- Open Access
Erratum to: Regulation of the hypoxic tumor environment in hepatocellular carcinoma using RNA interference
© The Author(s) 2017
- Received: 29 June 2017
- Accepted: 29 June 2017
- Published: 12 July 2017
The original article was published in Cancer Cell International 2017 17:3
After the publication of the original article , it was noted that the pre-revision version of this manuscript was mistakenly published. This erratum contains the correct, revised edition of this manuscript.
Hypoxia, which arises in tumor cells that have been deprived of oxygen, has been shown to play a role in tumor development in hepatocellular carcinoma. Hypoxia-inducible factors (HIFs) are transcription factors that regulate cellular homeostatic responses to oxidative stress and have been identified as key transcriptional activators of tumor angiogenesis, survival, and metabolism. Cytokines, such as IL-8, also influence survival and angiogenesis in endothelial cells. IL-8 is overexpressed under hypoxic conditions and has been demonstrated to induce tumor angiogenesis and growth. Regulation of these oncological factors using RNA interference-based tools, small interfering RNA (siRNA) and short hairpin RNA (shRNA), were investigated in vitro and in vivo. The conclusion based on multiple studies is that regulation of HIFs and IL-8 by si/shRNA results in modulation of tumor angiogenesis and apoptosis in the tumor microenvironment. This review summarizes the results of studies investigating regulation of the hypoxic tumor environment.
RNAi, HCC, HIF, IL-8, Angiogenesis, Apoptosis
Hepatocellular carcinoma (HCC) is the sixth most common, and the second most lethal, cancer in the world . Approximately 80–90% of cirrhotic liver disease cases resulting from chronic viral hepatitis B or C develop into HCC . Moreover, hypoxic induction of angiogenesis and tumor growth is observed in most cases of advanced HCC .
The most important factors that influence HCC progression are oxygen and nutrients . The liver is an organ with a specific blood supply. Approximately 25 and 75% of the total blood volume entering the liver does so via the hepatic artery and the portal vein, respectively. The latter drains into structures of smaller diameter called sinusoids. Vascular resistance is very low in these structures, and portal venous blood, which is loaded with food and large numbers of microbial antigens from the intestine, flows extremely slowly into the sinusoids. Thus, large amounts of nutrients and oxygen are required for HCC cell proliferation, resulting in localized hypoxia [3, 4]. This hypoxic environment causes tumor angiogenesis, i.e., the generation of new blood vessels from existing ones [3, 5]. Tumor angiogenesis overcomes oxidative stress  and the deficiency in oxygen-dependent energy production caused by hypoxia [3, 7]. The key factors responsible for regulation of angiogenesis during hypoxia are hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) [5, 8]. However, various studies have reported that angiogenesis is induced even during inhibition of HIF-1α during hypoxia , demonstrating that tumor angiogenesis is partially affected by various other factors . Increased expression of various factors, such as growth factors and tumor-stimulating factors, can induce angiogenesis during hypoxia, as they increase proliferation and ensure stabilization of endothelial cells. These factors are induced not only by HIF-1α, but also by cytokines such as interleukin (IL)-8 or by growth factors such as platelet-derived growth factor [10, 11]. In addition, these factors increase VEGF expression, as well as help increase and stabilize angiogenesis, by upregulating VEGF receptor on the surface of endothelial cells . Moreover, various solid tumors pass through the following three stages during their reproductive cycle: cell proliferation, hypoxia, and recovery tumor survival by angiogenesis [3, 13, 14].
Roles of HIF and IL-8 under hypoxic conditions
The hypoxia inducible factors (HIFs) are a family of heterodimeric transcription factors that act as master regulators of the homeostatic transcriptional response to hypoxia in virtually all cells and tissues . Active HIF consists of alpha and beta subunits [2, 15, 16]. Three alpha subunits, termed HIF-1α, HIF-2α, and HIF-3α, have been described in humans, mice, and rats; all bind to the common beta subunit HIF-1β, also known as aryl –hydrocarbon receptor nuclear translocator (ARNT) [17, 18]. Active HIF is named according to its alpha subunit; hence, HIF-1 consists of HIF-1α and ARNT, HIF-2 of HIF-2α and ARNT, and so forth . HIF-1 and HIF-2 are the major hypoxia-inducible factors in humans, mice, and rats .
Under conditions of normoxia, HIF-1α subunits are hydroxylated at proline residues by hydroxylases [3, 20]. Hydroxylation of HIF-1α and assembly of a protein scaffold consisting of the von Hippel–Lindau (VHL) tumor suppressor [21–24], along with other co-factors, result in rapid ubiquitination of the alpha subunit and subsequent degradation by the proteasome. Conversely, under conditions of hypoxia, HIFα subunits escape degradation and are free to dimerize with their binding partner, ARNT [3, 17]. HIF translocates to the nucleus where it affects the transcription of target genes, typically by binding to a hypoxia response element (HRE) in the upstream promoter region of such target genes, which include genes related to angiogenesis, apoptosis, metabolism, and survival [2, 3, 18].
Tumor cells have the ability to exploit the expression of various cytokines and their receptors for their own use. Cytokines secreted by tumor cells can act on the surrounding normal stroma, such as blood vessels, recruiting them to aid in tumor growth, survival, and metastasis. IL-8/CXCL-8 is a pro-inflammatory cytokine  and a key molecule influencing endothelial cell survival and angiogenesis . IL-8, which is a novel leukocyte chemotactic-activating cytokine [27, 28], is produced by various types of cells upon contact with inflammatory stimuli and exerts a variety of functions on leukocytes [29, 30], particularly neutrophils. IL-8 is also associated with several types of acute inflammatory reactions , including lipopolysaccharide (LPS)-induced dermatitis, LPS/IL-1-induced arthritis, and lung reperfusion injury . IL-8 can promote resolution of infection by inducing phagocytosis, oxidative burst, and the release of DNA webs known as neutrophil extracellular traps that trap and kill invading microbes . Conversely, IL-8 is also regulated under hypoxic conditions and directly regulates endothelial cells [31–34]. IL-8 has been shown to regulate pathological angiogenesis, tumor growth, and metastasis . 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 factors for tumor survival and development . Other studies have shown that a cell cycle-regulated apoptosis inhibitor, survivin, and the cell death-related gene products, Bcl-xL and Bcl-2 [19, 35], are associated with VEGF-induced angiogenesis [12, 34]. IL-8 and its receptors CXCR1 and CXCR2 have been shown to play a role in endothelial cell proliferation . Liver cancers, such as HCC, are dependent on angiogenesis; therefore, inhibition of angiogenesis could be a potential treatment modality to prevent the proliferation and growth of solid tumors [36, 37]. Thus far, efforts to treat solid tumors using angiogenesis inhibitors have yielded good results [37, 38]. However, these therapies affect not only solid tumors but also normal cells, which is an area of concern in cancer treatment . Furthermore, cancer therapies such as transarterial chemoembolization (TACE) delivered via blood vessels may not produce the desired effect and may even increase vascular proliferation and growth of malignant tumors by incomplete responses of TACE therapy . Correlations among hypoxia, cancer proliferation, angiogenesis, and tumor growth or development have been observed . Elucidation of the relationships among these processes may ascertain the basis for inhibition of tumor growth and metastasis. Management of the tumor can be achieved by dual control of HIF-1α and angiogenic factors . Innovative and more effective cancer therapies can be developed by regulating the expression of HIF-1α, which is the key factor in hypoxia, and by controlling the expression of IL-8 and other angiogenic stimulators, which restore the angiogenic processes caused by inhibition of HIF-1α expression .
HIF-1α knockdown directly represses tumor growth, whereas IL-8 knockdown indirectly represses tumor growth [1, 9, 36]. Combined knockdown of HIF-1α and IL-8 increased survival rates in mice . Conditioned media collected from HCC cells subjected to combined knockdown also decreased microvessel density and tumor volume in vivo . Similarly, combined knockdown of HIF-1α and IL-8 inhibited the angiogenic effects of HCC cell-conditioned media on tube formation and invasion by endothelial cells in vitro . Inhibition of HIF-1α and IL-8 upregulated the expression of apoptotic factors while simultaneously downregulating the expression of anti-apoptotic factors . Knockdown of HIF-1α and IL-8 increased the concentration of cytosolic cytochrome C and enhanced DNA fragmentation in HCC cell lines and human umbilical vein endothelial cells (HUVECs) . Moreover, apoptosis was induced in HUVECs treated with culture supernatant collected from HCC cell lines with silenced HIF-1α and IL-8 expression, under conditions of hypoxia . Silencing of HIF-1β expression suppressed tumor cell growth and inhibited the expression of tumor growth-related factors , such as VEGF, epidermal growth factor (EGF), and hepatocyte growth factor. Suppression of tumor cell invasion and migration was also demonstrated in HIF-1β-silenced HCC cell lines .
Comparing various RNA silencing methods
The ability of RNA interference (RNAi) or transient gene ‘knockdown’ to silence target genes with high efficiency and specificity has stimulated efforts to develop these molecules into therapeutic agents . RNAi can be classified into three types [44, 45], all of which have similar mechanisms of action. Firstly, long double-stranded RNAs (dsRNAs), approximately 500–1000 nucleotides in length, have been employed to evaluate gene functions . Following introduction of exogenous dsRNA into the cytoplasm of cells , it gets cleaved by the RNase III enzyme Dicer to produce a short dsRNA called small interfering RNA (siRNA) . The siRNA, which is 21–23 nucleotides in length, is loaded into a protein complex called the RNA-induced silencing complex (RISC) [47, 49]. The siRNA is then unwound and its sense strand degraded, while the surviving antisense strand guides the activated RISC to the target mRNA through full complementary binding . The mRNA is then cleaved by RISC, leading to silencing of the target gene . Since long dsRNA triggers an immunostimulatory response through the activation of Dicer-related antiviral pathways and induction of type 1 interferon [46, 52], it is less suitable for therapeutic use. In contrast, synthetic siRNA is a more promising gene silencing mediator, because it poses less risk of an immune reaction . Based on this principle, siRNA therapies have been investigated extensively in preclinical studies [43, 44, 48], and some siRNA agents have already been investigated in clinical trials for the treatment of cancer and several other diseases . To date, eight clinical trials of siRNA/dsRNA therapies for cancer have been reported [52, 54]. Various siRNA targets or pathways were evaluated in these trials, including polo-like kinase 1 [55, 56], KRAS (G12D) , protein kinase N3 , and VEGF [59, 60]. In addition, preliminary results have been released from a phase I trial of the first siRNA-based therapy targeting the oncogene MYC in patients with advanced solid tumors [43, 52].
Short hairpin RNAs (shRNAs) are delivered into cells using a DNA vector and then transcribed by either RNA polymerase II or III in the nucleus . The primary transcript is called primary shRNA (pri-shRNA), which contains a hairpin-like stem-loop structure . Pri-shRNA is processed into a 50–70-nucleotide-long loop-stem precursor shRNA (pre-shRNA) by a protein complex containing the RNase III nuclease Drosha and the dsRNA binding domain protein DGCR8 . It is then transported to the cytoplasm by a specialized nuclear membrane protein, exportin 5 (Exp5) . The loop sequence of the pre-shRNA is cleaved by Dicer to form a double-stranded siRNA [44, 47]. This endogenously produced siRNA is loaded into RISC and can induce RNAi through a similar process as the synthetic siRNA [47, 51]. Since shRNA expression units can be incorporated into viral vectors and continuously synthesized by host cells, shRNAs can induce long-lasting gene silencing effects [45, 47]. The RISC-loading process of shRNA is approximately 5- to 10-fold more efficient than that of siRNA, indicating that a lower dose of shRNA is required to maintain therapeutic efficacy with less off-target effects . However, the shRNA approach is a DNA-based strategy depending on the expression of shRNA-encoding genes, which often require viral vectors (such as adenoviral or lentiviral vectors) [47, 51, 61]. From a delivery perspective, the introduction of synthetic siRNA into the cytoplasm is a more straightforward method to induce RNAi . The use of viral vectors for delivery poses safety concerns in therapeutic applications [47, 61].
MicroRNAs (miRNAs) is a naturally occurring non-coding RNA molecules that play a key role in regulating gene expression . Primary miRNAs (pri-miRNAs) are transcribed by RNA polymerase II from endogenous miRNA genes in the nucleus . The hairpin-containing pri-miRNA is structurally similar to the pri-shRNA; therefore, miRNAs also have a similar function to shRNAs [45, 50]. The pri-mRNA is converted by the Drosha/DCGR8 complex into precursor miRNA (pre-miRNA), which consists of 70–100 nucleotides with interspersed mismatches, and adopts a loop structure [45, 63]. Pre-miRNA is subsequently transported to the cytoplasm by Exp5 and processed by Dicer into a mature miRNA of 18–25 nucleotides in length . In contrast to siRNA, the antisense strand of miRNA is only partially complementary to the target mRNA, leading to gene silencing via translational repression and/or mRNA deadenylation . Positions 2–7 at the 5′ end of miRNA is an essential sequence for target recognition, and the miRNA-binding sites of mRNA are located in the 3′ untranslated region [50, 63]. There are two major approaches in miRNA-based therapeutics: miRNA inhibition [64–66] and miRNA replacement [44, 67]. The former suppresses the activity of endogenous miRNA using an antisense oligonucleotide (anti-miR), and the latter introduces a synthetic miRNA (miRNA mimic) to restore the functions of endogenous miRNA [44, 49, 53, 67].
Inhibition of HIF-1α and IL-8 expression as a strategy for suppression of angiogenesis
Angiogenesis is essential for tumor growth and metastasis, and attempts to control tumor-associated angiogenesis may prove to be promising tactics for limiting tumor progression . Angiogenesis occurs during development and vascular remodeling as a controlled series of events leading to neovascularization, which supports changes in tissue requirements . Blood vessels and stromal components are responsive to pro- and anti-angiogenic factors that allow vascular remodeling during development, wound healing, and pregnancy [69, 70]. However, in pathological situations, such as cancer, the same angiogenic signaling pathways are induced and exploited.
Up regulation of tumor angiogenic factors under hypoxia
Increased vascular permeability
EC proliferation and migration
Increased vascular permeability
EC proliferation and migration
Degradation of extracellular matrix
Form the blood vessels
Increased EC differentiation formation of vascular structure
Formed vascular structure
Stimulate endothelial cell mitogenesis and cell migration
Enhances microvascular permeability
Regulates local vascular tone and integrity
Influences EC growth and survival
Increased vascular permeability
EC proliferation and migration
Antiangiogenic, inhibiting the proliferation and migration of endothelial cells
In cancer, multiple sources and modes of vascular remodeling contribute to disease progression . Targeting one aspect of this remodeling process may produce a short-term effect; nevertheless, suppression of a particular pathway could result in the promotion of another . The redundancy and diversity by which blood vessels can remodel might account for the poor efficacy or acquired resistance often observed in response to antiangiogenic therapies . Improving therapeutic responses thus requires consideration of the signaling pathways that regulate the multiple cell types comprising the vascular components of cancer . Once a tumor lesion exceeds a few millimeters in diameter, hypoxia and nutrient deprivation triggers an “angiogenic switch” to allow tumor progression [3, 19, 36]. Tumor cells exploit their microenvironment by releasing cytokines and growth factors to activate surrounding normal, quiescent cells, initiating a cascade of events that quickly becomes dysregulated .
Similar results have been obtained through animal studies; inhibition of HIF-1α expression rarely results in tumor reproduction in animal models. Moreover, apoptosis was not detected in existing tumors in these animals. However, in other animal models in which IL-8 expression was inhibited, tumor volumes were similar to those in other experiments, in which a shRNA targeting IL-8 was injected into tumors. These findings did not reveal any direct correlation between IL-8 function and tumor growth; however, IL-8 plays an important role in angiogenesis [33, 75, 77]. In addition, the results of experiments investigating angiogenesis, such as invasion, tube formation, and aorta sprouting assays, confirmed that simultaneous inhibition of two factors yields more favorable responses than does inhibition of a single factor . Experiments in animal models have also demonstrated apoptosis of existing tumors as well as high survival rates in the majority of animals in which both factors were inhibited. Moreover, various markers of blood vessel formation, such as CD31, CD34, and vascular endothelial-cadherin, were not observed . These findings suggest that in addition to controlling hypoxia, targeting the expression of angiogenesis-associated factors that act via different pathways can help inhibit angiogenesis.
Improved cancer therapies have been developed by overcoming the limitations of existing therapies. We hypothesize that if the symptoms associated with cancer treatment can be monitored and controlled, certain obstacles currently encountered during treatment can be eliminated. If it were possible to regulate tumor development, hypoxia, and angiogenesis simultaneously, cancer cells could be treated easily without peripheral damage. In other words, delivery of therapies that simultaneously inhibit factors controlling hypoxia and angiogenesis, while concurrently inducing apoptosis, may represent a more innovative anticancer treatment modality than those currently available.
Tumor escape from apoptosis under hypoxic conditions
Role of anti-apoptosis under hypoxia
Block BAX and BAD activity
Inhibited caspase activation
Inhibited cytochrome c release
Induces epithelial development
Initiate multiple cell proliferation
Downregulation of caspase-3 and 9
Regulates cell growth, differentiation, survival
HIF-1α, which is generated under hypoxic conditions, is an important anti-apoptotic factor [40, 79]. HIF-1, like tumor necrosis factor-α, activates the expression of FoxM1, which induces cancer cell growth in the liver and increases resistance to apoptosis . The expression of HIF-1 in liver cancer inhibits the expression of various caspases and reduces the expression of Bax and Bak, thereby leading to a higher intracellular concentration of cytochrome C . Increased expression of survivin and Bcl family members, which are important factors causing DNA fragmentation, can prevent hypoxia-induced apoptosis .
Tumors can be treated by various methods and several drugs ; the efficacy of these treatments has been confirmed via different experiments. Moreover, various studies have been conducted to develop potential anti-tumor treatments that regulate the tumor microenvironment or modulate various tumor growth factors as well as target the tumor itself [1, 31, 37, 73, 74]. Methods that inhibit the hypoxic mediator HIF-1α and the vascular endothelial cell activation factor IL-8, which have been implicated in tumor development, could potentially be used to develop a treatment that could directly regulate both tumor development and the tumor microenvironment [9, 18, 24, 41].
Although newly developed treatments for HCC employ various approaches to combat this disease, all are associated with significant side effects and complications . For example, TACE, which embolizes vessels to induce cancer tissue necrosis, also causes damage to surrounding tissues . Furthermore, any remaining embolization- or radiotherapy-resistant cancer tissue tends to be more malignant and can metastasize [89, 90]. Additionally, hypoxia induced by medical or surgical treatment induces the accumulation of HIF-1α inside tumor cells and its subsequent translocation into the nucleus, where it promotes the expression of angiogenesis-related genes and increases oxygen supply to the tumor . It also activates the expression of metastasis-related genes [16, 21, 40]. These hypoxia-induced processes reduce cellular injury and enable continuous tumor growth by ensuring that the tumor receives an adequate supply of oxygen .
In recent studies, inhibition of HIF-1α expression failed to block tumor-induced angiogenesis, allowing the tumor to survive and proliferate [9, 36]. The key factor involved in this process is IL-8, which is upregulated in response to hypoxic conditions during tumor proliferation [16, 90, 92]. IL-8 induces angiogenesis by activating vascular endothelial cells . HIF-1α directly regulates HCC development and IL-8-assisted tumor growth via regulation of angiogenesis in the vascular endothelial system . These findings might aid the development of effective treatments that do not harm normal cells. However, further studies must be conducted before any clinical applications can be performed. Although inhibition of HIF-1α and IL-8 had a significant effect on tumor angiogenesis in animal studies, this effect was restricted to specific hypoxic conditions. Since hypoxia destroys both tumor and normal cells, the expression of HIF-1α must be maintained in normal tissues.
Regarding tumor proliferation, hypoxia is an important condition for the initial growth of a tumor. It is thought that HIF-1α and HIF-1β expression regulates the initiation of tumor growth and induces more malignant growth under hypoxic conditions. Further studies will be required to determine other possible functions of HIF-1β, which are comparatively less known than those of HIF-1α, which has been the focus of most investigations at this point.
A local hypoxic microenvironment is one of the most important characteristics of solid tumors. Apoptosis is a critical mechanism in the development of organisms. However, apoptosis is also critical for inhibition of cancer cell growth. A significant survival mechanism in cancer cells is the suppression or prevention of apoptosis. Once apoptosis is induced, cancer cells induce expression of various anti-apoptotic factors, thereby suppressing apoptosis and promoting increased growth of cancer cells and tissues. Various anticancer agents or therapies have been rapidly developed to address this characteristic. TACE, which is widely utilized for the treatment of liver cancer currently, induces hypoxia and hypoglycemia in liver cancer cells, reducing the number of cancer cells. Radiation therapies also induce extended hypoxia in radiated areas, thereby promoting hypoxia-induced apoptosis of cancer tissues. Among the various treatments for liver cancer, most kill cancer cells via apoptosis.
The present study investigated the effects of induction or prevention of apoptosis in peripheral vascular cells, rather than the direct treatment of cancer cells [68, 74, 82]. Apoptosis of cancer cells was confirmed to influence apoptosis or growth of peripheral tissues in various experiments investigating apoptosis. Moreover, RNA expression was found to be regulated by various knockdown mechanisms when employing RNAi tools in vitro and in vivo . In vivo analyses used adenovirus-mediated shRNA directly injected into tumor tissues for effective knockdown of target genes. In vivo analyses also used lentivirus-mediated siRNA for effective knockdown of target genes in a rapid-growth cell-based assay [47, 94].
Improved cancer therapies have been developed by overcoming certain limitations associated with existing treatments. We hypothesized that if symptoms that occur during tumor treatment could be studied and controlled, obstacles that are currently encountered during cancer treatment could 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, a method that employs simultaneous inhibition of factors that potentially regulate hypoxia and angiogenesis while also inducing tumor apoptosis may represent a more innovative anticancer treatment modality than those currently available.
RNA: iribonucleic acid interference; siRNA: small interference RNA; shRNA: small hairpin RNA; HCC: hepatocellular carcinoma; HIFs: hypoxia inducible factors; IL-8: interleukin-8; HB(C)V: hepatitis B(C) virus; HUVECs: human umbilical vein endothelial cells; FACS-TUNEL: flow cytometry-terminal deoxynucleotidyl transferase dUTP nick end labeling; TACE: transcatheter arterial chemoembolization; VEGF: vascular endothelial growth factor; EGF: endothelial growth factors; bFGF: basic fibroblast growth factor
SHC contributed to analysis and interpretation of data and drafting of the manuscript. JYP contributed to study concept and design, confirmed and revised the manuscript for important intellectual content. Both authors read and approved the final manuscript.
We are grateful to Dong-Su Jang (Medical Illustrator, Medical Research Support Section, Yonsei University.
The authors declare that they have no competing interests.
Availability of data and materials
Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology (NRF-2011-0014537).
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