**CPT1A Over-Expression Increases Reactive Oxygen Species in the Mitochondria and Promotes Antioxidant Defenses in Prostate Cancer**

**Molishree Joshi 1, Jihye Kim 2, Angelo D'Alessandro 3,4, Emily Monk 2, Kimberley Bruce 5, Hanan Elajaili 6, Eva Nozik-Grayck 6, Andrew Goodspeed 1,4, James C. Costello 1,4 and Isabel R. Schlaepfer 2,\***


Received: 6 October 2020; Accepted: 9 November 2020; Published: 18 November 2020

**Simple Summary:** Prostate cancer (PCa) is the most common cancer in men and the second highest contributor to cancer deaths. Targeting lipid catabolism enzymes in PCa may offer new avenues for therapeutic approaches. During the last decade, carnitine palmitoyl transferase I (CPT1A) has been identified as a potential therapeutic target for a growing list of cancers. In this study, we have tested the hypothesis that excess CPT1A plays a key role in supporting adaptation to stress and antioxidant defense production in PCa cells. Specifically, we have studied molecular differences between CPT1A gain and loss of function models, revealing genetic and metabolic vulnerabilities that could be targeted to avoid progression to neuroendocrine differentiation, a lethal form of the disease. Examining public datasets, we have also found that excess CPT1A expression leads to worse progression-free survival in PCa patients.

**Abstract:** Cancers reprogram their metabolism to adapt to environmental changes. In this study, we examined the consequences of altered expression of the mitochondrial enzyme carnitine palmitoyl transferase I (CPT1A) in prostate cancer (PCa) cell models. Using transcriptomic and metabolomic analyses, we compared LNCaP-C4-2 cell lines with depleted (knockdown (KD)) or increased (overexpression (OE)) CPT1A expression. Mitochondrial reactive oxygen species (ROS) were also measured. Transcriptomic analysis identified ER stress, serine biosynthesis and lipid catabolism as significantly upregulated pathways in the OE versus KD cells. On the other hand, androgen response was significantly downregulated in OE cells. These changes associated with increased acyl-carnitines, serine synthesis and glutathione precursors in OE cells. Unexpectedly, OE cells showed increased mitochondrial ROS but when challenged with fatty acids and no androgens, the Superoxide dismutase 2 (SOD2) enzyme increased in the OE cells, suggesting better antioxidant defenses with excess CPT1A expression. Public databases also showed decreased androgen response correlation with increased serine-related metabolism in advanced PCa. Lastly, worse progression free survival was observed with increased lipid catabolism and decreased androgen response. Excess CPT1A is associated with a ROS-mediated stress phenotype that can support PCa disease progression. This study provides a rationale for targeting lipid catabolic pathways for therapy in hormonal cancers.

**Keywords:** CPT1A; prostate cancer; fatty acids; serine; androgen response; ROS; oxidative stress

#### **1. Introduction**

Prostate cancer (PCa) is the most common cancer in men and the second highest contributor to cancer deaths [1]. Although initially highly effective as a treatment for metastatic PCa, androgen deprivation therapy is characterized by a predictable emergence of resistance, a disease state termed castration-resistant prostate cancer (CRPC) [2]. An important feature of CRPC is the reactivation of androgen receptor (AR) signaling, an event reflected by progressive rises in serum prostate-specific antigen (PSA), a gene product regulated by the androgen receptor (AR) [3]. Substantial evidence has documented that the majority of AR-regulated genes (androgen-response hallmark genes) are re-expressed in most CRPCs, and several mechanisms capable of maintaining AR activity have been established [4,5]. Alternatively, disease progression can arise in the absence of a functional AR as an extremely aggressive and metastatic variant called small cell or neuroendocrine PCa [6,7]. Although genetic alterations are known to promote this aggressive state of the disease [8], we currently lack mechanistic insight as to which metabolic pathways play important roles in PCa plasticity, resistance and progression to lethal disease.

Mitochondrial fatty acid β-oxidation is the major pathway for the catabolism of fatty acids, and it plays an essential role in maintaining whole body energy homeostasis. The transfer of fatty acids into the mitochondria for oxidation happens in an organized, controlled way. The enzyme carnitine palmitoyl transferase I (CPT1A) resides in the outer mitochondrial membrane and catalyzes the reversible transfer of acyl groups between coenzyme A (CoA) and L-carnitine, converting acyl-CoA esters into acyl-carnitine esters. These acyl-carnitines can then enter the matrix where β-oxidation takes place [9]. The activity of CPT1A produces a greater number of acetyl groups, which can be used for energy, de novo lipid synthesis and acetylation reactions, including histone acetylation in the nucleus [10].

Support for a strong association between specific dietary lipids and PCa is still unclear. For example, a recent paper showed that a high fat diet promotes metastatic prostate cancer [11]. Although this is a great step forward in understanding the clinical role of dietary lipids promoting metastatic PCa, it does not address how tumor cells use the lipids to their benefit. Particularly, it is not clear how CPT1A activity could promote a metabolic environment conducive to cell transformation, cancer progression and drug resistance [12].

Targeting lipid catabolism enzymes in PCa may offer new avenues for therapeutic approaches [13]. During the last decade, CPT1A has been identified as a potential therapeutic target for a growing list of cancers [14]. In these cancers, CPT1A expression is increased, and/or its inhibition has been reported to have antitumor effects. Our group has shown that fatty acid oxidation is an important pathway in cancer metabolism in PCa cells when using the CPT1 inhibitor etomoxir [15–17]. The blockade of CPT1A with etomoxir produced unresolved endoplasmic reticulum (ER) stress leading to cell apoptosis and decreased tumor growth in vivo. Indeed, lipid oxidation in the mitochondria of aggressive cancer cells can provide adaptation to stress by promoting the generation of antioxidant molecules, like Nicotinamide adenine dinucleotide phosphate (NADPH) [18].

Loss of balance between the antioxidant defense and oxidant production in the cells, which commonly occurs as a secondary feature in many diseases, is loosely termed "oxidative stress". This balance is important because the intracellular redox environment must be more reducing than oxidizing to maintain optimal cell function. Oxidative stress and ER stress are linked to multiple pathologies, including metabolic, neurodegenerative, immune, and neoplastic diseases. Studies on these two cellular stresses have not only contributed to our understanding of disease, but also opened new avenues to next-generation therapies for these illnesses [19,20]. Their exact role in supporting cancer survival and drug resistance remains unknown.

Since oxidative stress is usually coupled with ER stress [21], the unfolded protein response (UPR) has evolved to handle both proteins folding defects and oxidative challenge. In fact, excess nutrients are proposed to induce ER stress and oxidative stress in pancreatic beta-cells, including glucotoxicity and lipotoxicity. For example, chronic excess glucose induces the pro-apoptotic UPR and oxidative stress in beta-cells cells in vitro and in mice models [22]. In addition, free fatty acids, such as palmitate, induce ER stress and oxidative stress and cause apoptosis in β cells [23]. Cancer cells, especially hormone-dependent cancers, like PCa, exploit these stress responses to deal with the oncogenic and metabolic reprograming, making them more adaptable to affront the insults from drug cancer therapies. Indeed, conditional knockout of the ER chaperone GRP78 (HSPA5) in the prostate of mice with PTEN inactivation suppressed prostate cancer growth [24]. The role of CPT1A in supporting adaptive ER stress responses and redox regulation has not been studied.

In this study, we have tested the hypothesis that excess CPT1A plays a key role in supporting adaptation to stress and antioxidant defense production in CRPC cells. We have used the LNCap-C4-2 model as it represents advanced CRPC that can grow in androgen depleted conditions and form metastasis [25]. We have also explored publicly available clinical databases to support our findings. Specifically, we have studied molecular differences between CPT1A gain and loss of function C4-2 models, revealing metabolic vulnerabilities that could be targeted to avoid progression to neuroendocrine differentiation.
