Background
Pancreatic cancer is the fourth leading cause of cancer-related death worldwide [
1]. Although the incidence of pancreatic cancer is relatively low, approximately 3.2% of all new cancer cases in the United States, the 5-year survival rate is 8.5% in patients diagnosed with pancreatic cancer. In addition, the detection rate of early pancreatic cancer remains low due to the lack of specific symptoms. Accordingly, most patients (52%) are diagnosed with distant metastasis [
2], and, unfortunately, the 5-year relative survival of patients with metastatic pancreatic cancer is less than 2% [
3]. Although smoking and health history can affect the risk of pancreatic cancer, the pathogenesis of pancreatic cancer development is not completely understood. Therefore, exploring the molecular features of pancreatic cancer growth and death is vital to control the disease progression and bring more clinical benefits to patients with pancreatic cancer.
The biological behavior of cancer is closely regulated by mitochondria [
4,
5]. Sufficient ATP supply, intracellular calcium homeostasis, metabolic signaling transduction, and cell apoptosis management are affected by mitochondria [
6‐
8]. In addition, mitochondria are also the key target of several chemotherapeutics and radiotherapies [
9]. A recent study has reported that pancreatic cancer death, proliferation and metastasis are modulated by mitochondrial homeostasis, especially mitochondrial fission [
10]. Excessive mitochondrial fission induces cancer cell oxidative injury and subsequently mediates mitochondrial ATP depletion; this effect impairs PANC-1 cell proliferation and evokes mitochondrial apoptosis [
10]. Notably, this conclusion is also supported by other studies. In colorectal cancer, the activation of mitochondrial fission is associated with SW837 cell apoptosis and migration inhibition [
11]. In gastric cancer, abnormal mitochondrial fission contributes to cancer cell oxidative stress and energy undersupply [
12]. In breast cancer, Drp1-mediated mitochondrial fission suppresses breast cancer cell invasion [
13]. This information indicates that mitochondrial fission has a well-characterized role in the regulation of cancer viability. However, the downstream molecular events of mitochondrial fission activation remain to be discovered.
Based on a previous study in a mouse model of cardiac ischemia reperfusion injury, the activation of mitochondrial fission promotes the formation of mitochondrial fragmentation, and these mitochondrial debris contain a decreased mitochondrial potential [
14]. In addition, mitochondrial fragmentation can activate cell death via two mechanisms [
15]; one mechanism is driven via HK2/VDAC1 disassociation-mediated mPTP opening, and the other involves mROS-induced cardiolipin oxidation. Notably, mitochondrial ROS (mROS) overloading, as a primary result of mitochondrial fragmentation [
16], has been noted in different disease models such as those of gastric cancer [
17], breast cancer [
18], and leukemia [
19]. Subsequently, excessive mitochondrial oxidative injury can activate the HtrA2/Omi-related apoptotic pathway in a manner that is dependent on caspase-9 activity [
11]. This evidence indicates that the downstream effectors of mitochondrial fragmentation include mROS overproduction, HtrA2/Omi upregulation, caspase-9 activation and mitochondrial apoptosis augmentation. Given these factors, we want to know whether mitochondrial fragmentation regulates pancreatic cancer viability via mROS-HtrA2/Omi-caspase-9 pathways.
To this end, erlotinib is the first-line anti-tumor drug for the treatment of pancreatic cancer in the clinic [
20]. Several human studies have verified the efficacy of erlotinib in improving the 5-year survival rate of patients with pancreatic cancer [
21,
22]. Molecular investigations report that several biological processes are modulated by erlotinib, including mTOR inhibition [
23], epidermal growth factor receptor downregulation [
24], and epidermal interstitial transformation (EMT) suppression [
25]. However, no study that explores the role of erlotinib in triggering mitochondrial stress has been conducted. In the present study, erlotinib was applied to activate mitochondrial fragmentation in a human PANC-1 pancreatic cancer cell line. Then, we explored the regulatory mechanism of mitochondrial fragmentation on cell viability in the presence of erlotinib.
Discussion
According to the previous findings, mitochondrial fission has been acknowledged as a potential target to reduce the proliferation, migration and survival of PANC-1 pancreatic cancer cells [
10]. Excessive mitochondrial fission promotes mitochondrial fragmentation [
15]. Fragmented mitochondria induce damage to mitochondrial structure and function, eventually interrupting the cellular ATP supply and activating the apoptosis response [
47,
48]. However, the detailed molecular mechanism by which mitochondrial fragmentation triggers mitochondrial damage and cellular apoptosis remains unclear. Our study provides an answer to this question. We used different doses of ERL to screen its proapoptotic effect in two types of cancer cell lines. Then, we used the minimal lethal dose of ERL to investigate its apoptotic mechanism, with a focus on mitochondrial damage. We observed the minimal lethal dose of ERL has an ability to induce the mitochondrial fragmentation and this finding may explain one of the mechanisms by which ERL mediated cancer cell apoptosis. Notably, whether higher dose of ERL could activate other signaling pathway to induce cell apoptosis requires further investigation. Our data illustrated that erlotinib treatment promoted mitochondrial fragmentation that occurred via increased mitochondrial fission and decreased mitochondrial fusion. Subsequently, excessive mitochondrial fragmentation triggered mROS overloading, leading to cellular oxidative stress and disordered energy metabolism. In addition, mROS overproduction was closely associated with cardiolipin oxidation and mPTP opening, favoring HtrA2/Omi liberation from mitochondria into the cytoplasm. As a consequence of HtrA2/Omi leakage, reduction of the mitochondrial potential and caspase-9 activation were noted, and these alterations were accompanied by an upregulation of proapoptotic proteins and a downregulation of antiapoptotic factors. Overall, we demonstrated for the first time that erlotinib-activated mitochondrial fragmentation mediated PANC-1 apoptosis via the mROS-HtrA2/Omi pathways. This finding fills the knowledge gap regarding how mitochondrial fragmentation induces mitochondrial damage and triggers the apoptotic pathway.
Mitochondrial fission and fusion are a part of mitochondrial dynamics. Under physiological conditions, the mitochondrial network undergoes moderate fission and fusion to fill the requirements for cellular metabolism [
49,
50]. Mild levels of mitochondrial fission help the mitochondria in generating daughter mitochondria, whereas moderate levels of mitochondrial fusion provides the energy for communication between the mitochondrial network [
51,
52]. Interestingly, uncontrolled mitochondrial fission generates massive amounts of fragmented mitochondria and disrupts mitochondrial homeostasis. Previous studies have identified mitochondrial fragmentation, which is produced by mitochondrial fission, as the apoptotic trigger in various disease models. For instance, in fatty liver disease, mitochondrial fragmentation promotes the apoptosis of hepatocytes and the progression of liver fibrosis by decreasing mitophagy [
53]. In neurodegenerative illness such as Alzheimer’s disease, excessive mitochondrial fragmentation disturbs mitochondrial energy metabolism and causes neuronal oxidative injury [
54]. In addition, in rectal cancer, activated mitochondrial fragmentation limits tumor proliferation and augments cancer apoptosis [
11]. In accordance with these findings, our data also illustrated the necessary role played by mitochondrial fragmentation in initiating pancreatic cancer PANC-1 cell death. Thus, mitochondrial fragmentation would be considered as a tumor-suppressor, and strategies to promote mitochondrial fragmentation are of significant importance in the design of anti-cancer drugs.
Although the proapoptotic effect of mitochondrial fragmentation has been well-documented, the detailed mechanisms by which mitochondrial fragmentation induces mitochondrial damage and activates cellular apoptosis are incompletely understood. In the present study, we found that mitochondrial fragmentation modulated mitochondrial homeostasis and cell viability through two mechanisms. One mechanism was driven by the promotion of mROS-mediated cell oxidative injury, and the other involved the HtrA2/Omi liberation-induced caspase-9 activation. First, mitochondrial fragmentation generated superfluous amounts of mROS, and the excess mROS induced cardiolipin oxidation and mPTP opening [
55]. Subsequently, oxidized cardiolipin and increased mPTP opening worked together to augment the liberation of HtrA2/Omi from mitochondria into the cytoplasm, where Htra2/Omi reduced the mitochondrial potential and induced caspase-9 activation. This information was also consistent with previous studies. In cardiac ischemia–reperfusion injury, excessive mitochondrial fragmentation-induced mitochondrial DNA damage evokes mROS overproduction and cardiolipin oxidation [
14,
15]. Additionally, in oral cancer, mitochondrial fragmentation-related cardiolipin oxidation and mPTP opening eventually contribute to caspase-involved cellular apoptosis [
56].
In the present study, we used erlotinib to activate mitochondrial fragmentation and found that erlotinib-mediated PANC-1 cellular apoptosis could be inhibited by Mdivi-1, which is an antagonist of mitochondrial fragmentation. To the best of our knowledge, this is the first study to investigate the role of erlotinib in mitochondrial stress. Although erlotinib has been tested in several human clinical studies [
57,
58], its pharmacological mechanism has not been adequately explored. Our study proposed that the anti-cancer property of erlotinib relied on the activation of mitochondrial fragmentation by upregulating mitochondrial fission and downregulating mitochondrial fusion. Notably, the dose selection of ERL was according to a previous study [
26] and this selection may be also relied on the types of cancer cell lines. In clinical practice, different doses of ERL have been used according to the tumor staging and pathologic grading. Further insight is required to figure out the appropriate concentration of ERL on different types of pancreatic cancer. Besides, there are several limitations in the present study. Although we used two pancreatic cancer cell lines to screen the role of erlotinib, an animal study is necessary to further support our finding. In addition, human evidence is also required to validate the tumor-suppressive effects of mitochondrial fragmentation in response to erlotinib treatment.
Authors’ contributions
JW, JC, and LW were involved in the conception and design, performance of experiments, data analysis and interpretation, and manuscript writing. KPW, XPH, YLZ, HK and SZ were involved in data analysis and interpretation. All authors read and approved the final manuscript.