Mitochondrial Protein SFXN1 Drives Bladder Cancer Spread by Blocking Cellular Cleanup

Bladder cancer (BLCA) is the tenth most common cancer globally, responsible for roughly 549,000 new cases and 200,000 deaths per year. While most cases are non-muscle invasive and manageable, the 25–30% that progress to muscle-invasive disease carry a poor prognosis, and metastatic spread remains the primary driver of mortality. This study from Nanjing Drum Tower Hospital set out to characterize the oncogenic role of SFXN1 (sideroflexin 1), a mitochondrial inner membrane protein previously known mainly as a serine transporter involved in one-carbon metabolism.

Using immunohistochemistry on 155 paraffin-embedded BLCA tumor tissues (including 21 matched normal tissue pairs and 13 fresh tissue pairs), the researchers found that SFXN1 was significantly overexpressed in tumor versus normal bladder tissue. UALCAN database analysis of TCGA data confirmed this upregulation, and high SFXN1 expression was positively correlated with advanced tumor stage and poor overall survival. These findings established a clinical rationale for deeper mechanistic investigation.

Functional experiments in T24 and J82 bladder cancer cell lines showed that SFXN1 knockdown (via siRNA) markedly reduced cell migration and invasion in transwell and wound-healing assays, and suppressed proliferation in MTT assays. Conversely, SFXN1 overexpression enhanced these metastatic behaviors. Critically, serine deprivation and formate rescue experiments demonstrated that SFXN1's pro-metastatic effects were independent of its classical serine transport function, pointing to a previously unknown mechanism.

Mechanistic investigation revealed that SFXN1 acts as a bridging factor between two inner mitochondrial membrane (IMM) proteases — PARL (presenilin-associated rhomboid-like protein) and MPP-β (mitochondrial processing peptidase-β) — to accelerate degradation of PINK1, the master initiator of mitophagy. Co-immunoprecipitation confirmed direct interactions among SFXN1, PARL, and MPP-β. In cells with high SFXN1, PINK1 protein levels were reduced, mitophagy markers (LC3B–mitochondria co-localization, mitochondria–lysosome co-localization) were suppressed, and transmission electron microscopy revealed accumulation of damaged mitochondria. Conversely, SFXN1 knockdown restored mitophagy flux. Treatment with CCCP (a mitophagy inducer) reversed the pro-metastatic effects of SFXN1 overexpression, while Mdivi-1 (a mitophagy inhibitor) negated the anti-metastatic benefits of SFXN1 knockdown.

With mitophagy blocked, dysfunctional mitochondria accumulated and mitochondrial ROS (mtROS) levels rose substantially, as measured by MitoSOX flow cytometry. Elevated mtROS activated TGF-β signaling, which in turn drove epithelial-to-mesenchymal transition (EMT), increasing expression of mesenchymal markers (vimentin, N-cadherin) and decreasing epithelial markers (E-cadherin). Scavenging mtROS with mitoTEMPO abrogated EMT activation and metastasis in SFXN1-overexpressing cells. In vivo, shRNA-mediated SFXN1 knockdown in T24 cells significantly reduced subcutaneous tumor growth and lymph node metastasis in nude mouse models over 40 days, validated by bioluminescence imaging and IHC. Together, these findings define a SFXN1 → PARL/MPP-β → PINK1 degradation → mitophagy arrest → mtROS accumulation → TGF-β/EMT → metastasis axis, identifying SFXN1 as a novel and druggable target in bladder cancer.