Regulation of the hypoxic tumor environment in hepatocellular carcinoma using RNA interference

The hypoxia inducible factors (HIFs) are a family of heterodimeric transcription factors that act as master regulators of a homeostatic transcriptional response to hypoxia in virtually all cells and tissues [3]. Active HIF consists of an alpha subunit and a beta subunit [2, 11, 12]. Three alpha subunits, termed HIF1a, HIF2a, and HIF3a, have been described in humans, mice, and rats; all bind to a common b subunit named, alternatively, HIF1b, or the aryl-hydrocarbon-nuclear receptor translocator (ARNT) [13, 14]. Active HIF is termed by its alpha subunit; hence, HIF1 is the active transcription factor consisting of HIF1α and ARNT, HIF2 is the dimer of HIF2α and ARNT, etc. [15]. HIF1 and HIF2 are the major hypoxia-inducible factors in humans, mice, and rats [13].

Under conditions of normoxia, HIF-1α subunits are hydroxylated at proline residues by hydroxylase enzymes [3, 16]. Hydroxylation of HIF1α and assembly on a protein scaffold consisting of the VHL tumor suppressor [17‐20], along with other cofactors, result in the rapid ubiquitination of the alpha subunit and subsequent degradation by the proteasome. Conversely, in conditions of hypoxia, HIFα subunits escape degradation and are free to dimerize with the binding partner, ARNT [3, 13]. The HIF trans-locates to the nucleus and affects transcription of target genes, typically by binding to a hypoxia response element (HRE) in the upstream promoter region of the target genes such as, angiogenesis, apoptosis, metabolism, survival related genes etc. [2, 3, 14].

Interlukine-8 (CXCL-8, IL-8) is a key factor of endothelial cell survival and angiogenesis [21]. IL-8 is also regulated under hypoxia and directly controlled endothelial cell [22‐25]. IL-8 has been shown to regulate pathological angiogenesis, tumor growth, and metastasis [24]. The mechanism(s) regulating IL-8-mediated endothelial cell survival are not well understood. Recent reports suggest that in addition to cell proliferation and migration, endothelial cell survival and death are also important components for tumor survival and development [25]. The other studies have shown that a cell cycle-regulated apoptosis inhibitor, survivin, and the cell death-related gene family products, Bcl-xl and Bcl-2 [15, 26], are associated with vascular endothelial growth factor (VEGF)-induced angiogenesis [10, 25]. IL-8 and its receptors CXCR1 and CXCR2 have been observed in endothelial cells and have been shown to play a role in endothelial cell proliferation [25]. Liver cancer, such as HCC, are dependent on angiogenesis; therefore, angiogenesis inhibition can be used as a potential treatment modality to inhibit the proliferation and growth of solid tumors [27, 28]. In addition, efforts to treat solid tumors using angiogenesis inhibitors have yielded good results [28, 29]. However, these therapies not only affect solid tumors but also normal cells, which is an area of concern in cancer treatment [27]. Furthermore, cancer therapies, such as transarterial chemoembolization (TACE) that uses blood vessels may not produce the desired results, and this may even increase vascular proliferation and growth into a malignant tumor by incomplete responses [30]. A correlation between hypoxia, cancer proliferation, and angiogenesis and the mechanism of growth or development of tumors has been observed [31]. If the link between any of the above can be elucidated, the basis for inhibition of tumor growth and excision can be ascertained. This can be achieved by dual control of HIF-1α and angiogenic factors [6]. Innovative and more effective cancer therapies can be developed by regulating HIF-1α expression, which is the key factor in hypoxia, and controlling the expression of IL-8 and other angiogenic stimulators, which restore the angiogenic processes, during inhibition of HIF-1α expression [7].

HIF-1α knockdown directly repressed tumor growth, whereas IL-8 knockdown indirectly repressed tumor growth [1, 7, 27]. Combined knockdown of HIF-1α and IL-8 increased survival rates of mice [7]. Conditioned media of Combined knockdown in HCC cells also decreased micro-vessel density and tumor volume in vivo [7]. Similarly, HIF-1α and IL-8 knockdown inhibited the angiogenic effects of HCC cell-conditioned media on tube formation and invasion by endothelial cells in vitro [7]. Inhibition of HIF-1α and IL-8 up-regulated the expression of apoptotic factors while down-regulating anti-apoptotic factors simultaneously [7]. Knockdown of HIF-1α and IL-8 increased concentration of cytosolic cytochrome C and enhanced DNA fragmentation in HCC cell lines and HUVECs [32]. Moreover, culture supernatant collected from the knockdown of HIF-1α and IL-8 in HCC cell lines induced apoptosis in HUVECs under hypoxia [32]. The silencing of HIF-1β expression suppressed tumor cell growth and inhibited the expression of tumor growth-related factors [33], such as vascular endothelial growth factor, epidermal growth factor, and hepatocyte growth factor. Suppression of tumor cell invasion and migration was also demonstrated in HIF-1β-silenced HCC cell lines [33].

Angiogenesis is essential for tumor growth and metastasis, and attempts to control tumor-associated angiogenesis may prove to be promising tactics for limiting progression [27]. Angiogenesis occurs during development and vascular remodeling as a controlled series of events leading to neovascularization, which supports changing tissue requirements [34]. Blood vessels and stromal components are responsive to pro- and anti-angiogenic factors that allow vascular remodeling during development, wound healing, and pregnancy [35, 36]. However, in pathological situations, such as cancer, the same angiogenic signaling pathways are induced and exploited.

Although an oncogenic event may allow tumor cells to evade surveillance or may enhance their survival, large-scale growth of a tumor ultimately requires a blood supply [27]. To obtain this blood supply, tumor cells can tilt the balance toward stimulatory angiogenic factors in order to drive vascular growth by attracting and activating cells from within the microenvironment of the tumor [37]. The magnitude and quality of the angiogenic response is ultimately determined by the sum of pro- and anti-angiogenic signals (Table 1) and, more specifically, their unique activities on multiple cell types [38]. Understanding how these various components are regulated is required for the design and development of effective anti-angiogenic therapies for cancer [27].

In cancer, multiple sources and modes of vascular remodeling contribute to disease progression [28]. Targeting one aspect of this remodeling process may produce a short-term effect; nevertheless, suppressing one pathway could promote another [7]. The redundancy and diversity by which blood vessels can remodel might account for the poor efficacy or acquired resistance often observed in antiangiogenic therapies [3]. Improving therapeutic responses thus requires consideration of the signaling pathways that regulate the multiple cell types involved in the vascular components of cancer [39]. Once a tumor lesion exceeds a few millimeters in diameter, hypoxia and nutrient deprivation triggers an “angiogenic switch” to allow the tumor to progress [3, 15, 27]. Tumor cells exploit their microenvironment by releasing cytokines and growth factors to activate normal, quiescent cells around them, initiating a cascade of events that quickly becomes dys-regulated [40].

Therefore, simultaneous inhibition of HIF-1α and IL-8 expression has proven to be more effective in hindering angiogenesis than inhibition of a single factor [7, 32, 33]. With regards to expression at a molecular level, studies have demonstrated that liver cancer is regulated more by HIF-1α; however, in vascular endothelial cells, such as human umbilical vein endothelial cells (HUVEC), the level of IL-8 regulation is similar to that of HIF-1α [2, 41, 42]. The growth of cancer cells and VEGF expression, which controls angiogenesis, has been observed to be regulated by HIF-1α, whereas IL-8 does not affect tumor growth or VEGF expression [7]. Alternatively, in HUVEC, when IL-8 expression is inhibited, angiogenic inhibition is observed at a level similar to HIF-1α inhibition (Fig. 1).

Similar results have been obtained through animal research; inhibition of HIF-1α expression rarely resulted in tumors reproduction in the animal models. Moreover, no apoptosis of existing tumors are observed in these animals; however, in other animal models, where IL-8 expression was inhibited, a tumor volume similar to the time when shIL-8 was injected into the tumors was observed. These findings did not reveal any correlation between IL-8 and direct growth of tumors; however, IL-8 plays an important role in angiogenesis [24, 41, 43]. In addition, the results of the experiments on angiogenesis, such as invasion, tube formation, and aorta sprouting assays, have confirmed that simultaneous inhibition of two factors yielded more favorable responses than inhibition of a single factor [7]. Experiments with animal models have also demonstrated apoptosis of existing tumors as well as high survival rates in a majority of animals in which both the factors were inhibited. Moreover, it was confirmed that various factors to test for blood vessel formation, such as CD31, CD34, and vascular endothelial (V-E) cadherin, were not observed [7]. These findings suggested that controlling hypoxia as well as the expression of angiogenesis-associated factors that act via different pathways can aid in the inhibition of angiogenesis.

Various treatments have been developed for cancer, and better therapies have been developed by overcoming the limitations of already-developed treatments. We hypothesized that if the symptoms that occur during tumor treatment can be studied and controlled, the obstacles currently encountered during cancer treatment can be eliminated. If simultaneous regulation of tumor development, hypoxia, and angiogenesis is possible, cancer cells could be easily treated without peripheral damage. In other words, simultaneous inhibition of the factors that potentially control hypoxia and angiogenesis during treatment to induce apoptosis may be a more innovative anticancer treatment modality.

A correlation exists between cancer and hypoxia [31]. Hypoxia during tumor development can destroy cancer cells; however, it acts as a key factor in excessive cancer proliferation [31, 44, 45]. Prevention of cancer via hypoxia treatment can be used as a highly effective anticancer therapy [2, 34, 46]. To date, studies have been carried out to induce apoptosis in various tumors [2, 34, 44, 47]. One potential treatment option involves reducing angiogenesis, typically by inhibiting VEGF, EGF, or bFGF, while an alternative option involves activating the intracellular intrinsic apoptosis pathway by inducing the expression of apoptotic factors and inhibiting the expression of anti-apoptosis factors [31]. Moreover, a method to stimulate an extracellular death signal in order to induce apoptosis could also be developed. Apoptosis, also referred to as programmed cell death, is one of the most important cellular functions [48]. In normal cells, a decrease in telomere length normally occurs with age; however, DNA damage, toxin exposure, and deprivation of growth factor also generate death signals in various pathways, which results in apoptosis [49, 50]. Hypoxic stimulation is also a crucial death signal; apoptosis is induced when the oxygen supply, required for the production of ATP, an important cellular metabolite, is suppressed [31]. However, in tumors, stimulation that induces apoptosis can be avoided [51]. During telomere length decrease, production of telomerase is promoted to restore the length of telomeres, and when DNA damage is induced, it can be repaired by mutation [31, 34, 52]. In hypoxic conditions, apoptosis can be avoided by inducing angiogenesis while increasing the expression of growth factors, thereby restoring oxygen supply, or by stimulating intracellular nitric oxide synthase (iNOs) through hematopoiesis and local vasodilation (Table 2) [6, 11, 53]. Apoptosis can be also be avoided by increasing anaerobic ATP production via glycolysis, which is facilitated by promoting GLUT1 or pyruvate dehydrogenase kinase activity [11].

HIF-1α, which is generated under hypoxic conditions, is an important anti-apoptotic factor [31, 45]. HIF-1, like tumor necrosis factor (TNF)-a, activates the expression of FoxM1, that in turn induces growth of cancer cells in the liver and increases resistance to apoptosis [50]. The expression of HIF-1 in liver cancer inhibits expression of various caspases and reduces expression of Bax and Bak, which lead to a higher concentration of cytochrome C inside the cells [32]. Increase in the expression of survivin and the Bcl-family, which are important factors that cause DNA fragmentation, can prevent hypoxic apoptosis [54].

Apoptosis in tumors is important because tumor angiogenesis, which is increased by tumor proliferation, can also be inhibited [34]. Apoptosis in tumors can also induce apoptosis in newly formed peripheral blood vessels, thereby ensuring the prevention of a relapse of cancer or cancer stem cell growth at an early stage [1, 27, 28]. The immunofluorescence-TUNEL technique has demonstrated that tumors in which HIF-1α expression is inhibited display increased DNA fragmentation [32]. Interestingly, although IL-8 does not exert a direct influence on tumor apoptosis, it plays a role in tumor apoptosis by controlling apoptosis in blood vessels [32]. Cultivated tumor cell lines with simultaneous inhibition of these two factors demonstrated increase in tumor apoptosis via the FACS-TUNEL technique. It was also confirmed that a vascular endothelial cell culture medium developed from a culture medium, in which apoptosis had been induced in tumor cells, promoted apoptosis in vascular endothelial cells without any stimulation (Fig. 2) [3, 32]. Apoptosis in tumors affects the surrounding tissues owing to the constant communication and transmission between cells. The blood vessels, which are essential for tumor growth, mutually communicate via various factors that are present in the vicinity of the tumor. Therefore, cancer treatment and anticancer drugs can induce apoptosis in tumors while simultaneously regulating the expression of an activation factor in vascular endothelial cells, a higher anti-tumor therapeutic efficacy can be achieved during treatment of tumors [3, 32, 37]. In addition, a more precise tumor treatment can be developed by eliminating the factors that support the growth of malignant tumors, which relapse due to various reasons after tumor treatment [27, 32].

Tumors can be treated using various methods and with several drugs [1]; the efficacy of these treatments can confirmed via different experiments. Moreover, various studies have been conducted to develop potential anti-tumor treatments that work by regulating the microenvironment of the tumor or controlling various tumor growth factors along with the existing tumor treatments [1, 22, 28, 39, 40]. Inhibition of the hypoxic mediator, HIF-1α, and the activation factor in V-E cells, IL-8, which is closely related to tumor development, can potentially be used to develop a treatment that can directly regulate tumor development as well as the microenvironment of tumors [7, 14, 20, 32].