Haptoglobin and Cancer: Various Tumours.
1.1. The acute phase response (APR) and haptoglobin (Hp)
Acute phase response is a stereotyped innate nonspecific reaction of the body proceeding specific immune reactions. It´s a systemic homeostatic reaction of the organism to local and or systemic disturbances caused by infections, tissue injury, trauma, immunologic disorders and neoplasias (Ron D
These acute phase proteins (APPs) can be positive (higher levels in plasma) or negative (lower levels in plasma). The alteration on mRNA in hepatocytes is due to simultaneous influence of systemic cytokines (IL1, IL6 and TNFα), glucocorticoids and catecholamines (Bowman BH 1993, Ron D
Haptoglobin together with fibrinogen, α-globulins with antiprotease-activity and lipopolysaccharide binding protein belong to the group of positive APPs that increase 3-fold in mammals (Trautwein C
Haptoglobin (Hp) is an acute phase α2 plasma glycoprotein that is a component of innate immunity, which also may influence acquired immunity. Through both types of immunity, Hp is involved in the pathogenesis of tumours and infections (Langlois MR and Delanghe JR 1996, Van Vlierberghe H.
2. Haptoglobin (Hp) synthesis, gene structure, variants and its geographic distribution
Haptoglobin locus is on chromosome 16q22 and its gene is transcribed and translated into a single peptide which undergoes post-translational processing resulting in a smaller α-chain and a longer β-chain linked by disulphide bridge (Giblett ER 1968, Langlois MR and Delanghe JR 1996, Wicher KB and Fries E 2007).
In 1955, Smithies, using thin layer starch gel electrophoresis identified the three phenotypes of Hp (1-1, 2-1, 2-2), corresponding to the α-chain length interindividual genetic variation.
The three genotypes are shown in electrophoresis in polyacrylamide gel electrophoresis (PAGE) (fig 1).
This genetic variation results from an internal duplication of a gene segment (exons 3 and 4), correspondent to α-chain of Hp1 giving rise to a larger one, characteristic of Hp2 (Maeda
This inter-individual variation is found only in humans and aroused about 100,000 years ago in Southeast Asia. The great majority of other mammals have only one band corresponding to the human Hp1-1, except the sheep, deer and cows (Ruminantia), which have only slow bands corresponding to Hp2-2 (Bowman BH and Kurosky A 1982, Wicher KB and Fries 2007).
The appearance of Hp2 can represent an important evolutionary genetic contribution for interpopulational diversity in human pathology (ER Giblett 1968, Maeda et al 1984, Wicher KB and Fries 2007). This allele is predominant in the human population (about 80% in some ethnic groups) and Hp1 allele is more predominant in populations subjected to malaria burden (Giblett ER 1968, Langlois MR and Delanghe JR 1996, Wobeto VP et al 2008, Levy AP et al 2010).
In close linkage with haptoglobin gene there is another one, 2-2 Kb downstream from Hp locus, coding for Hp related (Hpr) plasma protein with 91% sequence identity to Hp1. The α-chain of Hpr contains a hydrophobic signal peptide that may explain its association to lipoprotein particles (HDL) or membranes (Kuhajda FP
3. Haptoglobin locals of its synthesis and regulation
The Hp gene is expressed primarily in hepatocytes and more recently has been described in other locations, such as keratinocytes, airway epithelial cells of lung, leucocytes, fibrocytes, adipocytes and endometrial cells, particularly during the blastocyst implantation (Friederichs WE
Haptoglobin synthesis is induced by cytokines such as interleukin-6 (IL-6), interleukin-1 (IL-1) and tumour necrosis factor (TNFα) released by the macrophages, after activation of the innate immunity cells by PAMPs (pathogen associated molecular patterns) such as lipopolysaccharide, a TLR4 (Toll Like Receptor) activator (Raynes JG et al 1991, Kaisho T and Alkira S 2002).
Glucocorticoids and catecholamines activate haptoglobin synthesis previously induced by interleukins (increased), whereas insulin exerts an opposite action, despite the presence of these interleukins (Ron D et al 1990, Campos SP and Baumann H 1992 Nascimento CO et al 2004, Gruys E et al 2005 and XiaLi-xin et al 2008). Hypoxia also induces indirectly its synthesis (Wenger RH et al 1995, Oh Mi-Kyung et al 2011).
4. Haptoglobin metabolism, actions and respective mechanisms
Haptoglobin has a pronounced anti-inflammatory action, which is explained by its ability to bind to heme of haemoglobin, forming a Hp-Hb complex. This is characterized by stability and high affinity to its specific type scavenger receptor (CD163) located in the hepatocyte and the phagocytic-type cells such as circulating monocytes, resident macrophages (M2) and liver Kupffer cells. The CD163 is a membrane protein 130-kDa, whose long extracellular region has nine cysteine-rich domains of scavenger-type receptor (Graversen JH
After binding to its receptor the Hp-Hb complex is internalized in the form of endosome, followed by fusion with lysosomes, proteolysis of globin and intracellular release of heme to hemoxygenase (HO-1) with concomitant formation of biliverdin that is converted in bilirrubin, CO (carbon monoxide) and release of iron to ferritin where is compartmentalised (Graversen JH
The small protein Hp1-1 is excreted in the urine when occurs kidney damage, however, the Hp2-1 and Hp2-2 are always retained (Fagoonee S 2005). The clearance of free haemoglobin (Hb) after intravascular haemolysis by the haptoglobin is higher in individuals carrying the Hp1 allele (Giblett ER 1968, Langlois MR and Delanghe JR 1996, Moestrup SK and Møller H 2004, Nielsen MJ
The free Hb has the ability to catalyse the formation of hydroxyl radicals (OH·), from the hydrogen peroxide, with highly damaging effects to the cellular constituents and extracellular macromolecules (Sadrzadeh SMH 1984, Gutteridge JMC 1987).
The Hp-Hb complex, reduces the loss of Hb in urine and concomitant loss of iron and its transport is done mainly to the liver. As a result, the removal of free Hb has much important consequences for the organisms, preventing renal injury that may occur when the free Hb passes through the glomerular filter (Fagoonee S
Levels of haptoglobin in plasma or serum are lower in healthy infants than adults whose concentrations are between 0.38 and 2.08g/l (Langlois MR and Delanghe JR 1996). These steady state levels are consequence of haptoglobin half-life of 3.5 days and Hp-Hb complex of ten minutes (Sadrzadeh SMH and Bozorgmehr J 2004). The Hp can also be detected in urine and other organic fluids (Langlois MR and Delanghe JR 1996, Sadrzadeh SMH and Bozorgmehr J 2004). The half-life of Hp-Hb complex is phenotype dependent being Hp1-1 shorter than Hp2-2 (Levy AP
Haptoglobin levels are quantified by chemical and immunochemical methods, from these the most utilised are the immunonephelometric and immunoturbidimetric methods that are automated (Langlois MR and Delanghe JR 1996, Sadrzadeh SMH and Bozorgmehr J 2004).
The haptoglobin polymorphism is most commonly determined by starch or polyacrylamide electrophoresis (Fig 1). When plasma levels are lower than 0.10g/l PCR based assays are utilised (Linke RP 1984, Langlois MR and Delanghe JR 1996, Guerra A
The Hp-Hb complex also binds nitric oxide or nitrogen monoxide (NO), produced by cytokine activated macrophages, thus preventing their physiological and pathological actions (Langlois MR and Delanghe JR 1996, Azarov I
The Hp is also a potent endogenous inhibitor of prostaglandin synthesis, resulting in anti-inflammatory action. The inhibitory effects of Hp2-2 and Hp2-1 are less pronounced than those of Hp1-1 (Kendall PA
Haptoglobin has also bacteriostatic effects, because the capture and compartmentalization of the iron of Hb made it no longer available for bacterial growth. The Hp 2-2 is more efficient than the other phenotypes in this action against S
The role of Hp in angiogenesis has been identified as one of the factors for modulation of differentiation and proliferation of endothelial cells during the formation of new vessels (Cid MC
The local increased concentration of Hp in chronic inflammatory processes is important for the ischemic tissue reparation, promoting collateral vessel formation. Of the three genetic forms Hp2.2 is the most angiogenic (Cid MC
In resident tissues macrophages (M2 type), carbon monoxide (CO) resulting from the intracellular degradation Hp-Hb complex appears to be involved in anti-inflammatory effects of interleukin 10 (IL-10). The suppression of these immune and inflammatory responses results from its ability to decrease the antigen presentation and cytokine synthesis. This mechanism of regulation is more active in patients with the Hp1-1 phenotype that has a greater clearance of their complexes with their CD163 receptors present on monocytes, than for those carrying the phenotype Hp2-2 (Nielsen MJ
In macrophages, after the endocytosis of the Hp-Hb complex and CD163, increased levels of cytoplasmic iron ocurr, inducing the synthesis of ferritin, a primary iron storage, which can subtract it from inflammation site (Cozzi
Haptoglobin can also modulate the immune response by binding to receptors on immune cells, such as CD22 on B lymphocytes and β2 integrin (CD11b/CD18) in neutrophils or LFA-1 (lymphocyte function associated antigen-1) in T lymphocytes (EL Ghmati SM
Changes of the ratio of lymphocytes Th1 and Th2 are important for the determination of susceptibility to viral and parasitic infections, for allergies, for antitumor responses and autoimmunity (Gleeson ME 2006, Clerici M
5. Haptoglobin (Hp) and its related pathway as biomarkers in cancer
Genetic polymorphism of haptoglobin leads to its functional differences resulting in interindividual variation of the related intermediate phenotypes at the different biological levels that can constitute circulating biological markers of clinical importance not only for the susceptibility but also for the prognostic and response to treatment at the diverse levels of natural history of the neoplasia disease (Bicho MC 2011). We will review by organs and systems the studies that evidence those aspects.
In table 1, we describe the association of Hp polymorphism in several populations with CNS head and neck, lung, blood and skin malignancies.
For the central nervous system it was demonstrated that haptoglobin is transcribed and expressed (proteomic methods) in human glioblastome cells and it is significantly associated with greater plasmatic levels in the higher grades compared with lower ones and those of control subjects (Sanchez DJ
|Hp2 allele higher grades||Sanchez DJ |
|Head/neck squamous cell cancer||N=135/163||Hp2 allele tumour volume dependency||Chen C-B |
|N=134/49 Taiwan||Lee CC |
|Nasopharynx carcinoma||N=918/208 Belgium||Hp1-1 and Hp 2-1 less prone to positive EBV serology||Speeckaert R |
|Lung cancer||N=309 Sweden||Hp1 allele more frequent in adenocarcinoma in females||Beckman G |
|Acute lymphoid leukemia||N=2331/110 Sweden||No association||Fröhlander N and Stendahl U 1988|
|Associated Hp1-1 with ALL, AML, CML||Nevo S and Tatarsky I 1986|
|Acute myeloid leukemia||N=197/188|
|Campregher PV |
|ALL, AML, CML, IgA ML||N=134 Australia||Higher Hp 1-1 association||Mitchell RJ |
Germinis A et al 1983
|Squamous cell carcinoma (SCC)||N=300 Belgium||Hp phenotype 1.1 more prone to develop SCC in kidney transplanted patients||Speeckaert R |
|Kaposi´s sarcoma||Hp1.1 phenotype more prone||Speeckaert R |
Head and neck squamous cell cancer (HNSCC) is a term that collectively refers to cancer of oral cavity, salivary glands, larynx and pharynx. After a first study the authors whose objective is discovery of circulating biomarkers associated with those tumours, demonstrate in HNSCC in general and nasopharynx in particular, the haptoglobin overexpression, in a stage and tumour volume dependency (Chen C-B
In the eighties several studies of association with cancer of acute phase proteins in particular haptoglobin were done, that is the case for the lung cancer in 309 Swedish patients where the Hp1 allele is more frequent in women with adenocarcinoma (Beckman G
There are five references for blood malignancies such as acute and chronic lymphoid leukemia and acute myeloid leukemia from three different ethnic groups Sweden, Israel and Brazil (Caucasians and Afro-descendants) and only one sample of Ashkenazy Jews (Germinis A
Cutaneous malignancies and in particular squamous cell carcinoma (SCC)/Bowen´s disease are more frequent in kidney transplanted patients, that are more prone to disease when are carriers of Hp 1.1 phenotypes particularly after ten years of the transplantation (Speeckaert R
Tumours of gastrointestinal tract where also studied and the single reference to haptoglobin polymorphism in colon cancer refers to one association in 184 Greek patients of Hp1-1 phenotype (Archimandritis A
More recently it was shown that Hp is produced in a large molecular complex with the beta chain of urokinase in cancer cells as well as in capillary endothelial cells (Harvey S
|Association of Hp1-1 phenotype||Archimandritis A |
|Risk for Hp2-2 carriers||Jayanthi M |
|No association||Theodoropoulos G |
|Oesophageal cancer||Higher risk for Hp2-1 phenotype||Jayanthi M |
|Frequency of Hp 2-2 is higher||Deng R |
Geographic differences have been reported regarding the influence of the Hp alleles in cancer risk. In India, where the frequency of Hp2 allele is high at the population level (84%) the risk for gastric cancer of the Hp2-2 phenotype carriers is 4.04 and the risk for oesophageal is 3.86 for Hp2-1 phenotype carriers (Jayanthi M
Early references of the distribution of haptoglobin polymorphism in Greek patients with prostate carcinoma compared with prostate benign hypertrophy (BPH) patients failed to demonstrate any association (Germenis A
Serum levels of haptoglobin were elevated in kidney and bladder cancer concomitantly with a metabolite of Prostaglandin F2α, however only in bladder cancer was demonstrated in 264 Germans a statistically significant lower frequency of Hp2-2 genotype (Dunzendorfer U
|Failed to demonstrate any Hp association||Germenis A |
|Any association of the polymorphism with PSA and survival||Mavondo GA |
|Lower frequency of Hp 2-2||Benkman HG |
For breast cancer despite consistency of overrepresentation of Hp 1 allele in three earlier studies (Tsamantains C
The first references from the sixties of the last century about gynaecologic tumours indicate contradictory results between the authors when they were analyzed as a whole in what concerns to the frequency of Hp1 allele (Larkin MF 1967, Milunicova A
Cervical neoplasia is a good model that illustrates haptoglobin and its polymorphism influence in the several steps of its natural history interacting with oncogenic and non-oncogenic HPV (Human Papillomavirus) and other co-factors such as sexual steroid hormones and smoking habits (Bicho MC 2011).
Preliminary reports on the role of this haptoglobin polymorphism in the development of cervical cancer were conflicting, with two authors (Milunicova and Bartel) indicating that Hp1 allele carriers were at risk of cancer development. In opposition, Larkin et al report the Hp2 allele as the most represented in their cervical cancer cases (Milunicova A
In the case control study conducted in Canada (307 cases vs 358 control women), Mahmud et al examined the association of Hp phenotype with high grade cervical intraepithelial neoplasia (CIN III), a precursor lesion of invasive carcinoma (ICC). The control group had to present a normal cytology and HPV genotyping was performed to evaluate the HPV oncogenic type status. Accordingly, only when the risk analysis is restricted to the HPV positive women, an association was observed and Hp 1-1 carriers have almost a threefold increased susceptibility to the development of CIN III (OR=2.7, 95% IC: 1.0-7.2) (Mahmud SM et al 2007). In a recent study, we report an increased susceptibility for women that are Hp 1-1 carriers to develop ICC (OR=4.62, 95% IC: 1.86-11.48) (Bicho MC 2011). These results are consistent with another study performed in a different geographic localization (Ghana) and indicating a significant protective effect for the Hp2 allele in homozygous women (Quaye IK et al 2009). In another report, we studied the influence of Hp polymorphism on the risk for the development of HSIL and ICC (n=196) under the influence of sex steroid hormones. We found that the risk for an interaction is proportionally higher with the number of Hp 1 allele presents (Bicho MC et al 2009). However, when the interaction between Hp polymorphism with smoking habits was studied the Hp 2 allele in homozygoty increased the risk to develop HSIL and ICC (Bicho MC et al 2006).
|Overrepresentation of Hp 1 allele||Tsamantains C |
|Overrepresentation of Hp 1 allele||Kaur H |
|Overrepresentation of Hp 1 allele||Bartel U |
|Higher frequency of Hp 1 allele||Awadallah S|
Non familial (N=86)
|Higher frequency of Hp 1 and Hp 2 alleles||Atoum MF 2004|
|USA||No association||Hudson BL |
|No association||Gast M-C W |
|Associated Hp 1 allele||Dobryszycka W and Wavas M. 1983|
|Associated Hp2-1 phenotype with family history||Fröhlander N and Stendahl U 1988|
|Hp1 allele carriers at risk||Milunicova A |
|Hp1 allele carriers at risk||Bartel U |
|Hp2 allele as the most represented||Larkin M 1967|
|N=358/307 Canada||In HPV positive women, risk for Hp 1-1 is higher CIN III||Mahmud SM |
|In ICC women the risk for Hp 1-1 carriers is greater in steroid hormone ingestion||Bicho MC 2011|
|Protective effect of the Hp2 allele in homozygoty||Quaye IK |
During the first thirty years (from the sixties to nineties of the last century) of cancer association studies, genetic blood markers, including haptoglobin were concomitantly studied with descriptive studies of allele distribution in the different populations (Giblett ER 1968, Langlois MR and Delanghe JR 1996, Wobeto VP
Moreover, the lack of reproducibility of the several studies may also reflect methodological differences in the criteria of case definitions and selection of controls, and in what concerns to the influence of environment of factors such as microbiological (HPV, EBV, M. tuberculosis, H. Pylori, Plasmodia), smoking habits, sun exposition, xenobiotic and sex steroid hormones (Beckman G
The usual cross-sectional approach of these studies didn´t take into account the somewhat different influences of the genotypes on the natural story of the cancer that courses in multistep way (Zur Hausen H 2002). The great majority of the studies were done in patients with distant phenotypes (advanced stage cancer) and take not in consideration the subclinical disease. This isn´t evidenced in those times by lack of knowledge of physiopathology and lack of reliable biomarkers (circulating and imaging) that gives a more dynamic picture of the situation.
It was not common, the realization of measurements of serum and plasma levels of the Hp independently of phenotype in part due too time consuming of the technics (Langlois MR and Delanghe JR 1996). In these cases, not even the local processes are reflected in circulation but also it is demonstrated the existence of a local tissue environment in what Hp functions in paracrine and autocrine way (Yang F
The natural history of cervical cancer seems to be dependent of genetic polymorphism of haptoglobin in its interaction with HPV and cofactors such as sex steroid hormones and smoking habits (Bicho MC
Also there are reports of the different influences of Hp alleles in a context of familiar history for the breast and ovarian cancers (Fröhlander N and Stendahl U 1988, Awadallah S and Atoum MF 2004).
For the clarification of these issues a better knowledge of the physiopathology mechanisms of action of the Hp alleles is necessary.
Haptoglobin as a pleiotropic protein has several different functions being the Hp1 allele and correspondent genotypes Hp1-1 and Hp1-2, the more represented in the several cancers reviewed. The innate immune response of the host against the tumour is limited in the subject’s carriers of the Hp1 allele through several mechanisms, already reviewed.
It is accepted, in this pathway, the role of Hp-Hb, CD163, HO-1, CO, bilirubin, activation of anti-oxidant intracellular systems (including ferritin), and extrusion of iron through ferroportin. This pathway is characteristic of immunosuppressive tumor macrophages M2 types that are more active in Hp1 carriers inducing a switch for a Th2 antinflammatory cytokine profile characteristic of lesser Th1 type cytotoxic immune antitumor mechanisms (Van Vlierberghe
This switch can be also dependent of a stronger acute phase response characteristic of Hp1 carriers that can modulate immune cells activity after binding of Hp to its receptors CD22, β2 integrin and LFA located respectively in B cells neutrophils and T cells (EL Ghmati SM
An increased prevalence of Hp 2-2 genotype is observed in some tumours leading to the hypothesizes of haptoglobin involvement in the mechanisms associated with the carcinogenesis and tumorigenesis of the chronic inflammation (head and neck carcinomas, glioblastome, gastric carcinoma).
The lower antioxidative capacity and inhibition of prostaglandin synthase associated to the genotype Hp 2-2 are the best known explanatory mechanisms (Wen WN et al 2001, Saeed SA
Another mechanism involved is the withholding of iron in macrophages that is necessary for the proliferation of immune cell (Touitou Y
The effects of smoking habits are modulated by Hp2-2, because the effects in the nicotine down regulation of haptoglobin expression and also the effects of CO producing local hypoxia and the immune depression (Ye YN
More studies are necessary to complete our understanding about the role of this important acute phase protein, its levels variations, particularly the fucosylated isoforms and its regulation, the Hpr and its polymorphism and its immunomodulation role in cancer. Finally, future studies may focus in the importance of haptoglobin polymorphism conducting to a pharmacogenetic approach to chemoprevention.