Novel insights into molecular landscape of advanced renal cell carcinoma

Introduction

Renal cell carcinoma (RCC) is one of the 10 most common human cancers accounting for 2–4 % of all malignant diseases in adults, and up to 30 % of cases it is diagnosed in an advanced or metastatic stage [1]. In recent years, the increasing application of high-throughput technologies has resulted in the discovery of new therapeutic targets and a deeper understanding of the molecular basis underlying the development and progression of RCC. Numerous studies have shown that altered metabolism is involved in the development of clear cell renal cell carcinoma (ccRCC) – the most common subtype of RCC – and that many altered genes play a fundamental role in controlling cell metabolic activities in this tumor type [2], 3].

Metabolic landscape of renal cell carcinoma

RCC is a tumor characterized by immune and metabolic heterogeneity. Metabolic flux through glycolysis is differentially regulated and mitochondrial bioenergetics and oxidative phosphorylation are compromised [2]. Increased levels of metabolites in the upper chain of glycolytic reactions and a reduction in the lower part of glycolysis have been described [2]. This suggests differential partitioning of metabolic flux through glycolysis, rerouting sugars to the pentose phosphate pathway and triose phosphates to the Krebs cycle or one-carbon metabolism (Figure 1). The impaired mitochondrial bioenergetics and increased glucose utilization through the pentose phosphate pathway in ccRCC indicate that oncogenic signaling pathways promote cancer through altered glucose metabolism.

Figure 1:

Metabolic alterations of sugar metabolism in clear cell renal cell carcinoma. Modified from [2]

NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4-like 2 (NDUFA4L2) is a HIF-1 target gene highly expressed in ccRCC. It plays a pivotal role in bioenergetics, cell proliferation, migration, and angiogenesis. Knockdown of NDUFA4L2 decreased cell viability, improved chemotherapy drug susceptibility, inhibited autophagy, increased mitochondrial mass, and induced reactive oxygen species overproduction, particularly under hypoxic conditions [2]. These findings suggest that NDUFA4L2 regulates chemotherapy resistance in ccRCC and impacts mitochondrial function (Figure 2).

Figure 2:

Overexpression of NDUFA4L2 inhibits the mitochondrial complex I and blocks the electron flux through the electron transport chain of oxidative phosphorylation, promoting angiogenesis, chemoresistance, and mitophagy. Modified from [2]

Lipid metabolism is crucial in RCC pathogenesis. De novo lipogenesis, elongation, and desaturation processes contribute to the production of lipids essential for tumor growth and progression. Dysregulation of enzymes such as stearoyl-CoA desaturase (SCD1) and elongation of very long chain fatty acids proteins (ELOVLs) has been associated with enhanced cancer cell survival and migration, highlighting potential therapeutic targets [3].

Metabolomic profiling has shown promise in distinguishing RCC subtypes, staging tumors, and predicting drug responses. Future perspectives should focus on the standardization of methodologies for integrated multi-omics studies to advance the understanding of RCC pathways and optimize clinical applications, particularly in biomarker detection for disease management and treatment efficacy assessment.

Metabolic reprogramming has been associated with ccRCC progression. In a recent study, intricate intratumor heterogeneity (ITH) emerged across the genomic, transcriptomic, and metabolomic levels [4]. A distinct subgroup, termed de-clear cell differentiated (DCCD)-RCC, was identified and characterized by the absence of lipid droplets (LDs), enhanced nutrient uptake, different metabolic profiles, and a high proliferation rate. In ccRCC, HIF2A- and PLIN2-driven LD accumulation was associated with good prognosis in clinical cohorts. Indeed, LDs might restrict endoplasmic reticulum stress and provide energy through fatty acid oxidation pathways, potentially explaining the increased response to tyrosine kinase inhibitor (TKI) treatment in these tumors. The DCCD tumors showed increased metastatic potential, thus suggesting the need for extended surgical resection and post-nephrectomy targeted drug treatment, even at early stages given the poor prognosis. The study also evaluated treatment strategies and indicated that combined therapy with immune checkpoint blockade improved outcomes for DCCD patients, while TKI monotherapy was effective for patients without DCCD features. Furthermore, molecular subtyping was proposed as a guide for second-line treatment, with nivolumab (a PD-1 inhibitor) showing efficacy comparable to that of everolimus (an mTOR inhibitor) in a meta-checkmate cohort with combined RNA-seq data from CheckMate 009, 010 and 025. Overall, this study sheds light on a unique subtype of ccRCC with distinct metabolic features, offering new avenues for treatment in patients with treatment-resistant RCC [4].

Molecular landscape of tumor thrombus in RCC

Metastasis is a critical aspect of RCC progression, particularly through the formation of a tumor thrombus (TT) within the renal veins. Recent studies have highlighted the barriers faced in addressing RCC with TT, such as inter- and intratumoral heterogeneity, patient-specific treatment modifications, and different patterns of treatment resistance between the TT and the primary tumor [5], [6], [7]. Furthermore, temporal heterogeneity between the primary tumor, TT, and distant metastases was explored, along with the functional phenotypes of components in the tumor microenvironment (TME). Recent advancements in understanding genetic and nongenetic variations in tumors have led to a deeper understanding of tumor evolution and heterogeneity within the TME.

Multiregional sequencing is a valuable tool for studying the spatiotemporal intratumoral heterogeneity of RCC, providing insights into evolutionary trajectories and guiding targeted therapies. In a study by Kim et al. [5], a cohort of 83 RCC with TT patients was recruited to investigate the molecular events involved in the invasion mechanisms by comparing TT to primary RCCs. This study used a combination of multiregional genome sequencing, histological evaluation, and in vivo functional studies. Besides comparable overall mutational burden between TTs and primary tumors, similar mutation frequencies were observed in driver genes: VHL (74.3 % in primary tumor vs. 65.9 % in TT), PBRM1 (35.2 % vs. 23.7 %), SETD2 (14.3 % vs. 22.2 %), BAP1 (16.5 % vs. 20.0 %), PTEN (8.8 % vs. 11.9 %), and CSMD3 (6.6 % vs. 10.4 %). Four patterns of clonal evolution were found in this study, with 55.1 % of cases showing private driver genes in primary tumors and TTs, indicating divergent pathways. In some cases, driver gene mutations were exclusive to either TTs or primary tumors, while in others, the mutations were shared, suggesting coevolution or clonal expansion. Different evolutionary patterns were observed, including heterogeneous branching (45 %), linear homogeneous (35 %), and linear heterogeneous (20 %). This comprehensive analysis provides new insights into the genetic differences and evolutionary trajectories between TT and primary RCC.

In renal cell carcinoma with TT, the invasive pattern represents a unique challenge that warrants further exploration of the cellular and molecular mechanisms driving invasiveness. By transcriptomic analysis, group of transcription factors, including genes encoding elements of the AP-1 complex (FOSB, FOS, and JUNB and early growth response transcription factors), were found to be differently regulated between primary tumor and TT. These genes promote tumor invasion by inducing cellular plasticity through cyclic adenosine monophosphate-responsive element-binding protein (CREB). Enhanced CREB activity was associated with increased expression of immediate early genes and invasion in RCC cell lines. Additionally, the overexpression of the TGF-β-responsive gene PRRX1 in invasive TTs, along with TWIST1 (the epithelial–mesenchymal transition (EMT)-promoting transcription factor), contributes to cell invasion and dissemination. Furthermore, the upregulation of WT1 and cell cycle controlling genes in TTs compared to those in primary tumors, as well as in recurrent cases, suggests their involvement in tumor growth and potential as therapeutic targets. Pathway enrichment analysis indicated a significant contribution of these genes to EMT-associated pathways. Overall, the expression of immediate early genes modulated by the CREB/AP-1 pathway and the activation of EMT-related signaling pathways facilitate TT cell migration and tumorigenesis, highlighting the invasive nature of RCC with TT [6], 7].

Cellular landscape of tumor microenvironment in RCC

RCC is characterized by widespread infiltration of immune system cells combined with the activation of particular metabolic pathways regulating inflammatory response and angiogenesis [8], 9]. Moreover, features of the TME strongly affect cancer progression and may influence the response to systemic therapy [10].

In this scenario, The Cancer Gene Atlas database was adopted to define three multiomics subtypes (MoS1, MoS2, and MoS3) of ccRCC with different clinicopathological features and oncological outcomes [11]. The poorest prognosis was exhibited by the immune exhausted subtype MoS1 (plasma cells, memory B cells, CD4+ and CD8+T cells, and M0 macrophages). MoS2 had better overall survival and progression-free interval time. It was defined as the immune “cold” subtype because of the infiltration of proinflammatory M1 macrophages, γδ T cells, and eosinophils. MoS3 is the immune “hot” subtype which showed the highest infiltration of anti-inflammatory M2 macrophages and monocytes. As a result, different therapeutic strategies were suggested for MoSs subtypes: PI3K/AKT inhibitors for MoS1, sunitinib (a TKI) for MoS2 and anti-PD1 therapy for MoS3.

The use of spatiotemporal multi-omics technology with single-cell resolution holds promise for revealing the heterogeneity of RCC with TT and identifying key cellular populations influencing treatment efficacy. Understanding the molecular biology of RCC with TT is crucial for developing more precise and effective therapeutic strategies to improve long-term survival outcomes for patients with this highly malignant form of cancer.

In the context of TT treatment, tumor-infiltrating lymphocytes (TILs) seem to influence treatment response and prognosis. A study by Shi et al. identified various subpopulations of CD4+ T cells, CD8+ T cells, and NK cells in RCC with TT [12]. The authors reported a decrease in naive CCR7+ T cells and an increase in regulatory FOXP3+ T cells observed in primary tumors, pointing to an immunosuppressive microenvironment in the primary tumor. CD8+ T cells were categorized into tissue-resident, terminal exhaustion, circulating, and mucosa-associated invariant T cells, with an increase in resident cells in TTs compared to those in primary tumors. Then, a larger amount of exhausted CD8 T cells were described in primary tumors and TTs. Functional pathway analysis revealed an increased T-cell activation and lymphocyte differentiation at earlier stages, while cell cycle, ATP metabolism, and hypoxia-related pathways were enriched in the end stage of CD8+ T cells, indicating a transition to exhaustion in both primary tumors and TTs.

Tumor-associated macrophages (TAMs) along with other innate immune cells in the TME contribute to TT formation and resistance to immune checkpoint inhibitor (ICI) therapy. Single-cell RNA sequencing analysis revealed significant heterogeneity among myeloid cells in RCC patients with TT, with a higher number of TAMs in primary tumors and TTs compared to normal tissues. TAMs can differentiate into proinflammatory M1 and anti-inflammatory M2 subtypes, but distinguishing between these subtypes in RCC-TT based on known gene signatures has been challenging. TAMs in primary tumors showed enrichment in response to interferon-α/γ and antigen presentation pathways, indicating a proinflammatory phenotype, while also upregulating immune checkpoint and evasion markers associated with anti-inflammatory functions. This finding suggested that TAMs in RCC with TT exhibit both pro- and anti-inflammatory characteristics, impacting the TME and therapeutic responses. Additionally, TAMs were found to enhance extracellular structure organization and angiogenesis in tumor thrombi, highlighting their differential functions in primary tumors and TTs [13]. These findings suggest a complex interplay of immune cells within the tumor microenvironment, emphasizing the need for a comprehensive understanding to develop effective therapeutic strategies.

Conclusions and Future Prospects

Due to the lack of curative medical treatments for RCC, surgery remains the mainstay of therapy. In particular, the gold standard treatment for renal cancer with TT is complete surgical excision [14], [15], [16]. This surgery is complex and challenging for the surgeon, especially in the case of a tumor with a large neoplastic thrombus extending into the inferior vena cava and right atrium. Therefore, understanding the molecular mechanisms underlying neoplastic progression and the development of tumor thrombi is of the utmost importance. The progressive development of high-throughput technologies, the multi-omics data integration, and the introduction of spatiotemporal single-cell analysis, have provided a detailed picture about regulation processes of cancer cell metabolism, and novel molecular pathways are under investigation. In addition, the recent findings about the modulation of the TME and alterations in systemic immunity described in patients with advanced RCC, are defining the cellular dynamics underlying different responses to immunotherapy. The identification of diagnostic and prognostic molecular factors will play a prominent role in this tumor considering that to date up to 30 % of cases are diagnosed at an advanced stage and currently we have no specific biomarker that may help in risk stratification and therapeutic decision-making of patients with kidney cancer.