The study demonstrates that PF-8380 reduces cell viability and inhibits both EMT and autophagy in HCC. PF-8380 suppresses EMT by increasing E-cadherin and decreasing Snail, limiting cancer cell invasion. It also inhibits autophagy, as indicated by reduced LC3 and increased p62, impairing tumor cell survival. In an orthotopic HCC model, PF-8380 significantly reduced EMT, highlighting its potential as a therapeutic option, including for sorafenib-resistant HCC. Additionally, sorafenib treatment has been reported to induce hypoxia, which activates HIF-1α and upregulates ATX expression via ENPP2, leading to increased LPA production. This promotes cancer cell survival and therapy resistance through EMT and autophagy activation (Erstad et al. 2017). PF-8380’s ability to disrupt this ATX-LPA axis suggests its potential to counteract sorafenib resistance mechanisms.

ATX inhibitors, such as PF-8380, have been introduced as potential anticancer therapeutics due to their ability to suppress the production of LPA, a bioactive lipid known to facilitate oncogenic processes including cancer cell proliferation, invasive behavior, and metastatic dissemination (Bhave et al. 2013; Lee et al. 2018). ATX catalyzes the conversion of LPC to LPA, and by inhibiting ATX, PF-8380 effectively reduces LPA levels (Erstad et al. 2017; Perrakis and Moolenaar 2014). ATX inhibitors have been tested in a variety of cancers, including breast cancer (Tang et al. 2020), lung cancer (Bhave et al. 2013), colorectal cancer (Yun 2019), and melanoma (Jankowski 2011). Specifically, in a mouse model of breast cancer, ATX inhibitors were shown to increase the efficacy of radiotherapy and chemotherapy (Tang et al.). ATX inhibitors have also been shown to be effective in reducing the growth of lung cancer cells in vitro and in vivo (Bhave et al.). In addition, ATX inhibitors have been shown to induce apoptosis in melanoma cells (Jankowski). However, while LPA is known to play a substantial role in the pathogenesis of HCC (Balijepalli et al.; Erstad et al.; Magkrioti et al.; Memet et al.; Wu et al.), research investigating the application of ATX inhibitors as anticancer therapeutics for HCC remains markedly limited.

Sorafenib has been the primary therapeutic agent for advanced HCC, but its effectiveness is often limited by severe side effects, including hand-foot skin reaction, diarrhea, and hypertension, which negatively impact patient quality of life and long-term use (Kudo et al. 2018; Pang et al. 2022). More critically, the emergence of sorafenib resistance presents a significant clinical challenge, necessitating the development of novel therapeutic strategies to either replace or complement sorafenib in HCC treatment (Mir et al. 2017; Shen et al. 2010; Yang et al. 2023). In this study, we demonstrated that PF-8380, an ATX inhibitor, effectively suppresses both EMT and autophagy in sorafenib-resistant Huh7 cells. EMT and autophagy are key processes that contribute to tumor progression and therapy resistance, and their inhibition suggests a strong potential for therapeutic intervention. Notably, Huh7-R cells exhibited significant morphological differences from their sorafenib-sensitive counterparts, appearing larger and more elongated. These changes are commonly associated with increased migratory and invasive capabilities, features often observed in cancer stem cells (Li et al. 2020). Given the urgent need for effective treatment options for sorafenib-resistant HCC, our findings indicate that PF-8380 may serve as a promising therapeutic strategy. By targeting ATX and inhibiting the LPA-driven activation of EMT and autophagy, PF-8380 could play a role in reducing tumor aggressiveness and overcoming therapy resistance. This highlights the potential of PF-8380 either as a standalone agent or in combination with existing treatments to improve clinical outcomes in HCC.

PF-8380 suppresses both EMT and autophagy by inhibiting ATX-LPA signaling, which plays a key role in tumor progression (Fig. 6). Although this study did not directly assess the molecular interactions between LPA and these processes, previous research provides a strong rationale for how reduced LPA levels may contribute to EMT and autophagy inhibition. LPA is known to promote EMT by activating key signaling pathways such as PI3K/Akt and MAPK/ERK, which in turn increase the expression of EMT-associated transcription factors such as Snail and Twist (Fukushima et al. 2017). These transcription factors repress E-cadherin and upregulate N-cadherin, facilitating the transition to a mesenchymal phenotype and enhancing tumor invasiveness. Given that PF-8380 reduces LPA production, it is reasonable to infer that this inhibition disrupts LPA-driven EMT signaling, leading to increased E-cadherin expression and decreased Snail and N-cadherin levels, as observed in our study. Similarly, LPA has been reported to promote autophagy, which provides survival advantages to tumor cells under stress conditions. One of the mechanisms involves Beclin-1 activation, which facilitates the formation of the PI3KIII complex, a key component of autophagosome formation (Chen et al. 2017; Koike et al. 2009). Additionally, LPA has been shown to suppress mTOR, a well-known inhibitor of autophagy, further enhancing autophagic activity (Zhang et al. 2014). Since PF-8380 inhibits ATX and reduces LPA levels, it likely interferes with these autophagy-promoting mechanisms, contributing to the observed decrease in LC3-II and increase in p62, indicative of autophagy inhibition. Taken together, these findings suggest that LPA reduction through ATX inhibition disrupts EMT and autophagy-promoting pathways, thereby contributing to tumor suppression. While further studies are needed to fully elucidate these mechanisms, our results strongly indicate that PF-8380 effectively modulates these key oncogenic processes by targeting ATX-LPA signaling.

Schematic illustration of the PF-8380-mediated suppression of EMT and autophagy in hepatocellular carcinoma (HCC) cells. (1) In the tumor microenvironment, autotaxin (ATX) catalyzes the conversion of lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), leading to the activation of epithelial-mesenchymal transition (EMT) and autophagy. Increased EMT is characterized by decreased E-cadherin (E-Cad) and increased Snail and N-cadherin (N-Cad), while autophagy activation is indicated by increased LC3-II and decreased p62. These processes contribute to tumor progression. (2) PF-8380 inhibits ATX activity, reducing LPA levels, thereby suppressing EMT and autophagy. EMT inhibition is marked by increased E-cadherin and decreased Snail and N-cadherin, while autophagy inhibition is characterized by reduced LC3-II and increased p62. Consequently, tumor progression is suppressed in both sorafenib-sensitive (Huh7-S) and sorafenib-resistant (Huh7-R) HCC cells

Autotaxin plays significant roles in various conditions, spanning both nonmalignant and malignant diseases. Nonmalignant conditions include fibrosis and inflammatory disorders, while its role in malignancies primarily relates to cancer progression and metastasis. This study specifically investigated the effects of the autotaxin inhibitor PF-8380 on sorafenib-resistant HCC. However, the potential applications of PF-8380 could extend to other diseases where autotaxin is implicated. Future research is recommended to explore the broader therapeutic uses of autotaxin inhibitors like PF-8380 in these contexts. Such studies could provide valuable insights into the versatility and efficacy of PF-8380 across a spectrum of autotaxin-related diseases.

In conclusion, this study has unveiled the potential of PF-8380, an ATX inhibitor, as a viable therapeutic strategy for HCC, particularly in cases showing resistance to the standard treatment with sorafenib. PF-8380 demonstrated significant inhibitory effects on both EMT and autophagy, two critical processes that drive the pathogenesis and progression of HCC. This reinforces the potential of ATX inhibitors as significant contributors to the arsenal of anticancer therapeutics for HCC. However, it is important to acknowledge that the study was conducted in preclinical models and the results, though promising, need to be validated in a clinical setting to determine their translational potential. Further research is warranted to delve deeper into the underlying mechanisms of action and to optimize the therapeutic application of PF-8380 in the management of HCC.