Sirtuins and their role in ovarian aging-related fibrosis predisposing to ovarian cancer.

High-grade serous ovarian cancer (HGSOC) represents one of the most challenging and lethal forms of gynecological malignancies, characterized by its aggressive progression and often late-stage diagnosis. Genetic predispositions play a crucial role in the etiology of HGSOC. As recently shown, the prevalence of HGSOC subtype-specific survival varies by race 110 . Mutations in the BRCA1 and BRCA2 genes are the most significant genetic factors associated with an increased risk of developing ovarian cancer. Women carrying BRCA1 mutations face a 39%-44% lifetime risk, while BRCA2 mutation carriers have an 11%-17% lifetime risk of developing ovarian cancer (American Cancer Society). Besides BRCA mutations, other genetic factors such as mutations in BRIP1, RAD51C, and the genes associated with Lynch syndrome (PMS2, MLH1, MSH2, and MSH6) also contribute to the risk profile for HGSOC 111 – 113 . Familial aggregation of ovarian and other cancers, such as breast, pancreatic, melanoma, and colon cancers, in first-degree relatives, underscores the importance of genetic epidemiology in HGSOC. Understanding these genetic predispositions is essential for identifying high-risk individuals and implementing preventive measures such as genetic counselling and risk-reducing surgeries. Environmental and lifestyle factors are also implicated in the epidemiology of HGSOC. Obesity, use of hormone replacement therapy (HRT), and reproductive history, including lower parity, lifetime number of ovulatory cycles, and infertility, have been identified as significant risk factors for developing ovarian cancer 112 , 114 . While combined oral contraceptive use lowers the risk of HGSOC, the use of postmenopausal hormone therapy slightly increases the risk for HGSOC, highlighting the complex interplay between hormonal influences and cancer development 112 , 115 . Dietary factors, and physical inactivity are additional lifestyle factors that may contribute to ovarian cancer risk. For instance, diets high in fats and low in fruits and vegetables are associated with higher cancer risk, while regular physical activity may offer protective benefits 111 , 112 . Environmental exposures such as the use of talcum powder in the genital area and prolonged exposure to asbestos have also been studied for their potential to increase ovarian cancer risk 111 , 112 . The incidence of epithelial ovarian cancer, particularly endometriosis-associated ovarian cancer such as clear cell carcinoma and endometrioid carcinoma, has markedly increased in Japan 116 . It has also been reported that clear cell carcinoma and endometrioid carcinoma frequently co-exist with endometriosis, suggesting that endometriosis is a possible precursor lesion for these types of ovarian cancer. Endometriosis, a chronic gynecological condition affecting approximately 176 million ( ~10%) women of reproductive age worldwide, is another common risk factor for some OC types 117 . In a recent study in which 450 906 patients with and without endometriosis were analyzed, a history of endometriosis conferred a 4.2-fold increased risk for ovarian cancer 118 . Noteworthy, patients suffering from ovarian endometriomas and/or deep infiltrating endometriosis showed a 9.7-fold higher risk when compared to counterparts without endometriosis (Fig. 3 ) 118 . However, the risk for endometriosis patients to develop high-grade serous ovarian cancers is lower (2.7%) than to develop other histotypes, such as, endometrioid (7.96%) or low-grade serous (8.12%) ovarian cancer 118 . Women suffering from endometriosis are concerned about the increased ovarian cancer risk, and endometriosis-associated ovarian cancer is challenging for clinicians 118 . Moreover, the aforementioned patients are suffering from pain, anxiety and depression, impacting their psychological and social functioning 119 , 120 . Counselling programs for patients suffering from endometriosis should also be provided to test for specific gene mutations that could later in life cause the development of ovarian cancer 117 , 118 . Recent bioinformatic analyses have revealed a significant molecular overlap between PCOS and ovarian cancer, suggesting that PCOS may serve as a precursor condition for some OC subtypes. A set of 128 differentially expressed genes was found to be common to both PCOS and OC, with particular emphasis on OGN (osteoglycin) as a potential biomarker linking the two 121 . Lower OGN expression, frequently observed in both PCOS and OC tissues, was associated with altered hormone signaling and poor prognosis, potentially promoting tumor progression through dysregulation of FSHR and m6A methylation. These findings imply that PCOS, particularly when marked by hormonal imbalance and genetic susceptibility, may contribute to the molecular pathogenesis of ovarian cancer 121 . Fig. 3 Scheme showing the development of ovarian endometriosis into ovarian cancer and the primary tumor dormancy. Scheme showing the development of ovarian endometriosis into ovarian cancer and the primary tumor dormancy. In the context of OC, the role of sirtuins remains an active area of investigation. SIRT1 functions as an oncogene as well as a tumor suppressor, regulating cell cycle progression, apoptosis, cell senescence, and oxidative stress resistance. Several studies have linked SIRT1 to cancer stemness and resistance to conventional therapy. SIRT1 has been shown to play a complex and often contradictory role in cancer development and progression 122 . One study revealed that SIRT1 expression was significantly decreased in OC tissues compared to normal ovarian tissues, and that lower SIRT1 levels were associated with more advanced disease stages and poorer patient survival 123 – 125 . In another, SIRT1 overexpression increases chemoresistance, tumorigenesis and epithelial-to-mesenchymal transition (EMT) phenotype 126 , 127 . By extension, overexpression may promote a poorer prognosis for patients with OC 11 , 123 , 128 . SIRT1 inhibition generally promotes the survival, proliferation, and metabolism of cancer cells, playing an important role in their resistance to treatment. However, MHY2245, a new SIRT1 inhibitor, by inhibiting the activity and expression of SIRT1, leads to cell cycle arrest, apoptosis, and autophagy in cancer cells 129 . Extracellular vesicles derived from cancer-associated adipocytes (CAA-EVs) play a crucial role in ovarian cancer progression by modulating the immune response and tumorigenesis 130 . These vesicles carry SIRT1, which transcriptionally activates CD24 expression, leading to suppression of CD8 + T cell activity and promoting tumor immune escape 131 . Other studies indicate that SIRT1 plays a key role in inhibiting the progression of ovarian cancer by regulating the expression and acetylation of HMGB1. Importantly, overexpression of SIRT1 effectively reduced the migration and invasion of cancer cells and decreased angiogenesis, suggesting its therapeutic potential in the treatment of this aggressive disease 132 . Despite the original reports of an adverse effect of SIRT2 on OC prognosis 133 , several recent studies have indicated that it is an OC suppressor 11 , 123 . For example, lower expression of SIRT2 was associated with higher expression of cyclin-dependent kinase 4 (cdk4) 134 , and SIRT2 overexpression had a favorable effect on the prognosis of OC patients 123 . Metastatic spread is the main cause of death in epithelial ovarian cancer, yet the mechanisms remain unclear. Fn14 acts as a metastasis suppressor by inhibiting migration and invasion of EOC cells through downregulation of EMT 135 . Mechanistically, Fn14 promotes acetylation-dependent degradation of Slug, a key EMT transcription factor, by interfering with SIRT2. Fn14 binds SIRT2, preventing its nuclear entry and thus reducing Slug deacetylation and stabilization 135 . The third significantly down-regulated sirtuin in OC is SIRT3 11 . Since tumorigenesis destabilizes the cell’s energy economy, the role of SIRT3 may be crucial in OC. In one recent study, SIRT3 transcript levels in various OC subtypes were significantly lower than in normal tissues 123 . Moreover, as one of the most important proteins of mitochondrial metabolism, SIRT3, was identified as an independent favourable prognostic factor of OC 136 . Interestingly, downregulated SIRT3 has also been detected in pre-metastatic tissue 137 . SIRT3 is downregulated in metastatic ovarian cancer tissues and cells. Its knockdown enhances migration, invasion, and metastasis, while overexpression suppresses these processes. The mechanism involves inhibiting EMT by reducing the level of the protein Twist, with which SIRT3 directly interacts. The SIRT3/Twist axis may represent a novel therapeutic target for metastatic ovarian cancer 138 . Increased expression of mitochondrial proteins may occur in response to oxidative damage to cells in early tumorigenesis. SIRT5, which mainly acts in its territory, is also overexpressed in early tumorigenesis 139 . In addition, SIRT5 increases OC resistance to cisplatin 16 . Mechanistically, it suppresses cisplatin-induced DNA damage by reducing reactive oxygen species through activation of the Nrf2/HO-1 antioxidant pathway, thereby contributing to chemoresistance in ovarian cancer 16 . In some types of liver cancer, SIRT5 depression limits disease progression 140 . In ovarian cancer, reduction of SIRT5 expression, through upregulation of miR-27b-5p, reduced tumor progression in vitro 141 . The opposite dynamic is characterized by SIRT4, which maintains low escapes in OC 123 , 142 , but SIRT4 overexpression adversely affects OC prognosis 11 . The other sirtuins, nuclear SIRT6 and SIRT7, are lower in OC 123 . This may indicate that their function is impaired and that genome stability and DNA repair processes are impaired, which is characteristic of tumorigenesis progression 123 . Unfortunately, the results of other studies are inconclusive, on the one hand pointing to a suppressor character 143 , 144 and on the other, promoting OC progression 145 . Sirtuins, especially SIRT4 and SIRT6, play opposing roles in regulating ovarian cancer cell survival, making them potential competitive prognostic biomarkers. Bioinformatic analyses and immunohistochemical studies have shown that their high expression levels are associated with different prognoses and distinct impacts on tumor progression. SIRT4 is involved in the immune response during oocyte maturation, while SIRT6 participates in regulating mitochondrial processes and immune-related diseases, indicating their involvement in conflicting mechanisms influencing disease development 14 . In p53-mutant ovarian cancer, tumor cells under cisplatin treatment release exosomes containing the long non-coding RNA PANDAR (correlates with poor prognosis and promotes the development of cancer), which binds to the protein SRSF9 146 . After translocation to the nucleus, SRSF9 suppresses apoptosis and modifies gene expression, leading to an altered mRNA ratio of SIRT4/SIRT6 that promotes cell survival and the development of cisplatin resistance. This mechanism enables tumor cells to rapidly adapt to treatment-induced stress, hindering therapeutic efficacy 147 . Understanding this complex interaction between PANDAR, SRSF9, and sirtuins could open new therapeutic avenues for treating cisplatin-resistant ovarian cancers.

Ovarian fibrosis is a hallmark of reproductive aging and a common feature in pathological conditions such as premature ovarian insufficiency and polycystic ovary syndrome. One of the central mechanisms contributing to this fibrotic remodelling is oxidative stress (OS), which arises from an imbalance between the production of reactive oxygen species (ROS) and the cell’s ability to detoxify them. ROS can damage lipids, proteins, and nucleic acids, and their accumulation in ovarian cells impairs mitochondrial function, disrupts hormonal signaling, and promotes the activation of fibrotic and inflammatory pathways. A primary target of oxidative damage is telomeric DNA, which, due to its guanine-rich sequence and limited repair capacity, is especially susceptible to ROS-induced breaks. This damage activates the DNA damage response (DDR) via pathways involving γH2AX, XRCC6, and PARP1, which are upregulated in aging ovaries 70 – 73 . Telomere shortening and dysfunction consequently promote cellular senescence and apoptosis 74 , 75 , processes that drive tissue remodeling and fibrotic changes. Additionally, OS and DNA damage induces activation of p53, which transcriptionally regulates pro-apoptotic and pro-fibrotic genes, often through upregulation of CDKN1A (p21) and downregulation of the retinoblastoma protein (RB) pathway, leading to cell cycle arrest and promoting senescence-associated secretory phenotypes (SASP) 76 . Another major contributor to fibrotic remodeling under OS is p66Shc, a redox enzyme that promotes mitochondrial ROS production and upregulates profibrotic markers such as α-SMA and NLRP3 inflammasome components 77 . p66Shc levels increase with age and correlate with fibrosis in the ovary and other organs. Deletion of p66 in progeric mice (telomerase RNA component knockout mice) alleviates age-related phenotypes, indicating a potential role for p66 in the ageing process 78 . Similarly, dysregulation of the Keap1/Nrf2 antioxidant pathway under prolonged OS limits the cellular ability to activate antioxidant genes such as HO-1, SOD2, and CAT. Normally, oxidative stress leads to dissociation of Nrf2 from Keap1, its nuclear translocation, and activation of antioxidant response elements (ARE), but this axis is impaired in several ovarian pathologies including PCOS 79 – 81 . Mitochondrial dysfunction, another key element in fibrogenesis, is tightly coupled to cellular redox status. With aging, mitochondrial membrane potential and oxidative phosphorylation efficiency decline, NAD + /NADH ratios drop, and ATP production is reduced. This leads to an increase in mitochondrial ROS and subsequent oxidative damage, further driving fibrotic gene expression and cellular dysfunction 82 , 83 . In this context of pro-fibrotic and oxidative signaling, sirtuins act as crucial regulators that can suppress or reverse many of these pathological changes. These NAD + -dependent deacetylases respond to metabolic and redox status and coordinate protective mechanisms against OS and fibrosis. Among them, SIRT1, SIRT3, SIRT5, SIRT6, and SIRT7 have been most widely studied in ovarian tissue. SIRT1 regulates the DDR by deacetylating p53, thereby limiting its pro-apoptotic and pro-fibrotic activity 5 , 6 . Under oxidative conditions, SIRT1 expression is upregulated as a compensatory response, and its activity is further enhanced by compounds like celastrol and melatonin, which reduce the levels and activaion of γH2AX, XRCC6, and PARP1 84 , 85 . Moreover, the kinase TOPK promotes SIRT1 expression while repressing p53 acetylation, and its inhibition leads to apoptosis under inflammatory stress, indicating the importance of this regulatory axis in follicular survival 86 , 87 . SIRT1 also suppresses p66Shc expression, thereby attenuating ROS generation and inflammasome activation. In models of hyperandrogenism-induced ovarian fibrosis, resveratrol treatment upregulates SIRT1, reduces p66Shc levels, and ameliorates fibrotic changes 51 . SIRT6 complements this function by repressing p66Shc promoter activity and deacetylating histone H3K9Ac, reducing transcription of pro-apoptotic and pro-fibrotic genes 88 – 90 . SIRT3 and SIRT5, both localized in mitochondria, maintain mitochondrial integrity and redox balance. SIRT3 deacetylates and activates FOXO3a, which in turn upregulates antioxidant enzymes such as SOD2 and CAT 91 – 94 . It also regulates PGC-1α and TFAM, promoting mitochondrial biogenesis. In PCOS and aging models, SIRT3 expression is reduced, correlating with decreased antioxidant defense and increased fibrotic gene expression 95 – 97 . Resveratrol and melatonin restore SIRT3 levels and improve mitochondrial function, reversing these pathological changes 98 , 99 . SIRT5, through its desuccinylase activity, regulates mitochondrial enzymes involved in oxidative phosphorylation and supports redox homeostasis, although its specific role in ovarian fibrosis remains to be fully elucidated 100 . SIRT7, although less extensively studied in the context of ovarian function, has emerged as a key regulator of mitochondrial homeostasis and resistance to oxidative stress. It promotes mitochondrial ribosomal protein expression, supports mitochondrial translation, and limits the accumulation of ROS 101 . Importantly, the miR-17-5p/SIRT7 axis is a key regulatory factor in the DNA damage response in the ovaries, and its influence has been shown to reduce OS and the levels of γH2AX, XRCC6, and PARP1 activity 84 . Reduced expression of SIRT7 with aging may thus contribute to mitochondrial decline and the establishment of a pro-fibrotic environment. The interaction between sirtuins and FOXO transcription factors is central to the cellular oxidative stress response. SIRT1 and SIRT3 promote FOXO1 and FOXO3a activity, enhancing resistance to oxidative damage and inhibiting apoptosis 91 – 94 . Disruption of this interaction by miRNAs such as miR-132 and miR-181a leads to FOXO inactivation, increased apoptosis, and promotion of fibrotic and neoplastic changes 91 , 102 , 103 . Sirtuins also influence the Nrf2 pathway. SIRT1 deacetylates Nrf2, facilitating its nuclear translocation and activation of antioxidant genes. Natural compounds such as icariin and resveratrol amplify this effect, enhancing the expression of protective enzymes like HO-1 and restoring redox balance in oxidative ovarian environments 104 – 106 . Finally, declining NAD+ levels during aging limit sirtuin activity and thereby compromise antioxidant defenses and mitochondrial function. Supplementation with NAD+ precursors like nicotinamide riboside improves ovarian function by restoring NAD+ pools, increasing SIRT1 and SIRT3 expression, and improving mitochondrial energy metabolism 47 , 48 , 95 , 107 – 109 . Sirtuin-mediated pathways therefore intersect with multiple regulatory networks involved in oxidative stress, mitochondrial function, and the fibrotic remodeling of ovarian tissue. Their activity is tightly linked to cellular energy status and redox balance, positioning them as dynamic sensors and modulators of homeostasis in the ovarian microenvironment.

Sirtuins, through their extensive subcellular localization, exert important effects on energy (NAD + ) metabolism, on which cellular functions depend, and influence key signaling pathways directly related to ovarian fibrosis (Smads, TGF-β). As we have presented, the kinetics of SIRT1 and SIRT3 are the best understood so far. However, this is not surprising because of their indisputable effects on Smads pathway gene transcription (in the case of SIRT1) and mitochondrial metabolism (in the case of SIRT3). In the case of the remaining members, we can expect similar functions to SIRT1 from SIRT6 and SIRT7, due to their similar localization. The same situation applies to SIRT3 and SIRT5. While the functions of SIRT2 and SIRT4 in the ovary remain underexplored, future studies could investigate their potential roles by drawing parallels with their known activities in other tissues, such as SIRT2’s involvement in cell cycle regulation and microtubule dynamics, and SIRT4’s role in mitochondrial metabolism and stress response. What has been established is that inhibition of the major inflammatory and oxidative stress pathways NLRP3, NFkB reduced the expression of fibrosis markers (α-SMA) in ovarian tissue from aging mice, mice with induced fibrosis as well as ovarian tissue from postmenopausal women. In addition, sirtuins collaborate with key pathways that maintain mitochondrial fitness and biogenesis (PGC-1α), and transcription factors (Nrf2, FOXO) that ensure adequate expression of antioxidant enzymes. As a separate observation, there is an interesting relationship between sirtuins and AMPK, which translates, for example, into modifications of the macrophage population, making it possible to discover the true causes of ovarian fibrosis. In the context of tumorigenesis, an interesting relationship has emerged between sirtuins and important fibrotic pathways for OC. Indeed, sirtuins can model the EMT process, a key process for metastasis and overexpression of sirtuins was able to reduce collagen deposition in ageing mice. While therapeutic agents like metformin (an AMPK and SIRT1 agonist) demonstrate potential in mitigating ovarian fibrosis, and resveratrol may offer even stronger SIRT1 activation, further research is needed to address limitations such as side effects and the scarcity of robust human clinical data supporting their efficacy and safety. The action of resveratrol is limited by its low bioavailability, and its interaction with other drugs (due to the inhibition of cytochromes P450) is still poorly understood 148 . Metformin, on the other hand, can lead to numerous disorders of the digestive and endocrine systems 148 .

In the quest to comprehend the complexities of the aging phenomenon in the female reproductive system, early markers for ovarian aging, fibrosis, and cancer offer critical insights into the intricate cellular pathways that deteriorate as women’s ovaries age, presenting substantial implications for fertility treatments and reproductive health strategies (Fig. 1 ). Among various regulators of cellular homeostasis, the sirtuin (SIRT) family—comprising seven NAD⁺-dependent enzymes, SIRT1–SIRT7—acts as crucial sensors of energy and redox status 1 . Fig. 1 Scheme showing the phenomenon of ovarian aging and its effects on the decrease of the antral follicle count (AFC), and of the anti-Muellerian Hormone (AMH) in the bloodstream of women with advancing age. With increasing age, symptoms of fibrosis increase, and by this, the risk for tumorigenesis is elevated, too. With increasing age, symptoms of fibrosis increase, and by this, the risk for tumorigenesis is elevated, too. SIRTs are a family of seven enzymes: SIRT1-7, that play a key role in regulating protein function through post-translational modifications, primarily by removing acetyl groups 2 . Their roles in controlling oxidative stress, genomic stability, and cell fate position them as central modulators of both ovarian fibrosis and tumorigenesis. SIRT1 is widely recognized for its antifibrotic and tumor-suppressive effects through regulation of TGF-β, p53, and NF-κB pathways, although in certain cancers, its overexpression may support tumor progression 3 – 6 . SIRT2 regulates cell division and spindle integrity and has been shown to impact fibrotic signalling via DKK1 and Smad3, while also displaying tumor-suppressive properties in ovarian cancer 7 – 9 . SIRT3, the principal mitochondrial deacetylase, protects against oxidative stress and supports mitochondrial function; its decline with age promotes fibrosis and is associated with poor ovarian cancer prognosis 10 , 11 . SIRT4, though less well characterized, influences oocyte maturation and may contribute to mitotic stability; its aberrant expression is linked to both ovarian aging and malignancy 12 – 14 . SIRT5, through its desuccinylase activity, modulates metabolic enzymes involved in redox balance; it may promote chemoresistance in ovarian tumors and has been found altered in fibrotic ovarian conditions 15 – 17 . Nuclear sirtuins SIRT6 and SIRT7 are vital for chromatin remodeling and DNA repair; SIRT6 has been shown to counteract fibrotic matrix deposition and oxidative stress, while SIRT7 supports oocyte quality and genome maintenance, both showing deregulation in ovarian aging and cancer 18 – 22 . As NAD⁺ levels decline with age, the activity of all SIRTs diminishes, contributing to dysregulation of cellular stress responses, extracellular matrix remodeling, and ultimately creating a microenvironment that favors both fibrosis and tumor development 23 – 26 . Age-related ovarian dysfunction has been linked to decreased levels of SIRTs 27 , i.e., decreased ovarian reserve, which has been associated with decreased levels of SIRT1, SIRT3 and SIRT6 26 . Another feature of aging is ovarian fibrosis 28 , and, as mentioned earlier, fibrosis can promote conditions similar to the pre-metastatic niche 29 . Tumor-associated fibrosis may have a variety of functions, including a role in drug resistance. Indeed, targeting Transforming growth factor-β (TGF-β), which leads to inhibition of fibrosis, can sensitize the tumor to immunotherapies 30 , 31 . However, some studies claim that ovarian fibrosis may contribute to the development of a microenvironment favourable for tumor growth by mobilizing ECM components, primarily collagen, and activating fibrotic pathways, e.g. TGF-β, which are closely related to the potential of cells to undergo the EMT process. Interestingly, most malignant tumors are accompanied by severe stromal changes 32 , and increasing stiffness of ovarian tissue facilitates tumor invasion or metastasis 33 . Fibrotic ovaries are also encountered in postmenopausal and advanced-age women, a period of life that predisposes to the development of ovarian cancer (OC) 34 . The age-related accumulation of damage caused by increasing oxido-inflammatory stress contributes to worsening changes in the ovarian stroma and capsule, which may be linked to a higher predisposition to OC 29 . This review aims to explore the multifaceted roles of sirtuins in ovarian aging-related fibrosis and their contribution to ovarian cancer predisposition, integrating their molecular actions within key signaling pathways and physiological contexts.