Carnitine palmitoyltransferase 1A promotes mitochondrial fission and regulates autophagy by enhancing MFF succinylation in ovarian cancer

doi:10.21203/rs.3.rs-1951052/v1

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Carnitine palmitoyltransferase 1A promotes mitochondrial fission and regulates autophagy by enhancing MFF succinylation in ovarian cancer

https://doi.org/10.21203/rs.3.rs-1951052/v1

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Mitochondria are dynamic organelles and important for cell growth and proliferation. Dysregulated mitochondrial dynamics are highly associated with the initiation and progression of various cancers, including ovarian cancer. However, the regulatory mechanism underlying mitochondrial dynamics is still unclear and needs to be further studied. Previously, our study showed that Carnitine palmitoyltransferase 1A (CPT1A) is highly expressed in ovarian cancer cells and promotes the development of ovarian cancer. Here, we found that CPT1A regulates mitochondrial dynamics and promotes mitochondrial fission in ovarian cancer cells. In addition, autophagy induced by CPT1A knockdown was also related to mitochondrial dynamics. Further study showed that CPT1A regulates mitochondrial fission and function through MFF to promote the growth and proliferation of ovarian cancer cells. Mechanistically, CPT1A promotes succinylation of MFF at lysine 302 (K302), which protects against Parkin-mediated ubiquitin-proteasomal degradation of MFF. Finally, the study showed that MFF was highly expressed in ovarian cancer cells and that high MFF expression is associated with poor prognosis in patients with ovarian cancer. MFF inhibition significantly inhibited the progression of ovarian cancer in vivo. Together, CPT1A regulates mitochondrial dynamics through MFF succinylation to promote the progression of ovarian cancer. And MFF is a potential therapeutic target for ovarian cancer.

Carnitine palmitoyltransferase 1A

mitochondrial dynamics

MFF

succinylation

ovarian cancer

Mitochondria are highly dynamic organelles that continuously fuse and divide in the cell. The dynamics of mitochondrial fusion and fission are strictly regulated by some large GTPase proteins, such as Optic atrophy 1 (OPA1) [1], mitofusin 1 (MFN1) and mitofusin 2 (MFN2), which are involved in regulating mitochondrial inner and outer membrane fusion, and by dynamin-related protein 1 (DRP1) and its receptor proteins mitochondrial fission factor (MFF), Fis1, and MiD49/51, which are involved in regulating mitochondrial fission [2–4]. Once the dynamic balance of mitochondrial fusion and fission is abnormal, cells undergo pathological changes, such as tumorigenesis. Studies have shown that mitochondria in normal cells are mostly linear and reticular, while mitochondria in tumor cells are mostly fissioned and granular [5]. Our previous work also showed that enhanced mitochondrial fission or weakened fusion in non-small-cell lung cancer (NSCLC) played an important role in promoting the onset and development of NSCLC [6]. Abnormal mitochondrial dynamics play an important role in the onset, development and drug resistance of ovarian cancer. It has been reported that mitochondrial biogenesis is significantly enhanced in ovarian cancer cells compared to normal cells [7]. In serous ovarian cancer, mitochondrial morphology changes from a filamentous network to a single enlarged organelle with increasing malignancy [8]. In addition, in doxorubicin-acquired ovarian cancer cells, mitochondrial morphology and subcellular localization were significantly changed: mitochondria became rounder and more uniformly distributed in cells [9]. Although the role of abnormal mitochondrial dynamics in cancer development and multidrug resistance has been confirmed in many ways, the regulatory mechanism of mitochondrial dynamics is still unclear.

Carnitine palmitoyltransferase 1A (CPT1A), localized on the outer mitochondrial membrane, is a key rate-limiting enzyme for the intracellular transport of long-chain fatty acids to the mitochondrial matrix for oxidative degradation. Studies have shown that CPT1A is highly expressed in various malignant tumors, such as ovarian cancer, colorectal cancer, lymphoma and breast cancer, and is closely related to the development and migration of malignant tumors [10–13]. Inhibition of CPT1A activity has a significant inhibitory effect on these malignant tumors [14]. CPT1A not only promotes the adaptation of cancer cells to the abnormal tumor microenvironment through the production of ATP and NADPH by fatty acid β-oxidation (FAO) [15, 16] but also participates in the regulation of the expression of key pathways and factors that regulate cell gene expression and apoptosis [12, 17] and promotes the growth and proliferation of tumor cells. Recent studies have found that in addition to carnitine palmitoyltransferase (CPTase) activity, CPT1A itself also has lysine succinyltransferase (LSTase) activity [18] and can regulate the function and stability of substrate proteins by promoting the succinylation modification of substrate proteins [19–21].

Our previous study showed that CPT1A is highly expressed in ovarian cancer tissue cells and promotes the occurrence and development of ovarian cancer [22]. In this study, we found that CPT1A can promote the succinylation of MFF through its lysine succinyltransferase activity, regulate mitochondrial dynamics and promote the growth and proliferation of ovarian cancer cells. Meanwhile, the study showed that the autophagy caused by CPT1A knockdown was also closely related to the morphological changes of mitochondria. This study reveals the potential role and molecular mechanisms by which CPT1A regulates mitochondrial dynamics to promote ovarian cancer progression through modulation of MFF succinylation.

CPT1a regulates mitochondrial dynamics by MFF

CPT1A is highly expressed in a variety of types of tumors, and functions to promote the growth and proliferation of cells and to inhibit anoikis by promoting long-chain free fatty acid oxidation in mitochondria. Consistent with previous studies [22], knocking down CPT1A significantly inhibited the growth of ovarian cancer cells in vitro and in vivo (Fig. S1A-D). To test whether this growth inhibition is solely dependent on CPT1A-mediated transport of long-chain fatty acids, we overexpressed CPT1A-G710E, a carnitine palmitoyltransferase (CPTase)-deficient mutant [18, 23], on the basis of CPT1A knockdown in SKOV-3 cells. As shown in Fig. S1E-G, the expression of CPT1A-G710E did not restore cellular ATP but significantly rescued the growth and proliferation of ovarian cancer cells, suggesting that the decrease in cellular ATP by CPT1A knockdown may not be the only mechanism for the inhibition of cell growth and proliferation.

Mitochondria are highly dynamic organelles and important for cell growth and proliferation. To explore whether CPT1A is involved in the regulation of mitochondrial dynamics, we knocked down CPT1A in OVCAR-3 and SKOV-3 cells. As shown in Fig. 1A, CPT1A silencing signiﬁcantly promoted mitochondrial fusion and led to the elongation and hyperfusion of mitochondria. On the other hand, exogenously overexpressed HA-tagged CPT1A in A2780 cells caused mitochondria to appear more fragmented (Fig. 1B and 1C). Furthermore, knockdown of CPT1A significantly promoted mitochondrial fusion in xenograft tumors in vivo (Fig 1D). The effects of CPT1A on mitochondrial morphology were further conﬁrmed with transmission electron microscopy (TEM). Mitochondria showed a signiﬁcant increase in length, from an average of 0.45 to 0.81 μm after CPT1A knockdown (Fig. 1E). Finally, exogenous overexpression of CPT1A or CPT1A-G710E restored the fission of mitochondria caused by CPT1A knockdown (Fig. 1F) further suggesting that CPT1A regulates mitochondrial dynamics.

To explore the mechanism by which CPT1A regulates the dynamics of mitochondria, we analyzed the key players involved in mitochondrial fission and fusion. As shown in Fig. 2A, among all of the proteins examined, MFF was dramatically decreased after CPT1A knockdown in SKOV-3 and OVCAR-3 cells. In addition, exogenous overexpression of CPT1A in A2780 cells signiﬁcantly upregulated MFF (Fig. 2B). To verify whether MFF is the key mediator of CPT1A to regulate mitochondrial fusion and fission dynamics, we exogenously overexpressed MFF in the background of CPT1A knockdown (Fig. 2C). MFF overexpression significantly restored mitochondrial fission (Fig. 2D). Interestingly, SKOV-3 cells overexpressing CPT1A-G710E, the CPTase-deficient mutant, also restored the expression of MFF and rescued mitochondrial morphology (Fig. 2E, 1F). Together, our data indicated that CPT1A regulates mitochondrial dynamics by regulating MFF expression.

CPT1A promotes the growth and proliferation of ovarian cancer cells by regulating mitochondrial dynamics

As reported, the dynamics of mitochondria occur in a regulated manner to maintain cellular energy and metabolic homeostasis and play an important role in cell growth, proliferation, differentiation and apoptosis. As shown in Fig. 3A, 3B and 3C, MFF knockdown significantly inhibited cell proliferation and clone formation in SKOV-3 and OVCAR-3 cells. To investigate whether CPT1A promotes cell growth and proliferation by regulating mitochondrial dynamics, we overexpressed MFF in SKOV-3 and OVCAR-3 cells with CPT1A knockdown. The results showed that MFF overexpression rescued cell growth and proliferation (Fig. 3D and 3E). MFN2 is a mitochondrial fusion-related factor whose knockdown partly promotes the growth and proliferation of A2780 cells (Fig. S2A-C). Interestingly, we knocked down MFN2 in the background of CPT1A knockdown in SKOV-3 cells and found that mitochondrial fission was restored and cell growth was partially rescued (Fig. S2D). As we found CPT1A inactivation could induces G0/G1 cell cycle arrest and upregulation of p21 previously [22]. Here we observed that, similar to that of CPT1A knockdown, MFF inactivation also resulted in a significant upregulation of p21 expression (Fig. S2E, S2F). This suggests that the mitochondrial fusion and fission morphology is closely related to ovarian cancer cell growth and proliferation. And CPT1A might promote cell growth and proliferation by regulating mitochondrial dynamics.

The morphology of mitochondria is closely related to ATP production and ROS generation [25]. Similar to the knockdown of CPT1A, MFF knockdown significantly led to a decrease in cellular ATP (Fig. S2G). Meanwhile, MFF overexpression in SKOV-3 cells significantly restored the cellular ATP reduction caused by CPT1A knockdown (Fig. 3F). Furthermore, by utilizing the Seahorse Mito Stress Assay, we measured the overall oxygen consumption rate (OCR) and found that knockdown of CPT1A significantly reduced the maximum respiratory capacity and reserved capacity in SKOV-3 cells, which could be restored by exogenous overexpression of MFF (Fig. 3G-I), suggesting that CPT1A knockdown regulates mitochondrial dynamics and, thus, mitochondrial function through MFF.

Autophagy induced by CPT1A knockdown is related to mitochondrial dynamics

Autophagy is a self-protection mechanism for cells to survive by degrading their own structures or substances. It plays an important role in a variety of physiological and pathological processes, such as cellular senescence, immunity, tumorigenesis and neurodegenerative diseases. With CPT1A knockdown, LC3 II, a hallmarker for autophagosomes, was significantly accumulated in ovarian cancer cells of SKOV-3, OVCAR-3, A2780 and CAOV-3 cells (Fig. 4A). Using the mCherry-GFP-LC3 dual fluorescence indicator system to mark the expression of LC3, we found that CPT1A knockdown significantly promoted the enrichment of red fluorescence, even more than the etomoxir treatment (Fig. 4B), suggesting that knockdown of CPT1A promotes the occurrence of autophagy. Octanoic acid was added to SKOV-3 and OVCAR-3 cells with CPT1A knockdown. As a result, although cellular ATP was significantly restored compared with control cells (Supplementary Fig. 1H), a significant amount of autophagy still existed (Fig. 4B), suggesting that the autophagy caused by CPT1A knockdown is related to more than just the cellular ATP level. Furthermore, we found that the exogenous expression of CPT1A-G710E, although not able to restore cellular ATP (Supplementary Fig. 1E), significantly reduced the occurrence of autophagy (Fig. 4B).

To explore whether mitochondrial dynamics are involved in the occurrence of autophagy, we knocked down MFF. The results showed that MFF knockdown significantly inhibited cellular ATP (Fig. 4C), activated the expression of p-AMPK (Fig. 4D), and enhanced the expression of LC3 II (Fig. 4D). Knockdown of MFF significantly enhanced the aggregation of mRFP-LC3 (Fig. 4E). Similarly, knocking down DRP1 inhibited cellular ATP, activated p-AMPK, and enhanced the expression of LC3 II (Supplementary Fig. 3A-D). These results suggest that inhibiting mitochondrial division in ovarian cancer will induce autophagy. To investigate whether the autophagy induced by CPT1A knockdown is related to mitochondrial fission, we overexpressed MFF in the background of CPT1A knockdown in SKOV-3 and OVCAR-3 cells. As shown in Fig. 4B, 3F and Supplementary Fig. 3E-3G, the expression of MFF significantly restored cellular ATP and rescued the autophagy induced by CPT1A knockdown. Together, the above results indicate that the autophagy induced by CPT1A silencing is closely related to mitochondrial dynamics.

CPT1A stabilizes MFF by inhibiting its ubiquitination

To explore how CPT1A knockdown leads to MFF degradation, we treated SKOV-3 and OVCAR-3 cells with cycloheximide (CHX) for up to 9 hours in the presence or absence of CPT1A. The results showed that CPT1A knockdown significantly enhanced the degradation of MFF (Fig. 5A). The same results were also observed in SKOV-3 and ES2 cells exogenously expressing Flag-MFF (Fig. S4A and S4B), suggesting that CPT1A promoted the protein stability of MFF at the posttranslational level. In eukaryotic cells, protein degradation is mainly mediated through the ubiquitin–proteasome pathway or lysosomal proteolysis. To further confirm how CPT1A regulates the stability of MFF, we treated SKOV-3 cells with exogenous expression of Flag-MFF with the proteasome inhibitor MG132 or the lysosomal pathway inhibitor chloroquine separately. MG132 treatment significantly led to the accumulation of Flag-MFF (Fig. 5B), while chloroquine did not (Fig. 5C). The results were also observed in ES2 cells (Fig. S4C and S4D), suggesting that the degradation of MFF by CPT1A knockdown may be related to ubiquitin-proteasome degradation.

Then, Flag-MFF and HA-ubiquitin were exogenously overexpressed in SKOV-3 cells to verify whether CPT1A regulates the ubiquitination of MFF. The results showed that CPT1A knockdown significantly increased the ubiquitination level of MFF (Fig. 5D), while the exogenous overexpression of CPT1A significantly inhibited the ubiquitination of MFF (Fig. 5E), suggesting that the presence of CPT1A protein inhibits the ubiquitination of MFF and affects the stability of MFF.

To determine the ubiquitination type of MFF, we enriched MFF by immunoprecipitation (IP) for mass spectrometry analysis. As shown in Fig. 5F, the MFF K315 site was modified by ubiquitination, suggesting that the K315 may be a ubiquitination site regulated by CPT1A. The conservation of the K315 site was analyzed, and it was found that this site is highly conserved in MFF across various species (Fig. 5G). To examine whether the ubiquitination of K315 affects the stability of MFF, we mutated K315 to R. The mutation of K315R significantly decreased the ubiquitination of MFF and suppressed MFF degradation (Fig. 5H, 5I). Together, these results suggest that CPT1A inhibits the ubiquitination of MFF K315, thereby preventing MFF degradation.

Parkin promotes the ubiquitination of MFF

As reported, E3 ubiquitin ligases, including Parkin [26, 27]and March5 [28], promote the ubiquitination of MFF. We found that Parkin knockdown significantly inhibited the ubiquitination of MFF, while knockdown of March5 had little effect on the ubiquitination of MFF (Fig. 6A, 6B), indicating that Parkin may mediate MFF ubiquitination in ovarian cancer cells. Furthermore, Parkin knockdown rescued MFF expression in SKOV-3 cells in the background of CPT1A knockdown (Fig. 6C). And the MFF K315R mutation significantly reduced the ubiquitination modification of MFF (Fig. 6D), suggesting that Parkin may promote the ubiquitination of MFF at K315. To investigate how CPT1A affects the ubiquitination of MFF, we evaluated the interaction between Parkin and MFF by IP and found that CPT1A knockdown significantly enhanced the interaction between Parkin and MFF (Fig. 6E). This suggests that CPT1A regulates the ubiquitination of MFF by regulating the interaction between Parkin and MFF.

Our results show that CPT1A knockdown leads to the degradation of MFF and promotes mitochondrial fusion and autophagy. Next, we silenced Parkin by shRNA in the background of CPT1A knockdown in SKOV-3 cells. The results showed that Parkin knockdown restored the expression of MFF and decreased mitochondrial fusion caused by CPT1A knockdown (Fig. 6C, 6F). Meanwhile, the autophagy caused by CPT1A knockdown was attenuated as well (Fig. 6G). Furthermore, ATP content and the growth and proliferation of ovarian cancer cells were also significantly restored with Parkin knockdown (Fig. S5). Altogether, these results suggest that CPT1A promotes ovarian cancer cell proliferation by inhibiting Parkin-mediated ubiquitin-proteasome degradation of MFF.

CPT1A promotes MFF succinylation and inhibits its ubiquitin-proteasome degradation

Our studies showed that the presence of CPT1A interfered with the interaction of Parkin and MFF (Fig. 6E), thereby regulating Parkin's ubiquitination modification of MFF. As reported, protein ubiquitination could also be regulated by other types of protein posttranslational modifications, such as phosphorylation, acetylation, propionylation, butylation, glutarylation and succinylation [29]. To explore how CPT1A regulates Parkin’s ubiquitination modification of MFF, we analyzed the lipid acylation of cells after knockdown or inhibition of CPT1A and observed that CPT1A knockdown significantly reduced the succinylation modification of the total cell protein but had little effect on other acylation modifications, such as acetylation, propionylation, butylation, and glutarylation (Fig. 7A, Fig. S6A). As reported recently, CPT1A can promote the succinylation of lysine residues of its substrate [18]. The exogenous expression of CPT1A and Flag-tagged MFF in ES2 cells showed that CPT1A expression significantly increased the succinylation of immunoprecipitated MFF compared with that of the vector control (Fig. 7B).

As reported, CPTase activity and LSTase activity are independently regulated by CPT1A [18]. CPT1A-H473A, a catalytically inactive mutant that disrupts the putative binding pocket for the sulfur atom of the acyl-CoA thioester and lacks both CPTase activity and LSTase activity, and CPT1A-G710E, a CPTase-deficient mutant, were transfected into CPT1A-knockdown CAOV-3 cells separately. As a result, CPT1A-G710E restored the succinylation modification and inhibited ubiquitination and degradation of MFF (Fig. 7C). In contrast, CPT1A-H473A neither restored succinylation of cellular proteins nor restored MFF protein expression (Fig. 7D).

To determine the specific succinylated lysine (succK) in MFF, MFF were enriched by IP for In-Gel Protein Digestion and LC-MS/MS analysis. We found that MFF protein was succinylated only at lysine 302 (K302) in SKOV-3 cells (Fig. 7E). Similar to that reported by Kurmi et al. [18], the amino acids flanking the K302 succinylated lysines were enriched in nonpolar hydrophobic amino acids, such as leucine and isoleucine (Fig. 7E). We then analyzed the conservation of K302 and found that this site is highly conserved in MFF across various species (Fig. 7F).

We next confirmed whether CPT1A functions to succinylate MFF at K302. Flag-tagged wild-type (Flag-WT), K302R (Flag-K302R) and K302E (Flag-K302E) mutant MFF were transfected into SKOV-3 cells separately. As shown in Fig. 7G, both mutations decreased the succinylation levels of MFF, indicating that CPT1A can succinylate MFF at K302 and protect MFF from degradation.

To further analyze the role of K302 succinylation in MFF protein stability, we checked the ubiquitination modification of Flag-tagged MFF WT, K302R and K302E which were exogenously expressed in SKOV-3 cells. Compared with WT, the K302R (mimic of deletion) mutation enhanced MFF ubiquitination, while K302E, a mimic of the negatively charged succinyl lysine modification, decreased MFF ubiquitination and protected MFF from degradation (Fig. 7G). In addition, 293TN cells transfected with WT, K302R or K302E MFF were treated with cycloheximide for up to 12 hours. As shown in Fig. 7H, the K302E mutation significantly slowed the downregulation of MFF protein compared to the WT, while K302R decreased the half-life of MFF protein, indicating that succinylation of the K302 site protects MFF protein from ubiquitin-proteasome-mediated degradation.

To test whether the LSTase activity of CPT1A that stabilizes MFF may contribute to autophagy and cell proliferation in ovarian cancer cells, we exogenously overexpressed H473A in the background of CPT1A knockdown and found that CPT1A WT rescued mitochondrial fission and cellular ATP production, but H473A did not (Fig. 7I, Fig. S6B, S6C). As expected, H473A neither restored the inhibition of the mitochondrial oxygen consumption rate nor the inhibition of cell proliferation caused by CPT1A knockdown (Fig. S6D-G). It also did not reduce the occurrence of autophagy caused by the loss of CPT1A function (Fig. 7J), further suggesting that CPT1A regulates the stability of MFF through its LSTase activity and promotes the growth and proliferation of ovarian cancer cells.

MFF might be a target for ovarian cancer treatment

Our results suggest that CPT1A and MFF are positively correlated at the protein level due to posttranslational modification. We detected the protein expression of CPT1A and MFF in ovarian cancer cell lines and found that CPT1A and MFF were indeed positively correlated at the protein level (Fig. 8A). Given that the high expression of CPT1A is closely related to the onset and development of ovarian cancer and patient survival [22], we further explored the correlation of MFF and clinical outcomes of ovarian cancer patients. A tissue array with 100 paraffin-embedded samples, including 80 ovarian cancer tissues, 10 paracancerous tissues, and 10 normal ovarian tissues, were stained with MFF or CPT1A antibody by immunohistochemistry (IHC). Similar to the ovarian cancer cell line results, the expression of MFF and CPT1A showed a high positive correlation (Fig. 8B). Meanwhile, the results also showed that, similar to CPT1A, MFF had a higher IHC score for protein expression in endometrioid and mucinous ovarian cancers (Fig. 8C). Furthermore, Kaplan-Meier survival analysis from the TCGA data showed that ovarian cancer patients with high MFF expression correlated with a signiﬁcantly shorter overall survival (p=0.0017) and a shorter Progression-free survival (p=0.031) than those with low MFF expression (Fig. 8D). The results suggest that the expression of MFF in ovarian cancer patients correlates with poor clinical outcomes and that MFF could serve as an important prognostic marker.

To further examine the role of MFF in the onset and development of ovarian cancer, we next examined the effect of MFF knockdown on the tumorigenesis of SKOV-3 cells in nude mice. MFF knockdown strongly inhibited the initiation and development of subcutaneous xenografts in nude mice, as reﬂected by their growth curves and tumor weights (Fig. 8E, 8F). In addition, IHC staining showed that the positive rate of Ki-67 in tumor cells of the MFF knockdown group was signiﬁcantly lower, meanwhile the expression of p21 and LC3 were increased (Fig. 8G), indicating that MFF played a role in promoting carcinogenesis in ovarian cancer.

Mitochondrial dynamics are critical for regulating cellular homeostasis and survival. Disruption or imbalance in mitochondrial dynamics can lead to mitochondrial dysfunction, which in turn leads to various human diseases ranging from neurodegenerative diseases to cancer. Therefore, proteins that control mitochondrial dynamics are considered important regulators of mitochondrial function and mitochondrial quality control in health and disease. In this study, we found that CPT1A promoted MFF succinylation through the action of its lysine succinylase, thereby interfering with its recognition and binding to Parkin and inhibiting the ubiquitination and degradation of MFF. The high expression of MFF in ovarian cancer promotes mitochondrial fission and enhances mitochondrial function, thereby promoting the development of ovarian cancer. Furthermore, we also found that the regulation of autophagy by CPT1A is closely related to mitochondrial dynamics.

The balance of mitochondrial dynamics is controlled by the presence of fusion-promoting or fission-promoting macromolecules. How these macromolecules are regulated is the key to studying abnormal mitochondrial dynamics. Here, we showed evidence that CPT1A could regulate the stability of MFF at the posttranslational modification level, thereby promoting the fission state of mitochondria in ovarian cancer. As a constituent protein in the mitochondrial outer membrane, the expression of CPT1A is closely related to the shape and function of mitochondria. Luo et al. found that CPT1A overexpression increased the phosphorylation of DRP1 at Ser-637 to promote mitochondrial fusion and inhibited glioblastoma stem cell self-renewal [30]. Abnormal mitochondrial structure and dysfunction are characteristics of kidney disease pathogenesis [31]. CPT1A overexpression promotes mitochondrial biogenesis and prevents mitochondrial dysfunction [16]. Using a mouse ovarian surface epithelial model, Grieco et al. found that mitochondrial morphology changes from a filamentous network to a single enlarged organelle with increasing malignancy in serous ovarian cancer [8]. In view of the high expression of CPT1A in ovarian cancer tissue cells compared with normal tissue cells [22], our work suggests that the enhancement of mitochondrial fission in ovarian cancer tissue cells may be related to the high expression of CPT1A. CPT1A regulates mitochondrial dynamics.

As a key rate-limiting enzyme in mitochondrial long-chain free fatty acid uptake for beta-oxidation (FAO), CPT1A plays an important role in tumor progression, epithelial-mesenchymal transition (EMT) and migration [32]. It has also been reported to play an important role in the stemness maintenance and differentiation of embryonic brain neural stem cells and adult neural stem cells [33, 34]. These studies are based on the function of CPT1A-mediated FAO. Recently, studies have found that CPT1A itself has lysine succinyltransferase activity [18]. CPT1A regulates the expression and stability of its substrate proteins, such as S100A10 and enolase 1, through CPTase-independent lysine succinyltransferase activity, thereby promoting tumor cell growth, proliferation and migration [20]. Here, we had the novel finding that MFF is a substrate for CPT1A LSTase activity. Our results further confirmed that CPT1A has LSTase activity independent of its CPTase activity and also revealed a new modification and regulation mode of MFF through which CPT1A regulates mitochondrial morphology.

Under normal physiological conditions, mitochondrial fission leads to two outcomes: either the biogenesis of new mitochondria or the removal of dysfunctional mitochondria through mitophagy. Studies have shown that, unlike Fis1, MFF promotes mitochondrial fission mainly in the middle of the mitochondria, thereby promoting mitochondrial biogenesis and ultimately enhancing mitochondrial function and cellular ATP [35]. In this study, the high expression of CPT1A might not promote mitochondrial biogenesis but maintain mitochondrial fission to promote mitochondrial function through MFF, which is beneficial to the rapid metabolism of tumor cells. After knockdown of CPT1A or MFF, mitochondrial fusion is enhanced, and tumor cell growth is inhibited, partly because fused mitochondria lead to the accumulation of damaged or senescent mitochondria in cells, which is not conducive to cell survival. Interestingly, we noticed an increase in autophagy in cells after MFF or CPT1A knockdown. The increase in autophagy might be related to the lack of ATP in cells, and the increase in mitophagy may be related to the presence of Fis1 (Fig. 2A). The main role of Fis1 in promoting mitochondrial fission is thought to be mitophagy [36, 37].Of course, the effect of autophagy induced by CPT1A knockdown on the growth and proliferation of tumor cells is still unclear, and further study is needed.

Imbalances in mitochondrial dynamics, with enhanced fission or reduced fusion, have been found in patients with a variety of tumors and leads to morphological fragmentation of mitochondria. Increased expression of fission-related proteins or decreased expression of fusion-related proteins has been found in various tumors, such as liver cancer [38], breast cancer [39], and lung cancer [6], suggesting that these mitochondrial dynamics-related factors may be targets for tumor therapy. In this study, we confirmed that MFF is highly expressed in ovarian cancer cells. Knockdown of MFF significantly inhibited the growth and proliferation of ovarian cancer cells in vitro and in vivo, indicating that MFF can be a target for ovarian cancer therapy. Seo et al. found that MFF is overexpressed in NSCLC and forms homo- and heterodimeric complexes with voltage-dependent anion channel 1 (VDAC1) in the mitochondrial outer membrane. Targeted inhibition of MFF to disrupt the MFF-VDAC1 complex triggers cell death in multiple tumor types [40]. Therefore, MFF could be a therapeutic target in ovarian cancer.

Cell cultures

Human SKOV-3, A2780, OVCAR-3, OVCAR-5, CAOV-3, ES2, and DOV-13 cells were cultured in RPMI-1640 (Solarbio, Beijing, China) or DMEM (Gibco) accordingly and supplemented with 10% FBS (Biological Industries, Shanghai, China) and 1% penicillin-streptomycin (Solarbio) and were incubated in a humidiﬁed incubator at 37 °C containing 5% CO₂. Cells were obtained from ATCC or Shanghai Qincheng Biotechnology Co., Ltd. and confirmed by vendor via short tandem repeat profiling and were not reauthenticated by the authors. The cell lines were expanded at low passages and stored in liquid nitrogen after receipt. Cells were used within ten passages for experiments or resuscitated within 1 month.

RNA interference studies

For shRNA knockdown, the pGreenPuro shRNA plasmid was constructed and the lentiviruses were produced as previously described [22]. The shRNA target sequences are given in Supplementary Table S1.

Western blotting and immunohistochemistry (IHC)

Western blotting analysis was conducted as described previously [41]. Western blot results were quantified and statistically analyzed using ImageJ. Xenograft tumors were fixed with 10% neutral buffered formalin overnight and embedded in paraffin (FFPE). FFPE blocks were cut at 5-µm thickness, dried in a 60℃ oven overnight and stained with hematoxylin and eosin and antibodies. Ovarian cancer patient tissue microarrays were obtained from Avilabio (#DC-Ova11039, Avilabio Biotechnology, China) and stained with CPT1A and MFF antibodies. The detailed information is described in Supplementary Table S2. Each of the IHC-stained sections was scanned using a Pannoramic Digital Slide Scanner (Pannoramic MIDI, 3D HISTECH) and scored according to the percentage of immunostaining and the staining intensity (0, negative, 1+, weak, 2+, moderate, and 3+, strong) as described previously [42, 43]. An H-score (Histochemistry score) was calculated using the following formula: H-score= (percentage of weak intensity area ×1) + (percentage of moderate intensity area ×2) + (percentage of strong intensity area ×3). Antibodies for western blotting and IHC staining are shown in Supplementary Table S3.

Immunofluorescence and confocal microscopy

For mitochondria morphology analysis, cells expressing DsRed-mito (Miaoling, Wuhan, China) were infected with lentivirus of shRNA or exogenous overexpression plasmid of CPT1A, MFF, DRP1, MFN2, or Parkin. The infected cells were imaged using a confocal laser-scanning microscope (Leica, IL, USA). For autophagy analysis, cells expressing pCDH-mRFP1-EGFP-LC3B (Miaoling, Wuhan, China) were infected with lentivirus of shRNA or exogenous overexpression plasmid of CPT1A, MFF, Parkin, CPT1A-G710E, CPT1A-H473A or indicated. The infected cells were imaged using a confocal microscope (Leica).

Real-time PCR

Real-time PCR analysis was performed as previously described [6]. Specific primer sets used for this assay were given in Supplementary Table S4. GAPDH were detected as an internal control for normalization.

Cell proliferation and colony formation

SKOV-3 (1×10⁴), A2780 (1×10⁴) and OVCAR-3 (1×10⁴) cells seeded into 24-well plates were cultured for cell growth and proliferation analysis. Cells were harvested and stained with trypan blue, and live cells were counted. For colony formation assays, 500 cells/well of SKOV-3 cells, 500 cells/well of A2780 cells and 1000 cells/well of OVCAR-3 cells were seeded onto 12-well plates for 2-3 weeks. The cell culture media were replaced every 3-4 days. Colonies were stained with crystal violet and enumerated using ImageJ.

Transmission electron microscopy (TEM)

Cells were fixed with 2.5% glutaraldehyde for 2 h at 4°C. After being washed in 0.1 M PB three times and fixed in 1% osmic acid, cells were washed with ddH₂O and then dehydrated in 50, 70, 90, and 100% ethanol for 15 min each. After being embedded in a gradient propylene oxide and resin series, the samples were further embedded in Embed 812 resin. After cutting into 60 nm ultrathin sections, the samples were counterstained with uranyl acetate and lead citrate. Images were acquired via a HITACHI 7700 TEM (Tokyo, Japan).

ATP quantification

ATP content was measured using a firefly luciferase-based ATP assay kit (S0026, Beyotime) according to the manufacturer’s instruction. In brief, cells were lysed in 200 μl of lysis buffer/3 cm dish and collected for centrifuge at 12,000× g for 5 min at 4°C. The supernatants were collected. Two microliter were separated for protein concentration measurement and the remnant were incubated with the luciferin substrate and luciferase enzyme in the dark for 1 min to stabilize the luminescent signal. A fluorescence microplate reader was used to measure the bioluminescence intensity. The ATP content was calculated from the standard curve.

Xenografts study

The experimental protocols were approved by the Institutional Animal Care and Use Committee of Shaanxi Normal University. BALB/c nude Mice (female), 5-6 weeks old, were purchased from Beijing HFK Bioscience CO., LTD and used for experiments. Control and MFF-knockdown SKOV-3 cells (5×10⁶) were subcutaneously implanted into the right or left flank of nude mice. The tumor volumes were calculated and mouse body weight was recorded as previously described [6]. At the endpoint, the mice were euthanized and tumors were harvested for weight and immunohistochemistry analysis.

LC-MS/MS Analysis

LC-MS/MS analysis was performed by Hoogen Biotechnology as described [44]. Briefly, SKOV-3 cells were harvested and MFF was enriched by immunoprecipitation with anti-MFF antibody. After separation by SDS-PAGE, the MFF region was cut and digested, and separated using the EASY-nLC 1000 ultra-high performance liquid phase system. The resulting MS/MS data were processed using Proteome Discoverer 2.1. Tandem mass spectra were searched against NCBI human MFF sequence. The search parameters were: digestion protease was trypsin, modification of +114 Da on Lys selected as ubiquitination, modification of +100 Da on Lys selected as succinylation, mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Peptide confidence was set at high, and peptide ion score was set > 20.

Mitochondrial stress assay

The oxygen consumption rate (OCR) was measured using a Seahorse XF8 analyzer and an XF assay kit (Agilent Technologies). Cells were seeded at 2×10⁵ cells/well on an XF8 plate at 24 h before the assay. On the day of the assay, the medium was changed to XF Base Medium (0 mM glucose, Agilent Technologies) supplemented with, containing 10 μM/ml L-glutamine, 200 μM/ml glucose, 10 μM/ml sodium pyruvate, without serum, and the plates were incubated for 1 h in a non-CO₂ incubator at 37°C. The reagents for the assay were prepared using an XF Cell Mito Stress Test kit (Agilent Technologies) according to the manufacturer’s instruction, and injection was performed according to a standard assay protocol (Port A: oligomycin, Port B: FCCP, and Port C: rotenone/antimycin A).

Statistical analysis

All experiments were performed at least three times, and results were presented as the means ± SD. To determine the significance between the tested groups, Student’s t-test or GraphPad Prism 7 was used, where p values < 0.05 were considered as statistically significant.

Data Availability

All data generated or analyzed during this study are available from the corresponding author on reasonable request.

Acknowledgements

We thank Dr. Ling Guo (SNNU, China) for the technical support in TEM.

Author contributions

Huanjie Shao, Yaqin Zhu and Yue Wang conceived the project and designed the experiments. Yaqin Zhu, Yue Wang, Ying Li, Wenhui Kong, Shuting Chen, Liting Yan, Lenan Wang and Yunli Tong collected most of the data. Zhongqi Li supported the confocal microscopy analysis. Yaqin Zhu, Yue Wang and Huanjie Shao analyzed the results. Huanjie Shao and Yaqin Zhu wrote the paper. All authors read and approved the final manuscript.

Funding Statement

This work was supported by the National Natural Science Foundation of China (82072888, 81872250), the Natural Science Foundation of Shaanxi Province, China (2020JM-281), and the Student Innovation Training Program, Shaanxi Normal University (S202110718060).

Declaration of competing interests

The authors declare no competing interests.

Ethics approval

The experimental protocols were approved by the Institutional Animal Care and Use Committee of Shaanxi Normal University.

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Carnitine palmitoyltransferase 1A promotes mitochondrial fission and regulates autophagy by enhancing MFF succinylation in ovarian cancer

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