Abstract

Inactivation of the VHL gene, a master regulator of hypoxia-inducible factors (HIF), represents a critical event in the oncogenesis of clear-cell renal cell carcinoma (ccRCC), the most lethal histological subtype of kidney cancer. However, VHL loss alone is insufficient for malignant transformation, requiring secondary genetic alterations. Frequently, this secondary hit involves PBRM1, which encodes a pivotal subunit of the PBAF (polybromo-associated BAF) chromatin-remodeling complex. PBRM1 is fundamental to cellular homeostasis, modulating diverse metabolic pathways, cell adhesion, the hypoxic response, and cell-cycle progression. Consequently, a single somatic mutation in PBRM1 can significantly drive tumorigenesis. Dysregulation of this PBAF subunit alters the transcriptional landscape, notably inducing constitutive activation of the NF-κB signaling pathway. This upregulation promotes aberrant cell proliferation and accelerates tumor progression, highlighting the synergistic role of VHL and PBRM1 in ccRCC pathogenesis.

Content

Physiologically, PBRM1 is a human gene located on the short arm of chromosome 3p21. It encodes the BAF180 PBRM1 subunit, a critical component of the PBAF complex. As a prominent member of the SWI/SNF family, this complex specializes in chromatin remodeling by utilizing ATP hydrolysis as an energy source. ^{1 2 3}   The complex assembles from several proteins, including BAF180 PBRM1, BRD7, and SMARCA4, which serves as the catalytic ATPase subunit of PBAF. Chromatin remodeling facilitates essential genomic processes, including DNA replication, transcription, and repair, while also modulating cell proliferation. Furthermore, the PBAF complex contributes to the mitotic spindle checkpoint, ensuring rigorous control over cell division and chromosomal stability. ⁴

ROLES OF PBRM1 IN PHYSIOLOGICAL CONDITIONS
Wild-type PBRM1 functions as a tumor suppressor by regulating cell cycle progression through CDKN1A, thereby limiting cell proliferation. It is involved in the down-regulation of approximately 30 genes associated with the G1/S transition, along with numerous others that facilitate cell cycle arrest in the G1/G0 phase. ^{1 3}   Additionally, PBRM1 inhibits tumor growth by up-regulating STAT1 and promoting cohesin recruitment to centromeres, which prevents genomic instability. ^{1 5} It also mediates the transcriptional silencing of genes adjacent to DNA double-strand breaks and coordinates their repair through the accumulation of the H2AK119ub histone modification at the damage site. ¹ PBRM1 further acts as a key regulator of cellular metabolism. It governs carbohydrate metabolism by inhibiting genes encoding enzymes and receptors involved in glycolysis. This activity effectively modulates the insulin/PI3K signaling pathway, which would otherwise drive excessive proliferation. ^{6 5 7} Moreover, it serves as an effector of cholesterol homeostasis, maintaining low levels of oleate cholesterol relative to linoleate cholesterol, thereby preserving the physiological lipid composition characteristic of normal renal tissue. ^{6 5}   In addition, PBRM1 up-regulates genes associated with cell adhesion by reinforcing the cortical actin cytoskeleton, which inhibits tumor migration and the metastatic cascade. ^{6 8} Finally, PBRM1 is implicated in the regulation of apoptotic pathways, interferon responses, and cellular stress management. ⁶

PBRM1 AND THE GENES OF HYPOXIA
The relationship between PBRM1 and the hypoxic response is complex. PBRM1 regulates a specific subset of hypoxia-responsive genes; specifically, unmutated PBRM1 promotes the expression of HIF1A, which, together with ARNT, forms the heterodimeric HIF complex. While HIF1A is ubiquitous and highly sensitive to oxygen fluctuations, ARNT is constitutively expressed and primarily facilitates the assembly of the complex. Under hypoxic conditions, the heterodimer binds to Hypoxia Response Elements (HRE) in the DNA, up-regulating genes such as IGFBP1. This gene supports cell survival under hypoxic stress by modulating metabolism, angiogenesis, proliferation, and apoptosis, allowing for short-term cell cycle progression. Under conditions of extreme stress, however, it activates pathways related to cell cycle arrest, apoptosis, and the suppression of inflammation. ^{6 9 10}   Consequently, as a necessary factor for the transcription and activity of HIF1A, PBRM1 functions as a tumor suppressor. ^{9 11} Conversely, the activation of the oncogene VEGFA, which promotes tumor progression under hypoxia, appears independent of PBRM1 status. This suggests that PBRM1 is not essential for the activation of all hypoxia-related factors. ⁶

STRUCTURE OF BAF180 PBRM1 AND COMPLEX SWI/SNF
Regarding the architecture of BAF180 PBRM1, the protein contains an HMGB-like DNA-binding domain, two Bromo-Adjacent Homology (BAH) domains, essential for protein-protein interactions with other PBAF subunits, and six Bromodomains (BD). These BDs are specialized modules that recognize and bind acetylated lysine residues on the N-terminal tails of histones. Notably, mutations in BRD2 and BRD4 can impair the interaction between BAF180 PBRM1 and chromatin. ^{2 9 12}   Genomic mapping has revealed that PBRM1 and BRD7 operate coordinately on DNA, forming a functional unit within the PBAF complex. This unit binds downstream of the Transcription Start Site (TSS), predominantly between the +1 and +2 nucleosomes at the promoter level. The bromodomains specifically recognize the H3K14ac histone acetylation. ^{1 4 9} This mechanism functions independently of ARID2, which typically localizes upstream of the TSS and is frequently associated with enhancer regions. ¹

Figure 1 | Structural subunits of the PBAF complex. PBAF is a high-molecular-weight chromatin remodeling complex in eukaryotes, typically comprising 12–15 subunits. Key components include the defining subunit BAF180 PBRM1, along with BAF200 ARID2, BRD7, and the catalytic ATPase subunit SMARCA4.

SOMATIC MUTATIONS OF PBRM1
Cytogenetic rearrangements frequently trigger chromosomal instability. In clear-cell renal cell carcinoma (ccRCC), these alterations typically involve large-scale somatic events, such as the gain or loss of whole chromosomes or entire chromosome arms, rather than focal mutations. ¹³ Exome sequencing has identified somatically acquired mutations in PBRM1 that are specific to malignant cells, encompassing both coding sequence variants and splicing site alterations. These mutations characteristically occur in conjunction with the loss of heterozygosity (LOH) of chromosome 3p. ^{2 4}   Missense and nonsense mutations are the most prevalent; among the latter, truncating mutations are particularly frequent, resulting in a shortened BAF180 PBRM1 protein with compromised functional integrity. ² A widespread deletion involves an isoleucine codon (Ile 57) in the first bromodomain, which impairs the protein's ability to interact effectively with chromatin. Another recurrent mutation is an in-frame deletion of six amino acids in the second BAH domain, which disrupts protein-protein interactions within the PBAF complex. Furthermore, numerous frameshift mutations, arising from deletions, insertions, or in-frame alterations, lead to significant shifts in the amino acid sequence of BAF180 PBRM1. ^{2 4 12}   Specific missense mutations (e.g., p.T232P, p.A597D, and p.H1204P) have been identified as deleterious, as they localize to critical functional domains of BAF180 PBRM1. Interestingly, truncating mutations of PBRM1 have also been documented in breast cancer. Collectively, these data consolidate the role of PBRM1 as a vital tumor suppressor. ² Physiologically, PBRM1 modulates pathways associated with chromosomal stability and cell proliferation; for instance, it regulates CDKN1A, a key mediator of cellular senescence and growth control. ².

Figure 2 | Somatic mutations of PBRM1. The PBRM1 transcript encodes six bromodomains (BD1-6), two bromo-adjacent homology domains (BAH1-2), and an HMG-like binding domain (HMG). Stars denote nonsense mutations, dots represent missense mutations, and triangles indicate frameshift mutations (red for deletions, blue for insertions). Green triangles represent in-frame deletions.

PATHWAY NF-KB
NF-κB refers to a family of transcription factors essential for cellular homeostasis. Dysregulation or mutation of this pathway activates multiple cellular effectors that drive hallmark features of cancer, including accelerated proliferation, epithelial-mesenchymal transition (EMT), angiogenesis, evasion of apoptosis, and chronic inflammation. ¹⁴

NF-KB PROTEINS SUBUNITS
The family consists of five subunits: p65 RELA, RELB, REL, p50 NFKB1, and p52 NFKB2. These subunits assemble into various hetero- and homodimers to form distinct NF-κB complexes, each targeting specific gene sets. ^{15 16} Rel subunits possess both a DNA-binding domain (DBD) and a transcriptional activation domain (TAD), the latter being necessary for initiating transcription. ¹⁵ Conversely, the p50 and p52 subunits lack a TAD; therefore, they must dimerize with a Rel subunit to activate gene expression. When binding to DNA as homodimers, they typically act as transcriptional repressors. ¹⁶ The most prevalent heterodimer is NFKB1-RELA, which functions as a potent transcriptional enhancer for NF-κB target genes. ¹⁶   The family is divided into two subfamilies based on their C-terminal structures: the NF-κB proteins (p105 NFKB1 and p100 NFKB2), which are proteolytically processed into p50 NFKB1 and p52 NFKB2 respectively, and the Rel proteins. The precursor proteins contain ankyrin repeats (ANK) that inhibit transcriptional activity, whereas Rel proteins feature C-terminal transactivation domains. ¹⁷ All subunits share an N-terminal Rel homology domain (RHD), which mediates dimerization, nuclear translocation via a nuclear localization sequence (NLS), and binding to κB genomic sites to modulate transcription. ^{17 16}.

Figure 3 | Dimer subunits of NF-κB proteins. Five distinct subunits associate into heterodimers or homodimers to form functional NF-κB complexes. Their specific composition determines whether they activate or suppress target gene transcription.

NF-KB ACTIVATION PATHWAYS
NF-κB activation occurs through two primary pathways. The canonical (classical) pathway is triggered by pro-inflammatory cytokines and stress stimuli; for example, TNF-α binds to its receptor to induce the expression of CFLAR, a protein that regulates apoptosis and necroptosis to promote cell survival. Other activators include LPS and IL1B. ^{14 18 19}   The non-canonical (alternative) pathway is activated by a specific subset of ligands, primarily members of the TNF receptor superfamily that lack death domains, such as CD40, LTB, and TNFSF13B. ^{20 19} Various surface receptors, including TLR4 and ILR1, are responsible for transducing these signals. ^{17 19}   In the canonical pathway, ligand binding triggers a kinase cascade culminating in the activation of the IKK complex. This complex phosphorylates NFKBIA (an NF-κB inhibitor), leading to its degradation via the ubiquitin-proteasome system. Simultaneously, the proteolysis of ANK repeats occurs. These inhibitory elements normally sequester NF-κB complexes in the cytoplasm; their removal allows the dimers to translocate into the nucleus. ^{17 18 19} In the alternative pathway, ligand binding activates the kinase MAP3K14 (NIK), which phosphorylates the IKK complex, specifically activating the CHUK (IKKα) subunit. This triggers the processing of p100 NFKB2 into p52 NFKB2, enabling dimer formation. ²⁰ Once in the nucleus, NF-κB proteins bind to κB sites through their RHD to activate genes encoding inflammatory cytokines, chemokines, cell cycle modulators, and angiogenic factors that foster tumor development. ¹⁷

LOSS OF PBRM1 AND HYPER-ACTIVATION OF NF-KB
Mutations in PBRM1 typically result in the functional instability or loss of the BAF180 PBRM1 protein rather than the transcriptional silencing of the gene itself. ⁶ When PBRM1 is mutated, the interaction between BRD7 and SMARCA4 is compromised, destabilizing the PBAF complex. ¹ This leads to reduced association between the core subunits SMARCC1, SMARCB1, and ACTL6A. ¹ Interestingly, the association between SMARCA4 and other SWI/SNF subunits, such as ARID2, remains intact because SMARCA4 is spatially distant from BAF180 PBRM1. Consequently, the complex does not fully disassemble, but its functional integrity is profoundly altered. ^{1 21}   With mutated PBRM1, the PBAF complex exhibits aberrant DNA-binding patterns. ¹ SMARCA4 and ARID2 begin to bind more intensely to novel genomic regions; specifically, SMARCA4 reallocates from promoters to enhancers. ^{1 21} BRD7 also demonstrates altered binding affinities, showing decreased association with SMARCA4. ¹

HYPERACTIVATION OF NF-KB
The suboptimal synthesis of PBAF and the redirection of the remaining complexes to incorrect genomic sites, particularly NF-κB enhancers, drive the pathological phenotype. PBRM1 mutations primarily induce gene up-regulation, which subsequently hyperactivates NF-κB signaling. ^{1 20} While SMARCA4 is required to maintain NF-κB target gene activity in both wild-type and mutant cells, its role becomes critical in the presence of PBRM1 mutations. In these cases, SMARCA4 targets NF-κB enhancers, increasing chromatin accessibility for the RELA subunit. This sustained binding of RELA to DNA is essential for the constitutive activation of the NF-κB pathway. ¹   Loss of PBRM1 thus amplifies the activity of numerous RELA target genes. Even in physiological conditions, the SWI/SNF complex is indispensable for NF-κB binding to the HIF1A promoter, thereby modulating the transcription of hypoxia-related genes. ¹¹ ARID2 further contributes to this hyperactivation by binding to enhancers containing κB sites, where it overlaps with RELA. Conversely, the loss of ARID2 would impair colony-forming ability and further destabilize the PBAF complex. ¹ Meanwhile, BRD7 primarily targets regions regulating the UV response, apical junctions, and Wnt signaling. ¹

INVOLVEMENT IN CLEAR-CELL RENAL CARCINOMA
Renal cell carcinoma is among the ten most prevalent cancers in the United States, with clear-cell renal cell carcinoma (ccRCC) representing the most lethal subtype. The "clear cell" designation stems from the high accumulation of glycogen and lipids, reflecting a metabolic shift toward the Warburg effect. ^{6 22 23} Genetic analysis has revealed a complex landscape of at least 19 significantly mutated genes, resulting in high intra-tumoral heterogeneity. ¹³   A fundamental prerequisite for ccRCC development is the loss of the VHL (Von Hippel-Lindau) tumor suppressor gene on chromosome 3p25. This gene encodes pVHL VHL, an E3 ubiquitin ligase responsible for the degradation of HIF transcription factors (HIF1A and EPAS1) under normoxic conditions. VHL mutations can be hereditary and predispose individuals to ccRCC, though they are insufficient for full malignancy, typically leading only to preneoplastic cysts. ^{24 25 26}   Other genes mutated in less than 15% of ccRCC cases include KDM6A, KDM5C, BAP1, and SETD2, which encode epigenetic enzymes that modulate histone H3 methylation and chromatin accessibility. ^{2 27 28} Notably, PBRM1 is the second most frequently mutated gene in ccRCC (~40% of cases), following VHL. In cells lacking VHL, the subsequent loss of PBRM1 specifically disrupts the PBAF complex while preserving BAF and ncBAF complexes. ²⁹   The physical proximity of these genes on chromosome 3p likely explains the high frequency of LOH in this region. ² Based on these genetic profiles, ccRCC can be classified into subtypes: the ccA subtype (associated with better prognosis) often involves the m1 group of chromatin remodeling genes (primarily PBRM1), while the ccB subtype involves m2/m3 genes such as PTEN. A fourth group (m4) involves DNA repair genes. ¹³ Metabolic alterations in ccRCC are stage-dependent and hallmark features of the disease. ¹³

CONSEQUENCES OF MUTATIONS
The landscape of PBRM1-related mutations is heterogeneous, giving rise to various clinical subtypes. ⁶ These alterations drive tumor progression and metastasis by helping cells overcome replicative, oxidative, and nutritional stress via the HIF response, making the tumor highly dependent on neoangiogenesis. Consequently, tumors with PBRM1 alterations often show increased sensitivity to anti-angiogenic therapies. ¹   In ccRCC, the loss of VHL creates a state of pseudo-hypoxia; the absence of functional pVHL VHL prevents the degradation of HIF factors, leading to the stabilization of HIF1A. ^{25 26} The loss of PBRM1 integrity further distorts the epigenetic landscape. Since the SWI/SNF complex is required for NF-κB binding to HIF promoters, its alteration disrupts the balance between HIF1A and EPAS1 (HIF-2α) in favor of the latter. EPAS1 overexpression is a critical driver of malignancy in VHL syndrome, stimulating genes that promote angiogenesis, metabolic reprogramming, apoptosis inhibition, and metastasis. ^{11 10}   ccRCC is further characterized by suppressed Krebs cycle activity and up-regulated aerobic glycolysis (the Warburg effect), supported by the HIF response and the PI3K signaling pathway. This metabolic axis represents a promising therapeutic target. ^{6 7 23} Additionally, cancer cells exhibit an increased reliance on the pentose phosphate shunt. ^{13 30} The characteristic "clear cell" morphology is due to massive lipid accumulation, specifically an exponential increase in oleate cholesterol and a decrease in linoleate cholesterol. Enhanced fatty acid production and glutamine-dependent lipogenesis further define this metabolic phenotype. ^{13 30} Finally, during EMT, cells lose apico-basal polarity and undergo actin cytoskeleton remodeling, forming dynamic lamellipodia that facilitate invasion and systemic spread. ³¹.

Figure 4 | The VHL-HIF-VEGF axis. The illustration depicts the consequences of EPAS1 accumulation under hypoxic conditions. Hypoxia leads to the loss of prolyl hydroxylase activity, preventing the binding of pVHL VHL to EPAS1. This inhibits oxygen-dependent ubiquitination and subsequent proteasomal degradation, resulting in the stabilization of the transcription factor.

CONCLUSIONS
In conclusion, we have demonstrated the critical importance of the PBRM1 gene, which encodes a fundamental subunit of the PBAF complex. Its loss compromises the structural integrity of the complex and its ability to accurately target specific genomic regions. This dysregulation leads to the pathological hyperactivation of the NF-κB signaling pathway, which plays a determinant role in the progression of ccRCC, a malignancy typically initiated by primary genetic alterations, most notably the loss of VHL.   Elucidating these molecular mechanisms is essential for the development of targeted therapeutic strategies. Given the significant heterogeneity of this carcinoma, pharmacological intervention must focus on specific components of the PBAF complex or the NF-κB pathway. For instance, the targeted inhibition of RELA could mitigate clonal proliferation and tumor expansion. Additionally, the clinical application of angiogenesis inhibitors remains a highly effective strategy in managing this disease.   Physiologically, the NFKBIA protein family sequesters NF-κB in the cytoplasm, preventing its activation. Upon signaling, NFKBIA is targeted for proteasomal degradation, thereby releasing NF-κB. Consequently, one promising therapeutic approach involves the use of Bortezomib, a proteasome inhibitor. By preventing the degradation of NFKBIA, Bortezomib maintains the inhibitory bond with NF-κB, thereby suppressing the pathway hyperactivation that contributes to malignant transformation.   This represents just one example of how a detailed understanding of the SWI/SNF and NF-κB complexes can guide the adoption of therapeutic strategies capable of arresting ccRCC progression, potentially improving clinical outcomes for many patients.   The future of oncological research depends on the development of increasingly precise therapies designed to circumvent resistance mechanisms. Translating our knowledge of molecular biology into clinical practice is fundamental to improving the prognosis and long-term survival of patients affected by clear-cell renal cell carcinoma.