5. ATPase Inhibitory Factor 1, the Physiological Inhibitor of the ATP Synthase
As mentioned above, the control of the overall ATP synthase activity is not only exerted at the protein level, but also by regulation of the content and activity of IF1, its inhibitor protein. For many years, it has been considered that under normal physiological situations, i.e., when the mitochondrial matrix pH is above neutrality, IF1 forms inactive oligomers by masking the inhibitory regions that bind to the F
1 domain of the enzyme (
Figure 2b)
[104][105][175,176]. Only when mitochondria become de-energized, and matrix pH drops below neutrality, IF1 is considered to become activated by depolymerization and binds to the ATP synthase to inhibit its hydrolase activity (
Figure 2b)
[104][105][175,176].
Studies in metabolic reprograming in human carcinomas revealed that normal lung, colon and breast tissues express negligible quantities of IF1
[33][85] but showed a sharp increase in the expression of the protein in the carcinomas and in cultured cancer cells
[30][31][33][74,75,85]. These findings raised the reasonable hypothesis that IF1 might represent a sort of mitochondrial “oncogene”
[106][178], not only to control the hydrolytic activity of the ATP synthase, as it was postulated many years ago, but also its ATP synthetic activity. In other words, the overexpression of IF1 could be mediating the partial inhibition of OXPHOS contributing to the metabolic reprogramming experienced in oncogenesis. Indeed, the overexpression of IF1 in several cancer cells
[30][31][33][107][108][74,75,77,78,85], mouse models of gain of function of IF1
[32][34][35][36][37][44][79,80,81,82,84,89] and pharmacologic in vivo approaches
[109][76], amply demonstrated that IF1 overexpression inhibits the ATP synthetic activity of ATP synthase in isolated mitochondria, diminished tissue ATP levels, activated AMPK and promoted the metabolic rewiring of cells and tissues to an enhanced glycolytic phenotype, i.e., induced the Warburg phenotype. Conversely, silencing or knocking out IF1 in cancer cells
[68][110][20,179] and mouse models of loss of function of IF1
[34][45][82,83] resulted in and enhanced activity of the ATP synthase.
Stem cells, iPSC and cancer cells have a predominant glycolytic phenotype whereas OXPHOS prevails in differentiated cells
[111][112][180,181]. Interestingly, IF1 also plays a relevant role in metabolic reprogramming during differentiation of human mesenchymal stem cells (hMSC) and in the maintenance/acquisition of stemness
[113][182]. In fact, hMSC also present high levels of IF1, altered mitochondrial structure and molecular composition, low rates of OXPHOS and an enhanced glycolytic metabolism
[113][182].
Remarkably, the results in tissues of mouse models with regulated expression of IF1 further suggest that mitochondria in non-stressed physiological conditions of some cellular types such as cardiomyocytes
[109][76], neurons
[34][82] and intestinal epithelial cells
[45][83] contain a fraction of IF1-bound and -inhibited ATP synthase, in agreement with recent cryo-EM studies of mammalian heart ATP synthases
[46][47][90,91].
The regulation of IF1 activity as an inhibitor of ATP synthase is also exerted by covalent modification of the protein by phosphorylation (
Figure 2b). Phosphoproteomic studies indicated that serine residues of IF1 are phosphorylated in cancer cells
[114][115][116][185,186,187].
Indeed, the phosphorylation of IF1 has been observed when there is an increase in energy demand in the heart in vivo or in cells in cultures when they are forced to increase OXPHOS
[109][76]. Likewise, IF1 is phosphorylated when cells progress through the OXPHOS-dependent G1-phase of the cell cycle
[109][76]. In contrast, hypoxia and progression through the reductive phases of the cell cycle (S/G2/M) promote dephosphorylation of IF1, the reduction of OXPHOS and an enhanced glycolytic flux
[109][76]. Consistently, dephosphorylated IF1 is the prevalent form of the inhibitor protein in lung, colon, and breast carcinomas, contributing to the inhibition of ATP synthase and the reprogramming of metabolism to an enhanced glycolysis and fermentation
[109][76].
Most available evidence suggests that regulation of IF1 expression is exerted at post-transcriptional levels by controlling the rates of its synthesis and degradation
[113][182]. In fact, the increase in protein content of IF1 in carcinomas
[33][85] or its sharp reduction during cellular differentiation
[113][182], are exerted in the absence of relevant changes in the abundance of IF1-mRNA. In fact, the IF1 protein has a very high turnover rate (2–3 h), both in cancer cells
[33][85] and in differentiated osteocytes
[113][182], which is much faster than the turnover of many other subunits of ATP synthase in cancer cells (18 h)
[117][193]. IF1 degradation is mediated by several mitochondrial serine proteases
[33][113][85,182] and metalloproteases
[118][194], but the specific genes involved have not been identified yet
[113][182].
6. Tissue-Specific Activity of IF1: Tumor Promotor and Tumor Suppressor
A fundamental characteristic of IF1 is that it is a tissue-specific and species-specific expressed protein in mouse and human tissues
[42][87]. Human normal tissues such as the heart, brain, kidney, stomach, endometrium and liver have a high IF1 content, whereas breast, colon, and lung epithelia contain negligible levels of IF1
[33][42][85,87]. Likewise, in human carcinomas the expression of IF1 is also variable
[33][85]. Whereas oncogenesis in epithelial cells of the endometrium, stomach and kidney does not promote an increase in IF1 expression, carcinomas in the colon, lung and breast show a very sharp increase in the content of IF1
[33][85].
In agreement with the pro-oncogenic role of IF1, studies in human hepatocellular carcinomas (HCC) showed that high levels of IF1 predict worse overall and progression free survival for the patients (
Figure 5)
[43][88]. Mechanistically, IF1 activates the non-canonical NFκB pathway, driving angiogenesis and metastasis through the activation of SNAI1 and VEGF
[43][88]. Consistent with these findings, transgenic mice overexpressing in the liver the constitutively active mutant IF1-H49K promoted the inhibition of OXPHOS and a state of metabolic preconditioning guided by the activation of the stress kinases AMPK and p38 MAPK
[36][80]. Diethylnitrosamine-induced liver carcinogenesis in IF1-H49K transgenic mice contributed to an enhanced liver carcinogenesis by augmenting proliferation and apoptotic resistance of carcinoma cells
[36][80]. Likewise, the overexpression of IF1 in bladder carcinomas
[119][190] and in gliomas
[120][191] also predict a worse prognosis for the patients and a shorter time to disease recurrence (
Figure 5). Mechanistically, the silencing of IF1 diminished the rates of proliferation, migration and invasive capacities of the cells, preventing epithelial mesenchymal transition (EMT) (
Figure 5)
[43][119][120][88,190,191]. In contrast, the overexpression of IF1 in these cancer cells promoted angiogenesis and activation of EMT, increasing the expression of E-cadherin and diminishing that of vimentin, to promote their metastatic capacity (
Figure 5)
[43][119][120][88,190,191].
Figure 5. The role of IF1 as tumor promotor or tumor suppressor. In bladder carcinomas, hepatocarcinomas and gliomas, high expression levels of IF1 (red) predict a poor prognosis for the patients when compared to carcinomas with low levels of IF1 (blue). In these carcinomas, IF1 acts as a tissue-specific tumor promotor facilitating proliferation, angiogenesis, epithelial mesenchymal transition (EMT) and metastasis. In contrast, in breast, colon and lung carcinomas, high expression levels of IF1 (red) predict a good prognosis for the patients when compared to carcinomas with low levels of IF1 (blue). In these carcinomas, IF1 acts as a tissue-specific tumor suppressor preventing epithelial mesenchymal transition (EMT) and metastasis by favoring cell death upon cellular detachment and immune surveillance.
7. Mitochondria as s Promising Target for Cancer Treatment
Novel therapeutic approaches based on personalized medicine are required to minimize the social and economic burden caused by cancer. The enzymes that control metabolism are promising targets to combat progression of the disease [3][121][122][123][124][9,14,26,200,201]. In this regard, several glycolytic inhibitors have been proposed and some of them are actually in clinical trials [3][69][124][125][126][127][128][26,51,152,201,202,203,204]. Mitochondrial metabolism also offers other effective targets to restrain tumor growth [124][127][129][130][131][132][201,203,205,206,207,208]. In this regard, antibiotics such as doxycycline that target the mitochondrial ribosome and inhibit translation prevent mitochondrial biogenesis which is required for the clonal expansion and survival of cancer stem cells (CSCs) [133][209]. Doxycycline has also been used in combination with other anticancer therapies [134][135][136][137][210,211,212,213] and their effectiveness lies in their ability to prevent metastasis by inhibiting the growth of CSCs.
In this line, it recently found that nebivolol, a third-generation β1-blocker, halts colon and breast tumor growth in vivo by a severe inhibition of OXPHOS [138][60]. Mechanistically, nebivolol binds to β1-adrenergic receptors in cancer cells, causing the increase in IF1 content bound to the ATP synthase and promoting its inhibition. Concurrently, nebivolol prevents the phosphorylation of NDUFS7, a Complex I subunit, which promotes partial inhibition of the activity of the enzyme [138][60]. In addition, nebivolol also arrests proliferation of endothelial cells by blocking β1-adrenergic receptors impeding tumor angiogenesis [138][60].