Introduction

A body is made up of many living cells, which grow, multiply and generate new cells and die after some time in an accordingly manner called apoptosis. In case of cancer, body cells start growing out of control. They cannot die and grow deliberately to form new cells and form their own blood vessels through angiogenesis. Subsequently, cancer cells metastasise into the nearby organs, when their growth is not diversified or arrested. At this stage, cancer cells replace normal tissues and form new mass termed as tumor. There are eight endorsements of cancer participating in the physiology of cancer: self sufficient pro growth signalling, loss of sensitivity to antigrowth signals, resistance to cell death, replicating without limit, angiogenesis, invasion and metastasis, reprogrammed metabolism and evading immune surveillance [1, 2].

Cells manage a wide range of functions including growth, movement, housekeeping and many more which requires energy. The energy is liberated from the chemical bond in food molecules and serves as fuel for the cells through a string of oxidation reduction process. Sugar molecule plays a very paramount role, as they are oxidized in small steps to carbon dioxide and water. A large array of mechanisms are involved in the breakdown/catabolism of sugars to produce adinosine tri phosphate (ATP), nicotinamide adenine di phosphate (NADPH) and other molecules [3].

The normal cells and cancer cells are peculiar from each other in their intermediatry metabolism, which helps the cancerous cells to survive in hypoxic microenvironment. Cancer cells get energy from the process of glycolysis rather any other process, as more ATP is generated via glycolysis (Warburg effect) [4]. During glycolysis, two molecules of pyruvate with a net gain of two molecules of ATP are generated from the metabolism of one molecule of glucose. Under normoxic condition, the end product of glycolysis, i.e., pyruvate get converted into acetyl CoA which works as the starting material for citric acid cycle and oxidative phosphorylation, yielding about 34 ATP molecules. During tumor hypoxia (low oxygen level), cells goes through shift to glycolytic pathway for the stipulation of energy (Fig. 1) [5]. Thereby, tumor hypoxia and hypoxia inducible factors (HIFs) are the important pathway for tumorigenesis and angiogenesis. HIF can also get triggered under normal condition of oxygen (normoxia) by the loss of Von Hippel Lindau tumor suppressor protein (pVHL) [6].

Fig. 1
figure 1

Energy requirement for normal cell and for cancer cell. Metabolic differences between normal and cancer cells. Normal cells, primarily metabolizes glucose into pyruvate(end product of glycolysis) for survival and growth followed by citric acid cycle and as a result thirty-four ATP molecules are generated. In cancer cells, there is increased glucose uptake but there is inefficient utilization of glycolytic end product and pyruvate is converted into lactate which may leads to biomass incorporation and cancer cell proliferation. This effect is termed as “Warburg effect”

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Hypoxic microenvironment in tumor cell is a result of high oxygen consumption within the tumor vasculature by rapidly procreating tumor cells. To support the unceasing growth in the hypoxic microenvironment, cancer cells are known to stimulate alternate metabolic pathways including anaerobic metabolism, increased signalling of growth factors, diversified regulation of cell cycle, cellular proliferation and protein catabolism. During hypoxia, cancer cell derive energy first and foremost through glycolysis as oxygen is not available to derive acetyl CoA from pyruvate. This change in metabolic pathway during normoxic condition is conventionally termed as “Warburg effect.”

HIF activation also vitiates mitochondrial respiration by making HIF a key regulator of cancer cell metabolism along with stimulation of glycolysis [7, 8]. It has been reported that there are alterations in lipid metabolism and high rates of de novo fatty acid biosynthesis in tumor cells [9]. Fatty acid synthase (FASN) was identified as the breast tumor associated protein and there are diversified studies to show that FASN is overexpressed in tumor cells including mammary gland carcinomas. According to various clinical and preclinical studies, it is clear that human cancer cells have the capacity to synthesize their own fatty acid which is autonomous of the regulatory signals. The mechanism which is culpable for FASN up regulation in cancer is unknown [10–13]. Nonetheless, FASN appears to be an attractive target in the management of lipoidal tumors and thereby has aimed increased response. Notwithstanding, the present review was ventured to elucidate the various aspects of tumor hypoxia arbitrated regulation of FASN along with the present and future aspects of modulating FASN overexpression through prolyl hydroxylase (Phds).

Tumor hypoxia

Tumor hypoxia is a situation where the level of oxygen is decreased in tumor cells. Oxygen is the primitive requirement for a cell to grow [14–16] and most of the solid tumors are hypoxic in nature due to the limited amount of vasculature, poor tumor vessels with chaotic architecture [17]. In normal cells, hypoxia is associated with decreased proliferation and differentiation. Hypoxia decreases the production of energy and cell viability, it arrests cell growth and induces cell death [18, 19]. Hypoxic environment is also responsible for the activation of anaerobic metabolism, signalling of growth factors, regulation of cell cycle, cell proliferation and protein catabolism. These processes enable tumor cells to survive in a poorly oxygenated conditions [20, 21]. Therefore hypoxia enhances the expansion of tumor cells with diminished apoptotic potential [22]. HIF is the main effecter of oxygen homeostasis, allocating various genes involved in biological processes like angiogenesis [23, 24]. HIF also binds to the vascular endothelial growth factor gene which is obligatory for transactivation of vascular endothelial growth factor (VEGF) in response to hypoxia. This type of binding promotes the proliferation, which accompany the formation of blood vessels. HIF-1α plays a very imperative role in metabolic angiogenesis and is proclaimed to be highly expressed in a wide variety of human cancers [25].

Under normoxic condition, prolyl hydroxylation allows the interplay of HIF-1α to pVHL and acts as recognition component of E3 ubiquitin ligase and subsequently HIF-1α goes through proteosomal degradation [26–28]. In hypoxic condition, HIF-1α fails to interact with pVHL mediated degradation and relocates into the nucleus to form a complex with HIF-1β (Fig. 2). This complex can actuate the transcription of target genes with the help of core hypoxia response element (HRE) [29, 30]. HIF-1α also interacts with p300/CBP co-activator complex which is regulated by oxygen and Asn 803 (human HIF-1α). The HIF C terminal transactivation domain is also catalyzed by another 2-OG oxygenase named as factor inhibiting HIF (FIH), who blocks the HIF p300 interaction [31, 32]. Henceforth, two factors that can control the HIF activity: Phds and FIH [27, 33, 34]. HIF-1α is forthwith perceived as a major molecular target for anti-cancer therapy [35]. Notwithstanding, the Phds and FIH are foreseen as the factors, who can control the HIF activity, making HIF-1α as a major molecular target for anticancer activity.

Fig. 2
figure 2

Effect of normoxic and hypoxic condition on cellular proliferation. In the presence of oxygen, Phd-2 oxidize HIF-1α which is recognized by Von Hippel Linadu protein (pVHL) and then HIF-1α get ubiqutinized followed by proteosomal degradation of HIF-1α. Conversely, when Phd-2 is not active in hypoxic condition then it is not recognized by pVHL and HIF-1α get translocates to the nucleus and forming a complex with HIF-1β and stimulates a number of processes like vascular endothelial growth factor (VEGF) induced angiogenesis

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Prolyl hydroxylase

Phds domain proteins are the group of enzymes, who can hydroxylate HIF-1α and supervise its activity. Phds belongs to a 2-oxoglutarate (2OG)-dependent dioxygenase superfamily [36, 37]. Along with 2OG, these enzymes require Fe2+, oxygen and ascorbate to modify the functions of HIF-1/2α. There are four isoforms of this enzyme, named as Phd-1, Phd-2, Phd-3 and Phd-4 [38]. In relation to HIF-1α, Phd-1 and Phd-2 are found to be very comparable (407 and 426 amino acid residue proportionately) while Phd-3 is found to be short (only 236 amino acid residues). Phd-4 is recorded to be the larger one with 502 amino acids [39, 40]. Phd-4 has been found to be appended with membrane and its active site is located into the lumen of endoplasmic reticulum [41]. Phds possess a double stranded β-helix core fold in which Fe2+ is bound with catalytic centre [42]. Phd-2 is ubiquitously expressed, while Phd-1 is expressed in placenta and Phd-3 in the heart. Role of Phd-2 in tumor angiogenesis is controversial [43, 44]. Some authors have reported that, tumor vasculature is regulated by Phd-2. Whereas others observed that Phd-2 expression is reduced and lead to the normalization of blood vessels with reduced tumor metastasis [45, 46]. A modest set of researchers have also divulged that Phd-2 deficiency is associated with cell invasion and metastasis in pancreatic tumor cells, thereby suggesting its tumor promoting nature [46, 47]. By and large, Phd-2 is perceived to reduce tumor metastasis and few of the activators of Phd-2 have been reported to manifest anticancer activity [48, 49].

Fatty acid synthase (FASN)

FASN is a major enzyme for lipogenesis. It is a complex multienzyme protein that contains seven catalytic domains [50–54]. The major function of FASN is to catalyze the condensation of acetyl CoA and malonyl CoA to produce palmitic acid in the presence of NADPH. FASN is allocated in two classes: type I and type II. FASN I is a multifunctional polypeptide and very common in mammals and fungi. FASN I produces palmitic acid and cooperate with FASN II for the production of lipid products [55]. FASN II is commonly found in archaea and bacteria and it is symbolized to be a multifunctional enzyme [56]. In highly lipogenic tissues like liver, lactating breast and adipose tissues, FASN have three major functions:

  1. I.

    Storage of profound excess energy in the form of fat.

  2. II.

    If the diet is low in fat then the synthesis of fat from carbohydrate or protein.

  3. III.

    Synthesis of fat for lactation.

Analogues to normal cells, the majority of fatty acid in cancer cells are derived from de novo synthesis rather than dietary fats [57, 58]. Upsurge in lipogenesis is reflected as an inflated activity of lipogenic enzymes such as ATP citrate lyase and FASN [59, 60]. The FASN level is overexpressed in lipoidal tumor cells and is one of the most common molecular changes which occur in cancerous cell [60–64]. Generally, glycolysis increases the uptake of oxygen in cancer cells away from blood vessels and consumes glucose which results in accumulation of pyurvate and lactate. This exhaustion of glucose and agglomeration of lactate is the main cause of hypoxia induced apoptosis [65]. A decent set of FASN inhibitors like cerulenin, C75 and orlistat [66] are reported to urge cell death in a variety of tumors mainly lipiodal tumors [60–64]. FASN inhibitors can cause cell death and are considered to be a particularised target for the treatment of cancer. It also has been proclaimed that the essential parts of FASN act as energy substrates for the cells. Thereby inhibition of FASN induces decrease in the production of lipids in the tumor cells by which the tumor cells cannot get the energy for their propagation [67, 68].

FASN and mammary gland carcinoma

A unique pathophysiological microenvironment including hypoxia, low pH and nutrient starvation implicates in the mammary gland tumors [69]. This type of environment triggers several intracellular signalling pathways and can induce the level of FASN [66]. It was reported that hypoxic microenvironment can upregulate sterol regulatory element-binding protein (SREBP), which is a major transcriptional regulator of FASN gene through Akt phosphorylation [70]. FASN overexpression is a well reported phenomenon in the preclinical and clinical cases of mammary gland carcinoma [60].

Researchers have reported increased expression of FASN in MNU and DMBA induced mammary gland carcinoma in mouse model and thereby considered it as a viable target for chemoprevention [71–73]. Inhibition of FASN has been reported to be associated with reduced synthesis of some essential fatty acids requisite for the cell growth [74, 75]. In line to the animal studies, FASN is reported to be highly expressed in hormone independent SKBR3 and hormone dependent MCF-7 and ZR-75-1 cell lines as well [75–77]. Over expression of FASN has been linked with poor prognosis and reduced disease free survival in many cancer types including mammary gland carcinoma [13, 78, 79]. Infact, several cohort studies have linked the FASN overexpression with poor patients survival and chemoresistance [79–81]. Authors would also like to mention that chemoresistance in cancer patients have been linked with FASN overexpression mediated palmitate overproduction [82]. Clinical studies revealed that the stage 1 of breast cancer patients with high levels of FASN expression have fourfold increased risk for death [59]. Together with previous findings for FASN as a poor prognostic marker for breast cancer patients, we suggest that FASN has a key role to play in drug resistance and can be considered as an ideal target for chemosensitization in breast cancer chemotherapy.

HIF and tumor microenvironment

Hypoxia plays a paramount role in the embryonic development and human physiology [83–85]. Almost all cancerous cells are reported to be hypoxic in nature and their proliferation is resolved through HIF-1 activation [86, 87]. Henceforth, HIF-1α plays a momentous role in tumor proliferation [88]. Energy is implemented to the cells with the help of angiogenesis which fulfil the oxygen supply [89]. This energetic shift in cancerous cell coordinates with the variety of enzymes, which are diversified in the process of glycolysis for example glucose transporters (aldolase A and pyruvate kinase) [90]. These transporters help the cells to produce energy in the hypoxic micrenvironment [89]. This constitutive activation of anaerobic metabolism in tumor cells provides a relationship of cell response to the oxygen deficiency [91, 92]. Thus HIF-1α function in hypoxic environment is involved in VEGF arbitrated angiogenesis, increased glycolysis and other steps in tumor progression [93].

Regulating Phd-2 to counteract tumor hypoxia and FASN over expression

Initially it was found that Phd-2 is the most critical hydroxylase in a variety of cultured cells and could be attributed to the fact that Phd-2 is more profoundly expressed than other Phds [38, 94]. The activity of Phds can be restrained by post translational mechanisms including proteosomal degradation and modulation of Phds activity by collaborating proteins. It has been reported that all Phds can downregulate HIF-1α in vitro, however the specific role of Phds in cancer is still inconclusive [17]. In tumor hypoxia, there is a shift to glycolytic mechanism as respiration is not feasible without oxygen. Tumor hypoxia and activation of HIF-1α is an important pathway that contributes to so many processes like tumorigenesis, angiogenesis, up surged glycolysis and tumor cell survival. HIF-1α is a key regulator of cancer metabolism [6, 7] and glycolysis is a major target of cancer cells for the stipulation of energy. The end product of glycolysis is pyruvate which can be persuaded into cytosolic lactate which is secreted or converted into mitochondrial acetyl CoA and which is mediated into citrate within the mitochondria. Citrate can be processed by the citric acid cycle or exported to the cytoplasm where it is cleaved by ATP citrate lyase and generate cytosolic acetyl CoA which is a building block for fatty acid biosynthesis.

Hypothesis

Notwithstanding, herewith it is hypothesized that the chemical activators of Phd-2 would cause accelerated deterioration of HIF-1α through proteosomal degradation which can leads to the favourable outcomes against cancer. With the help of Phd-2 activators, the activity of HIF-1α will be curtailed and this will cause decrease in glycolytic pathway and subsequent diminution in the FASN level which is recorded to be over expressed in tumor cells (Fig. 3). On the account of curtailed glycolysis, one can expect decreased pyruvate and subsequently lactate formation. As mentioned earlier, the lactate formation significantly participates in the biomass formation in the cancerous cells. FASN is a multifunctional enzyme participating in citric acid cycle and is overexpressed in the fastly growing cancerous cells to meet the energy requirements. Pyruvate (end product of glycolysis) is converted into citric acid (starting material of citric acid cycle). Diminshed glycolytic activity due to HIF-1α proteosomal degradation (due to Phd-2 activation) can lead to scarcity in the pyruvate formation subsequently citric acid synthesis in cells would be curtailed leading to abatement in the citric acid cycle and FASN overexpression. All in all, we hypothesize that activation of Phd-2 can affect cancerous cells in a multidirectional way. Firstly, by inhibiting the activation of HIF-1α (a well established target in cancer chemoprevention); secondly, decreased production of lactate may help to cut down the biomass formation and lastly, by downregulating the FASN overexpression through curtailing the citric acid cycle (Fig. 4).

Fig. 3
figure 3

Regulatory pathway for FASN and its modulation by HIF-1α and Phd-2. In normoxic condition, HIF-1α interacts with pVHL followed by proteosomal degradation. In hypoxic condition HIF-1α cannot recognize by pVHL and translocates into the nucleus and complex with HIF-1β. HIF-1α is regulated by iron and 2-oxoglutarate dependent dioxygenase family termed as Phd-2. In nucleus the glycolysis takes place along with Pentose Phosphate Pathway (PPP) which results in pentose sugar and Nicotinamide adenine dinucleotide phosphate (NADPH). The end product of glycolytic pathway is pyruvate which gets converts into citrate. The citrate participates in the citric acid cycle and the resultant product is 16 C polyunsaturated palmitic acid in the presence of fatty acid synthase (FASN)

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Fig. 4
figure 4

Hypothesized outcomes of activation of Phd-2. In hypoxic condition, HIF-1α escapes proteosomal degradation and translocates into nucleus where targets the glycolytic pathway and fatty acids for the requirement of energy and the resultant is overexpression of FASN. Whereas in normoxic condition, Phd-2 gets activated and HIF-1α is degraded so decrease in glycolytic activity and decrease in pyruvate and lactate formation and the resultant is downregulation of FASN expression. The hypothesized outcomes of activation of Phd-2 is inhibition of VEGF mediated angiogenesis, decrease in biomass production and decrease in lipid production and palmitate mediated chemoresistance so tumor cells cannot propagate

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Present scenario and future directions

As elaborated above, modifying the Phd-2 could be a viable target for cancer prognosis, which can be contemplated to have a momentous effect on the glycolytic pathway and thereby FASN expression in lipoidal cancers. It is to be noted that only three activators of Phd-2 namely KRH102053, KRH102140 and R59949 has been reported till date, with KRH102053 and KRH102140 having a conspicuous effect on angiogenesis [95, 96]. On the contrary, the R59949 (diacyl glycerol kinase II inhibitor) was also recorded with Phd-2 activating potential without much affecting the tumor progression [97]. It would be important to pen down that apart from activating the Phd-2, other physiological effects of KRH102053 and KRH102140 are unelucidated till date. In short, the underlying mechanism of these compounds is poorly understood, which we are in opinion could be transmutations in pathways of energy production and subsequent alteration in FASN over expression. We would also like to mention that the effect of KRH102053 and KRH102140 on glycolytic pathway and subsequent FASN overexpression could be a research question for future endeavours.

Down regulating the FASN over expression in lipoidal cancer cells to customize tumor growth is a well studied and established phenomena. In fact, a sufficient number of FASN inhibitors (e.g., cerulenin-3-derivative C75, β-lactone orlistat, green tea polyphenol epigallocatechin-3-gallate (EGCG) and other naturally occurring flavonoids as well as antibiotic triclosan) are proclaimed to have a profound effect on cell proliferation, angiogenesis, metastasis and apoptosis [98].

Most of the reported compounds are broad spectrum FASN inhibitors and mediate their action through ameliorating β-ketoacyl synthase. The compounds are well documented for their anti-cancer activity owing to their FASN inhibiting potential [10]. However, repercussion of FASN inhibition on glycolytic pathway, regulation of HIF-1α and consequent effect on Phd-2 needs to be elucidated to full and stands still as a research question.

Additionally, most of the FASN inhibitors have several metabolic and pharmacological limitations which restrict their transformations from preclinical to clinical phase of drug discovery. Authors are in opinion that these impediments are the repercussion to the fact that FASN overexpression is the later consequence of hypoxic situation in cancerous cells and merely prohibiting the FASN, will not cater compelling detrimental effects on the tumor progression. In fact, we are not hesitant to put on records that activation of Phd-2 appears to be a more viable and constructive targets in terms of overseeing the hypoxic microenvironment and FASN overexpression in expeditiously dividing tumor cells.

With all above, authors are in strong accredit that activating Phd-2 could be a generous target to look after cancer progression and is hypothesized to be effectuated through down regulation of FASN over expression.