaping the immunosuppressive microenvironment are well established.

On the other hand, it is unclear why the authors decided to focus on the basal compartment when there is a wealth of literature suggesting that luminal cells are if not exclusively, surely one of the cells of origin of prostate cancer and responsible for recurrence upon antiandrogen treatment. As a result, most of the later shown data has to be taken with caution as it is not known if the same phenomena occur in the luminal compartment.

Reviewer #2 (Public Review):

Summary:

In this study, Sha and Zhang et al. reported that androgen deprivation therapy (ADT) induces a switch to a basal-stemness status, driven by the TNF-CCL2-CCR2 axis. Their results also reveal that enhanced CCL2 coincides with increased macrophages and decreased CD8 T cells, suggesting that ADT resistance may be related to the TNF/CCL2/CCR2-dependent immunosuppressive tumor microenvironment (TME). Overall, this is a very interesting study with a significant amount of data.

Strengths:

The strengths of the study include various clinically relevant models, cutting-edge technology (such as single-cell RNA-seq), translational potential (TNF and CCR2 inhibitors), and novel insights connecting stemness lineage switch to an immunosuppressive TME. Thus, I believe this work would be of significant interest to the field of prostate cancer and journal readership.

Weaknesses:

(1) One of the key conclusions/findings of this study is the ADT-induced basal-stemness lineage switch driving ADT resistance. However, most of the presented evidence supporting this conclusion only selects a couple of marker genes. What exacerbates this issue is that different basal-stemness markers were often selected with different results. For example, Figure S1A uses CD166/EZH2 as markers, while Figure S1B uses ITGb1/EZH2. In contrast, Figure 1D uses Sca1/CD49, and Figure 2B-C uses CD49/CD166. Since many basal-stemness lineage gene signatures have been previously established, the study should examine various basal-stemness gene signatures rather than a couple of selected markers. Moreover, why were none of the stemness/basal-gene signatures significantly changed in the GO enrichment analysis in Figure 6A/B?

(2) A related weakness is the lack of functional results supporting the stemness lineage switch. Although the authors present colony formation assay results, these could be influenced simply by promoted cell proliferation, which is not a convincing indicator of stemness. To support this key conclusion, widely accepted stemness assays, such as the prostasphere formation assay (in vitro) and Extreme Limiting Dilution Analysis (ELDA) xenograft assay (in vivo), should be carried out.

(3) Another significant concern is that this study uses concurrency to demonstrate a causal relationship in many key results, which is entirely different. For example, Figure S4A and S4B only show increased CCL2 and TNF secretion simultaneously, which cannot support that CCL2 is dependent on TNF. Similarly, Figure 5A only shows that CCL2 increased coincidently with a rise in TNF, which cannot support a causal relationship. To support the causal relationship of this conclusion, it is necessary to show that TNF-KO/KD would abolish the increased CCL2 secretion.

(4) Some of the selective data presentations are not explained and are difficult to understand. For example, why does CD49 staining in Figure S3A have data for all four time points, while CD166 in Figure S3D only has data for the last time point (day 21)? Similarly, although several TNF_UP gene signatures were highlighted in Figure 4B, several TNF_DN signatures were also enriched in the same table, such as RUAN_RESPONSE_TO_TNF_DN. What is the explanation for these contrasting results?

Reviewer #3 (Public Review):

Summary:

The current manuscript evaluates the role of TNF in promoting AR targeted therapy regression and subsequent resistance through CCL2 and TAMs. The current evidence supports a correlative role for TNF in promoting cancer cell progression following AR inhibition. Weaknesses include a lack of descriptive methodology of the pre-clinical GEM model experiments and it is not well-defined which cell types are impacted in this pre-clinical model which will be quite heterogenous with regards to cancer, normal, and microenvironment cells.

Strengths:

(1) Appropriate use of pre-clinical models and GEM models to address the scientific questions.

(2) Novel finding of TNF and interplay of TAMs in promoting cancer cell progression following AR inhibition.

(3) Potential for developing novel therapeutic strategies to overcome resistance to AR blockade.

Weaknesses:

(1) There is a lack of description regarding the GEM model experiments - the age at which mice experiments are started.

(2) Tumor volume measurements are provided but in this context, there is no discussion on how the mixed cancer and normal epithelial and microenvironment is impacted by AR therapy which could lead to the subtle changes in tumor volume.

(3) There are no readouts for target inhibition across the therapeutic pre-clinical trials or dosing time courses.

(4) The terminology of regression and resistance appears arbitrary. The data seems to demonstrate a persistence of significant disease that progresses, rather than a robust response with minimal residual disease that recurs within the primary tumor.

(5) It is unclear if the increase in basal-like stem cells is from normal basal cells or cancer cells with a basal stem-like property.

1. In the Hi-MYC model, MYC expression is regulated by AR inhibition and is profoundly ARi responsive at early time points.

Reviewer #4 (Public Review):

In this manuscript by Sha et al. the authors test the role of TNFa in modulating tumor regression/recurrence under therapeutic pressure from castration (or enzalutamide) in both in vitro and in vivo models of prostate cancer. Using the PTEN-null genetic mouse model, they compare the effect of a TNFα ligand trap, etanercept, at various points pre- and post-castration. Their most interesting findings from this experiment were that etanercept given 3 days prior to castration prevented tumor regression, which is a common phenotype seen in these models after castration, but etanercept given 1 day prior to castration prevented prostate cancer recurrence after castration. They go on to perform RNA sequencing on tumors isolated from either sham or castrate mice from two time points post-castration to study acute and delayed transcriptional responses to androgen deprivation. They found enrichment of gene sets containing TNF-targets which initially decrease post-castration but are elevated by 35 days, the time at which tumors recur. The authors conduct a similar set of experiments using human prostate cancer cell lines treated with the androgen receptor inhibitor enzalutamide and observe that drug treatment leads to cells with basal stem-like features that express high levels of TNF. They noticed that CCL2 levels correlate with changes in TNF levels raising the possibility that CCL2 might be a critical downstream effector for disease recurrence. To this end, they treated PTEN-null and hi-MYC castrated mice with a CCR2-antagonist (CCR2a) because CCR2 is one receptor of CCL2 and monitors tumor growth dynamics. Interestingly, upon treatment with CCR2a, tumors did not recur according to their measurements. They go on to demonstrate that the tumors pre-treated with CCR2a had reduced levels of putative TAMs and increased CTLs in the context of TNF or CCR2 inhibition providing a cellular context associated with disease regression. Lastly, they perform single-cell RNA sequencing to further characterize the tumor microenvironment post-castration and report that the ratio of CTLs to TAMs is lower in a recurrent tumor.

While the concepts behind the study have merit, the data are incomplete and do not fully support the authors' conclusions. The author's definition of recurrence is subjective given that the amount of disease regression after castration is both variable (Figure 8) and relatively limited, particularly in the PTEN loss model. Critical controls are missing. For example, both drug experiments were completed without treating non-castrate plus drug controls which raises the question of how specific these findings are to castration resistance. No validation was performed to ensure that either the TNF ligand trap or the CCR2 agonist was acting on target. The single-cell sequencing experiments were done without replicates which raises concern about its interpretation. At a conceptual level, the authors say that a major cause of disease recurrence in the immunosuppressive TME, but provide little functional data that macrophages and T cells are directly responsible for this phenotype. Statistical analyses were performed on only select experiments. In summary, further work is recommended to support the conclusions of this story.