Researchers at Fudan University Shanghai Cancer Center, writing in the journal Holistic Integrative Oncology, have assembled a comprehensive transcriptional landscape of prostate cancer progression by re-analyzing single-cell RNA sequencing data from 44 patient samples drawn from three prospective clinical trials. Their meta-cohort captured three clinically distinct stages of the disease: 13 samples of localized, treatment-naïve prostate cancer; 25 samples of hormone-sensitive prostate cancer obtained while patients were actively undergoing androgen deprivation therapy; and 6 samples of metastatic castration-resistant prostate cancer, collected from metastatic lesions in the prostate, lymph nodes, bladder, bone, and liver. The hormone-sensitive samples were particularly valuable, having been obtained 13 to 15 weeks after the initiation of a standardized ADT regimen, offering a snapshot of tumor cells in the act of adapting to therapeutic pressure rather than merely before or after it.

The technical challenge at the heart of the study was to reliably distinguish malignant epithelial cells from the benign cells that surround them within each tumor. The team applied a battery of quality-control filters, excluding cells with more than 25 percent mitochondrial gene expression, cells with fewer than 500 detected genes, and potential doublets exhibiting co-expression of incompatible lineage markers. Epithelial cells were identified by high expression of canonical markers such as EPCAM and KRT18, together with the absence of immune or stromal markers. The researchers then turned to inferCNVpy, a computational tool that infers single-cell copy number variation profiles by comparing each epithelial cell against a reference baseline built from 800 immune cells and 800 stromal cells. Clusters showing large-scale copy number aberrations consistent with genomic instability were classified as malignant, while those lacking such patterns were deemed normal. After these stringent steps, the final analytical dataset comprised 35,365 malignant tumor cells from 39 samples: 13 treatment-naïve, 22 hormone-sensitive, and 4 castration-resistant.

The first major finding concerns what happens inside tumor cells during the early months of ADT. Comparing malignant cells from hormone-sensitive samples with those from localized, untreated tumors, the researchers documented extensive transcriptional remodeling. Canonical androgen-responsive genes, including KLK3, the gene encoding the widely used clinical biomarker prostate-specific antigen, were significantly downregulated, confirming that the therapy was doing precisely what it was designed to do: suppressing androgen receptor signaling. Gene set enrichment analysis showed that the hallmark androgen response pathway was markedly suppressed in tumor cells under ADT, consistent with the known pharmacological effects of the treatment.

But the suppression of androgen signaling was only part of the story. In a finding with potentially far-reaching clinical implications, the team observed that ADT induced the activation of immune-associated transcriptional programs within the malignant cells themselves. Hallmark interferon-alpha and interferon-gamma response pathways were significantly enriched, alongside pathways involved in antigen processing and presentation. Over-representation analysis of the most upregulated genes in hormone-sensitive tumor cells further highlighted biological processes related to leukocyte differentiation, humoral immune response, and inflammatory signaling. In parallel, apoptosis-related pathways were significantly enriched, reflecting therapy-induced cellular stress and diminished survival signaling. Crucially, these immune programs were detected within the tumor cells themselves, independent of immune cell infiltration, indicating a direct transcriptional response of malignant cells to androgen deprivation rather than a secondary effect of a changing tumor microenvironment.

This observation aligns with a growing body of evidence suggesting that hormonal therapy can transiently enhance tumor immunogenicity. Prior work has shown that androgen receptor inhibition can increase the expression of major histocompatibility complex class I molecules on prostate cancer cells, potentially making them more visible to the immune system. The new single-cell data provide tumor-cell-intrinsic molecular support for this idea and carry an important practical implication: the timing of combination strategies that pair ADT with immune checkpoint inhibitors may matter enormously. If androgen deprivation opens a transient window of heightened immune visibility in tumor cells, then delivering immunotherapy during that window, rather than after resistance has fully established itself, could maximize therapeutic benefit. Clinical trials are already exploring this logic in metastatic castration-sensitive disease, and the new findings add molecular granularity to the rationale.

The second major finding shifts the focus to the endgame of the disease. When the researchers compared malignant cells from metastatic castration-resistant samples with those from localized, treatment-naïve tumors, they uncovered a strikingly different transcriptional profile, one dominated by genes involved in protein synthesis, mitochondrial function, and oncogenic signaling. Among the most prominently upregulated genes were ribosomal proteins such as RPL41 and RPSA, mitochondrial-encoded genes including MT-ND1 and MT-ND3, and FOLH1, the gene that encodes prostate-specific membrane antigen, or PSMA, a clinically important diagnostic and therapeutic target in advanced prostate cancer. These changes collectively point to an enhanced biosynthetic and metabolic capacity in castration-resistant tumor cells, a cellular state geared toward rapid growth under hostile conditions.

Pathway-level analysis crystallized this picture around a single dominant theme: MYC. Gene set enrichment analysis identified significant enrichment of MYC target gene sets in castration-resistant tumor cells, while androgen response pathways remained significantly downregulated relative to treatment-naïve samples. Over-representation analysis supported the same conclusion through an independent statistical route. To guard against the possibility that the finding was an artifact of their particular cohort, the researchers validated it in an independent dataset, GSE264573, and again found higher hallmark MYC pathway scores in tumor cells from castration-resistant samples compared with castration-sensitive ones. Together, these converging lines of evidence position MYC-driven transcriptional programs as a central feature of tumor cell adaptation in castration-resistant disease.

The emphasis on MYC is biologically coherent with prior mechanistic studies. In localized prostate cancer, MYC has been reported to cooperate with RAS signaling to promote bone metastasis and castration resistance, and a MYC/RAS co-activation signature has demonstrated predictive value across clinical cohorts. In neuroendocrine prostate cancer, an aggressive variant marked by loss of androgen receptor signaling, MYC activation has been associated with neuroendocrine differentiation, and alisertib, an Aurora-A kinase inhibitor that disrupts N-MYC stabilization, has shown clinical activity in a subset of neuroendocrine prostate cancer patients. Still, the authors are careful to note the limits of translation. MYC is not yet an established clinical biomarker in prostate cancer, its activity is not reliably captured by any single genomic alteration, and further studies are required to validate it as a robust prognostic and predictive tool. Moreover, other pathways, including PI3K/AKT signaling, may also contribute to castration resistance even if they were not prominently enriched in this dataset, reflecting the biological heterogeneity of metastatic disease, in which different tumors may rely on different dominant escape routes. The MYC-associated program identified here likely represents one major transcriptional state within a subset of patients rather than a universal mechanism.

The study also carries a molecular rationale for PSMA-targeted medicine. The significantly higher expression of FOLH1 in castration-resistant tumor cells provides single-cell-level support for the clinical efficacy of PSMA-directed therapies, most notably lutetium-177 PSMA-617 radioligand therapy, which has transformed the management of metastatic castration-resistant disease. The authors acknowledge the limitations inherent in their design: the number of castration-resistant samples was small, a constraint that reflects the genuine difficulty of obtaining metastatic specimens suitable for single-cell analysis, and functional validation will be needed to establish causal relationships between the observed transcriptional states and resistance. The analysis also did not systematically link tumor-cell changes to immune infiltration, clonal expansion, or T-cell receptor dynamics, information not available in the underlying dataset. Even so, by defining a transcriptional roadmap that spans from treatment-naïve disease through early hormonal therapy to full castration resistance, the study highlights two actionable features of tumor cell adaptation: a transient immune activation during ADT that could be exploited with timed immunotherapy combinations, and MYC-driven programs in resistant disease that may point toward new biomarker and drug development strategies aimed at delaying or preventing the emergence of lethal, treatment-resistant prostate cancer.

Subject of Research: Single-cell transcriptomic evolution of prostate cancer cells during androgen deprivation therapy and progression to castration resistance

Article Title: Single-cell analysis reveals tumor cell evolution from ADT-naïve to castration-resistant prostate cancer

Article References: Wei, Y., Wu, J., Wang, B., Gu, W., Qin, X., Dai, B., Lin, G., Gan, H., Wan, F., Zhu, Y., & Ye, D. (2026). Single-cell analysis reveals tumor cell evolution from ADT-naïve to castration-resistant prostate cancer. Holistic Integrative Oncology, 5(1), Article 39. https://doi.org/10.1007/s44178-026-00261-0

Image Credits: AI Generated

DOI: 10.1007/s44178-026-00261-0

Keywords: prostate cancer, single-cell RNA sequencing, androgen deprivation therapy, castration resistance, MYC signaling, PSMA, FOLH1, interferon response, tumor evolution, immunotherapy timing, androgen receptor, metastatic cancer

The clinical stakes are considerable. Renal cell carcinoma accounts for roughly 3.8 percent of adult cancer diagnoses, and clear cell carcinoma represents about 75 percent of those cases. According to the 2023 Cancer Statistics Report, the United States was projected to see 81,800 new kidney cancer cases and 14,890 deaths. Surgery remains the mainstay of treatment, but more than half of patients develop metastasis after nephrectomy, and the five-year survival rate for metastatic disease languishes below 12 percent. Against this grim backdrop, the search for molecular markers that can stratify risk and reveal actionable vulnerabilities has taken on real urgency.

RPL28 belongs to a family of proteins that assemble into ribosomes, the molecular factories that translate messenger RNA into protein. Eukaryotic ribosomes comprise a large 60S subunit containing roughly 46 ribosomal proteins and a small 40S subunit with about 33 more. Yet ribosomal proteins are increasingly recognized as multitaskers, engaging in extraribosomal functions that include regulating proliferation, apoptosis, DNA repair, and cell cycle control. Recent work has implicated them in tumorigenesis across multiple cancers, and the new study asked whether RPL28, located on chromosome 19q13.42, plays a similar role in kidney cancer, whose distinct molecular landscape of VHL-driven pseudohypoxia, metabolic reprogramming, and frequent chromosome 3p alterations could give ribosomal components entirely different significance than in colorectal or liver tumors, where RPL28 had previously drawn attention.

The researchers began by mining RNA sequencing data from The Cancer Genome Atlas, comparing 535 clear cell renal cell carcinoma specimens with 72 adjacent normal kidney tissues. RPL28 expression was markedly elevated in the tumors, a finding independently confirmed in the International Cancer Genome Consortium’s RECA-EU cohort of 91 tumors and 45 normal samples. Crucially, the team also validated the result at the protein level, using immunohistochemistry on paired tumor and normal tissue from 12 patients treated at Meizhou People’s Hospital, where the mean patient age was 62.3 years. A pan-cancer survey through the TIMER2.0 database further revealed RPL28 overexpression in breast, bladder, colon, liver, and lung cancers, positioning the protein as a broad marker of malignant behavior.

When the TCGA cohort was split into high- and low-expression groups at the median, the differences were striking. Elevated RPL28 correlated significantly with male sex, tumor presence, higher histological grade, advanced T stage, distant metastasis, and advanced TNM stage. Survival analysis drove the point home: patients with high RPL28 expression had significantly worse overall survival, with a hazard ratio of 1.5, and worse disease-specific survival, with a hazard ratio of 1.9. Multivariate Cox regression confirmed that RPL28 expression stood as an independent prognostic factor alongside histological grade and TNM stage. The team then built nomograms incorporating RPL28 to predict one-, three-, and five-year survival, achieving area under the curve values of 0.856, 0.798, and 0.753 respectively, outperforming any single clinical factor in both ROC and decision curve analyses.

The study also probed how RPL28 relates to the molecular subtypes defined by kidney cancer’s characteristic driver mutations. Among the genes examined, including VHL, PBRM1, BAP1, KDM5C, MTOR, TP53, PTEN, PIK3CA, and ARID1A, only SETD2 showed a significant association, with RPL28 expression significantly higher in SETD2-mutant tumors. Because SETD2 regulates chromatin and transcription, the authors caution that this exploratory finding is hypothesis-generating rather than proof of direct regulation, but it hints that RPL28 overexpression may arise within a specific epigenetic context rather than being a generic byproduct of the common 3p deletions that dominate the disease.

Given that clear cell renal cell carcinoma is among the most immune-infiltrated solid tumors and responds clinically to immunotherapy, the researchers examined whether RPL28 expression tracked with immune features. Using xCell algorithms adjusted for tumor purity, they found that RPL28-high tumors harbored significantly different levels of regulatory T cells, NKT cells, and mast cells. Checkpoint gene analysis added nuance: the RPL28-high group showed higher expression of PDCD1 and LAG3 but lower levels of CD274 and PDCD1LG2, the genes encoding PD-L1 and PD-L2. A pharmacogenomic analysis using CTRP-trained models further suggested that RPL28-high tumors might be more sensitive to several targeted agents, including axitinib, sunitinib, sorafenib, pazopanib, cabozantinib, and temsirolimus, though the authors stress these computational predictions await experimental validation.

The most compelling evidence came from the laboratory. The team silenced RPL28 in two clear cell renal cell carcinoma cell lines, 786-O and OSRC-2, using lentiviral short hairpin RNA, confirming efficient knockdown by quantitative PCR. Cell Counting Kit-8 assays showed that RPL28 silencing significantly impaired proliferation in both lines, while wound-healing assays, quantified over 24 hours in serum-reduced conditions to exclude proliferative effects, revealed a marked drop in migratory capacity. Gene set enrichment analysis supplied a mechanistic framework: elevated RPL28 expression was strongly associated with epithelial-mesenchymal transition, the developmental program that endows cancer cells with mobility and invasiveness, as well as with TGF-beta and MYC signaling, two pathways known to drive EMT. The correlation between RPL28 and EMT reached a coefficient of 0.335 and held across multiple cancer types in the pan-cancer analysis.

These results fit into a growing body of work showing that individual ribosomal proteins can steer cancer behavior. RPL23 has been linked to cisplatin resistance in ovarian cancer through EMT activation, RPS7 overexpression promotes growth and migration in prostate cancer, and RPL14 drives proliferation and invasion in nasopharyngeal carcinoma. The concept of ribosome heterogeneity, in which tumors assemble ribosomes with varying protein compositions to preferentially translate mRNAs supporting proliferation, stress adaptation, and therapeutic resistance, offers a plausible explanation for how a single ribosomal protein could exert such outsized influence in a tumor defined by metabolic and translational plasticity.

The authors are candid about the study’s limitations. Survival analyses rested primarily on a single cohort, immune infiltration was inferred computationally from bulk RNA sequencing, myeloid-derived suppressor cells were not directly estimated, drug sensitivity was predicted rather than tested, and the functional experiments were confined to cell cultures. Multicenter validation, in vivo models, and mechanistic dissection will be needed before RPL28 reaches the clinic. Nevertheless, the convergence of evidence, from overexpression in tumors to independent prognostic power to demonstrable effects on cancer cell proliferation and migration, makes RPL28 a rare example of a ribosomal component emerging as a credible biomarker and drug target in one of oncology’s most challenging cancers.

Subject of Research: The role of the ribosomal protein RPL28 in the progression, prognosis, and potential treatment of clear cell renal cell carcinoma

Article Title: Knockdown of RPL28 Inhibits the Progression of Clear Cell Renal Cell Carcinoma

Article References: Luo, R., Wu, G., Xie, Z., Xiao, Y., Zhu, W., & Jiang, H. (2026). Knockdown of RPL28 Inhibits the Progression of Clear Cell Renal Cell Carcinoma. Cancer Reports, 9(9), Article e70683. https://doi.org/10.1002/cnr2.70683

Image Credits: AI Generated

DOI: 10.1002/cnr2.70683

Keywords: RPL28, clear cell renal cell carcinoma, kidney cancer, ribosomal protein, prognostic biomarker, epithelial-mesenchymal transition, TCGA, SETD2, immune infiltration, drug sensitivity, TGF-beta signaling, MYC signaling