EASY APPROACHES TO CANCER TREATMENT USING CELLS’ CYCLE INHIBITORS.

CHAPTER ONE

LITERATURE REVIEW

1.1 Breast Cancer Epidemiology

1.1.1 The Global burden of breast cancer

For a large number of women newly diagnosed in the world, it has been ascertain that, breast cancer is a neglected disease in terms of other numerically more frequent health problems. It has also been described as an orphan disease, in the sense   that the very detailed knowledge about tumor characteristics and the necessary host biology capable of providing basic care is absent. Current international cancer policy and planning initiatives are irrelevant to breast cancer, with the exception of nutritional recommendation. However, progress with declines in mortality in some developed countries has been reported (Ginsburg et al., 2011).

Breast cancer is the most prevalent cancer in the world (4.4 million survivors up to 5 years following diagnosis) and the second most common cause of cancer related mortality in women wide world (Parkin et al., 2005). It also accounts for 23% (1.38 million) of the total new cancer cases and 14% (458,400) of the total cancer deaths in 2008 and ranks second most common cancer overall (10.9% of all cancers) but ranks fifth as cause of death  (Ferlay et al., 2010).  1.15 million new breast cancer cases were recorded in 2004 and over 500,000 deaths reported around the world and more than half of all cases occurred in industrialized countries (Parkin and Fernandez, 2006). Breast cancer incidence rates vary from 19.3 per 100,000 women in Eastern Africa to 89.7 per 100,000 women in Western Europe. They are normally high in developed regions of the world (except Japan) and low in most of the developing regions. Due to more favorable survival of breast cancer in developed regions, the range of mortality rates is very much less, approximately 6-19 per 100,000. Notwithstanding, it is still the most frequent cause of cancer death in women in both developing (269 000 deaths, 12.7% of total) and developed regions, where the estimated 189 000 deaths is almost equal to the estimated number of deaths from lung cancer (188 000 deaths) (Ferlay et al., 2010).

For some time now, there have been some encouraging in both breast cancer incidence and mortality trends with the incidence of new cases stabilizing as well as death rates falling in some high income or developed countries. However, this appears to be vice versa in developing countries (Kanavos, 2006). Notably, breast cancer incidence rates have leveled off since 1990, with a decrease of 3.5%/year from 2001 to 2004 (Li et al., 2003). In the same manner, breast cancer mortality rates have also declined by 24%, with the greatest impact among young women and as well as women with estrogen receptor (ER)-positive disease (Berry et al., 2005).  Also, both incidence and mortality declined in the United States; between 1999 and 2006, incidence rates decreased by 2.0% per year, and mortality decreased by 1.9% annually between 1998 and 2006 (Horner et al., 2006).  The decline in breast cancer mortality has been largely attributed to the combination of early detection with screening programs and the advent of more efficacious adjuvant systemic therapy.

Breast cancer is common in women both in the developed and the developing countries, comprising 16% of all female cancers. Although it is thought to be a common cancer in the developed countries, a majority (69%) of all breast cancer deaths occurs in developing world. Indeed, increase life expectancy, increase urbanization and adoption of western lifestyles have increased the incidence of breast cancer in the developing countries (Kanavos, 2006). Eventhough it is now the most common cancer both in developed and developing regions with around 690 000 new cases estimated in each region, much of the burden of incidence, morbidity, and mortality will occur in the developing world with population ratio of 1:4 (Ferlay et al., 2010). As developing countries succeed in achieving lifestyles similar to those in advanced economies, they will also encounter much higher cancer rates, particularly cancers of the breast. This forms part of a larger epidemiological transition in which the burden of chronic, non-communicable disease once limited to industrialized nations, is now increasing in less developed countries (Kanavos, 2006).

A report by Stewart et al (Stewart and Kleigues, 2003), mentioned that many of the new cancer cases are now occurring among women from low and middle income countries, where the incidence is increasing by as much as 5% per each year and there are about three fourths of breast cancer deaths occurring worldwide. Of the 411,000 breast cancer deaths around the world in 2002, 221,000 (54%) occurred in low- and middle-income countries (LMCs). The incidence of breast cancer rose from 126,227 cases in 2002 in China (IARC: Cancer Epidemiology Database, GLOBOCAN. 2002) to over 169,000 in 2008 (IARC: Cancer Epidemiology Database, GLOBOCAN. 2008).

 

Figure 1.1: Chart showing worldwide prevalent Breast Cancer cases (X1000), Adult population.

(GLOBOCAN, 2008)

1.1.2 Differences in Population of Breast Cancer

Breast cancer  variation among population, or the regional differences in the types  have  been attributed to the following: prevalence of major risk factors, availability and use of medical practices such as cancer screening, availability and quality of treatment, completeness of reporting, and age structure. However, geographic areas, and counties and parishes within countries also determine the frequency of the most commonly diagnosed cases or deaths (Garcia M et al., 2007). The highly penetrant but rare susceptibility genes, BRCA1 and BRCA2 (Fackenthal et al., 2007) and more prevalent, but lower penetrance genes, CHEK2 and FGFR (Easton et al., 2007) have been indicated to be the key inter-individual and inter-group differences in the distribution of reproductive risk factors. Countries with massive economic development over the past 50 years, such Japan, Singapore, and urban areas of China have experience an increase in breast cancer incidence (Horn-Ross  et al., 2000).

Age-standardized incidence rates for breast cancer 1998–2002 were 110 (nonHispanic Caucasians, California), 82.3 (Ontario, Canada), 41.3 (Hong Kong) and 14.7 (Jiashan, China) (Curado et al., 2007). Reports on migration studies reveal that the incidence of breast cancer changes significantly over one to two generations to more closely reflect the breast cancer risk in the adopted country (Ziegler  et al., 1993), which seems to occur in parallel with dynamics in diet and certain indicators of acculturation (Porter, 2008). Notably, evaluation of  differences in risk factors and natural history of all tumor types, would permit for comparisons based on geographical regions, socioeconomic status and levels of industrialization (Ginsburg et al, 2011).

Other differences in population of breast cancer are outlined below:

In a study by Li et al (Li  et al., 2002), it was showen that the majority of breast tumours from Asian women are estrogen receptor (ER) negative. Also it has been indicated that both pre-and postmenopausal Asian women with breast cancer, are likely to have ER positive tumors as Caucasians (Uy et al., 2007). In addition, greater proportion of ER+ tumors in a Vietnamese cohort, has been found in a studies on ER positivity among premenopausal breast cancer cases as compared with the comparison group of Caucasian women in Australia. (Tran and Lawson, 2004).

Considerably, variation in the gene profiles of tumors from populations of different genetic/ethnic backgrounds have also been reported. About 15% of sporadic breast cancer, which are BRCA1 origin in Caucasian women appears to have the basal phenotype. On the other hand, other studies have also suggested that breast cancer in women of African ancestry may have a higher proportion of basal phenotype (Carey  et al., 2006). In similar manner among Nigerians, a high frequency of basallike tumors was observed, where 87 of 148 (59%) breast cancer cases were both ER- and HER2-

(Olopade et al., 2004).

1.2 General characteristics of Breast Cancer

1.2.1 The normal Breast

 

Figure 1.4: Picture showing lobes and ducts inside the breast and lymph nodes near the breast (National Cancer Institute Booklet)

There are about 15 to 20 sections called lobes in a female’s breast and each lobe is made of many smaller sections known as lobules, which in turn have groups of tiny glands or milk-producing glands that can make milk. It is also made up of ducts, tiny tubes that carry the milk from the lobules to the nipple, and stroma fatty tissue and connective tissue surrounding the ducts and lobules, blood vessels, and lymphatic vessels  (American Cancer Society booklet).

1.2.2 The lymph (lymphatic) system of the breast

The lymph system  is very important in breast cancer research in that, it is one way breast cancers can spread and has several parts. Lymph nodes are small, bean-shaped collections of immune system cells that are connected by lymphatic vessels. These vessels are like small veins, except that they carry a clear fluid called lymph in place of blood away from the breast. They also contain Lymph tissue fluid and waste products, in addition to immune system cells. Breast cancer cells can enter lymphatic vessels and begin to grow in lymph nodes. Most lymphatic vessels in the breast connect to lymph nodes under the arm (axillary nodes). Some lymphatic vessels that connect to lymph nodes inside the chest are called internal lymph nodes, and those either above or below the collarbone are called  supraclavicular or infraclavicular nodes (American Cancer Society booklet).

There are several types of breast cancer, but some of them are quite rare. Currently, majority of all breast cancers worldwide are the ductal and lobular subtypes .However, the ductal subtype accounting acounts for the majority of the diagnosed cases ,constituting for about  40–75% (Rakha  et al., 2006).  In addition, several linear models of breast cancer initiation, transformation and progression, as depicted in Fig. 1.5 have been formulated. There are two models for the ductal subtype The first ‘ductal’ model, reported by Lerwill (Lerwill, 2008) are as follow: First of all, it recognizes  flat epithelial atypia (FEA), to atypical ductal hyperplasia (ADH) and then ductal carcinoma in situ (DCIS) as the nonobligate precursors of the advanced invasive and metastatic ductal carcinoma. In the second model, usual epithelial ductal hyperplasia (UDH) was proposed as an intermediate stage of progression between FEA and DCIS (Page et al., 1985). In the case of lobular subtype, atypical lobular hyperplasia (ALH) and lobular carcinoma in situ (LCIS)  was also proposed as the non-obligate precursor lesions to invasive lobular carcinoma. (Boecker et al.,  2002).

Figure 1.5:  Models of breast cancer progression. (A) The classic model of breast cancer progression of the ductal type proposes that neoplastic evolution initiates in normal epithelium (normal), progresses to flat epithelial atypia (FEA), advances to atypical ductal hyperplasia (ADH), evolves to ductal carcinoma in situ (DCIS) and culminates as invasive ductal carcinoma (IDC). Immunohistochemical, genomic and transcriptomic data strongly support the evidence of a continuum from FEA to ADH, DCIS and IDC, indicating FEA as the potential non-obligate precursor of ADH. In contrast, the alternative model of breast ductal cancer progression (B), which was mostly based on epidemiological and morphological observations, proposes usual ductal hyperplasia (UDH) instead of FEA as the direct precursor to ADH. However, recent studies have shown that UDH has a distinct immunohistochemical and molecular profile from FEA and probably represents a biological dead end. (C) The model of lobular neoplasia proposes a multi-step progression from normal epithelium to atypical lobular hyperplasia, lobular carcinoma in situ (LCIS) and invasive lobular carcinoma (ILC). (Bombonati and Sgroi, 2011)

1.2.3 Risk factors for Breast Cancer

The aetiology of breast cancer is multifactorial and from descriptive epidemiological data it has clearly emerged that breast cancer is a disease of affluent societies which have acquired the Western lifestyle, characterized by a high-caloric diet rich in animal fat and proteins, combined with a lack of physical exercise. Regions which have featured this lifestyle for a long period of time (North America, Nort hern Europe, Australia) have reached a plateau of an incidence rate of 70 to 90 new cases per 100,000 population/year while countries that have more recently become industrialized and affluent show a marked increase in incidence and mortality. (ACS booklet) Various risk factors identified include age,hereditary,dietary (diet and obesity), gynecological (oral contraceptives, hormone replacing therapies,endogenous hormone levels,age of menarche and menopause,parity and mammographic density), life style (physical activity,smoking and alcohol) ,oxygen reactive species,radiation and environmental pollutants. (Fig. 1.6).

With age,breast cancer incidence is known to drastically increase up to the age of 50, after which it increases slowly (Mitruen and Hirvonen, 2003). Hereditary factors are observed in about one fourth of the total cases of breast cancer,which involves two classes of genes; high and low penetrance genes. High penetrance genes with allelic variants that are relatively rare,such as BRCA1/2,tumor protein 53 gene (TP53) and ataxia telangiecttasi mutated gene (ATM).  Low penetrance genes such as the genes encoding for the enzymes involved in estrogen and carcinogen metabolism as well as in the detoxification of reactive oxygen species,for instance P450 cytochrome and Gluthioone-Stransferases (GSTs) on the other hand are more common and allelic variants confer low risk of breast cancer (Mitruen and Hirvonen ,2003).

In the case of dietary, the human diet contains variety of natural carcinogens and anticarcinogens. Uptake of fruits and vegetables which are rich in antioxidants reduces the risk (McKeown, 1999) whereas increase in polysaturated fatty acids (Bartsch et al., 1999) and meat consumption (Zheng et al., 1998) increases the risk of braest cancer. Obesity has been reported to be associated with an increase in estrogen levels and as well as a risk in postmenopausal women,who have most of their estrogen derived from the conversion of androgens,in the adipose tissue,as a result of aromatse enzyme activity  (Hunter et al., 1993). However, in premenopausal women, it has been indicated that, obesity can have  a protective effect, due to the higher period of frequent ovulation which reduces estrogen levels (Mannisto et al., 1996). Physical activity is a lifestyle factor which is considered as a breast cancer risk factor. It is considered protective against breast cancer because it reduces the regualar ovulatory cycles and increases the level of catechol-O-methylated estrogens (Henderson et al., 1985).

Cigarette smoke is very rich in carcinogens and reactive oxygen species and may be considered as one with high risk in breast cancer (Mitruen et al., 2003). Its function is controversial ,in that it can serve as a protective against cancer.It may contain antiestrogenic effect,such as nicotine wich inhibit aromatase. Further more,women who smoke tend to reach menopause earlier than nonsmokers (baron et al., 1990). Another fact to consider is alcohol. 15% of alcoholic women have the risk of developing breast cancer (Kuper et al., 2000). It has been stipulated alcoholic women have higher levels of estrogen than non-alcoholic (Reichman et al., 1993).

The use of oral contraceptive increases breast cancer risk but disappears after ten years of cessation, whereas hormone replacement therapy disappears in five years. However, breast cancer cases in hormone replacement therapy (HRT) tend to be less advanced at the time of diagnosis, and biologically less agressive compared to women who never used such therapy (Holli et al., 1997).

Relationship between endogenous estrogen levels and breast cancer has been indicated. High estrogen levels in the serum or urine, and low levels of sex hormone binding protein (SHBG), resulting in high bioavailability of free estradiol also point for an important role for endogenous and exogenous estrogens in the risk of breast cancer (Kristensen et al., 2000).

Another strong marker of breast cancer risk is the degree of mammographic density. It has been indicated that, the risk in women with denser breast is four to six times higher than those with less dense breast (Boyd et al., 1995). Evidence suggest that, the etiology of mammographic density may be due to the exposure to steroid hormone, since it decreases with age (Boyd et al., 2002) as well as in women on tamoxifen (Cuzick et al., 2004) and also increases in women who are on hormone replacement therapy (Rutter et al., 2001).

Environnmental pollutants similar to hormones can also interfere in the control of a large family of nuclear hormone receptors, which in turn can upregulate various genes involved in the cell cycle,such as TP53, Retinoblatoma (RB)  and the serine.threonineprotein kinase proto-oncogene RAF by transcriptional activation induced by ligand (Kristensen et al., 2000).

These polluatnts are designated xeno-estrogens, which include pesticides, dyes, food preservatives and other polluatnts and can play a role in the etiology of breast cancer, since they interfere with the activity of endogenous estrogens (Garner et al., 2000). Ionizing radiation (John and Kelsey, 1993) and history of benign breast cancer (Mitruen et al., 2003) have also been established to increase breast cancer risk.

 

Figure 1.6:  Aetiological factors involved in the development of breast cancer (Tavassoli and Devilee, 2003)

1.2.4 Breast Cancer treatment and survival

There are various treatment plan for cancer patient depending on the type and the stage at diagnosis. Surgical treatment for breast cancer has to do with breast-conserving surgery (BCS) or mastectomy and when this is done properly for localized or regional cancers, long-term survival is the same as with mastectomy (Jatoi et al., 2005). With early stage (I or II) breast cancer, 57% of women undergo BCS, 36% have mastectomy, 6% undergo no surgical treatment, and about 1% do not receive any treatment (Fig. 1.7). On the other hand, with women of late stage (III or IV) breast cancer, 13% receive BCS, 60% undergo mastectomy, 18% do not have surgery, and 7% do not receive any treatment. Most of the early stage breast cancer women who undergo BCS receive adjuvant treatment; nearly one-half undergo radiation therapy alone and one-third receive both radiation therapy and chemotherapy. Contrary, most of them diagnosed with late stage disease undergo chemotherapy in addition to surgery and other therapies. (Siegel et al., 2012).

Figure 1.7: Female Breast Cancer Treatment Patterns by Stage, 2008. BCS indicates breast conserving surgery; RT, radiation therapy; chemo, chemotherapy (may include common targeted therapies). Percentages do not sum to 100% due to rounding. (Siegel  et al., 2012)

Patients can expect to be cured or to experience at least long-term survival of more than 10 years. There has been an improvement for the overall 5-year relative survival rate for female breast cancer patients, from 75.1% between 1975 to 1977 to 90.0% for 2001 through 2007. And this is attributed to the fact that there has been an improvement in chemotherapy and hormone therapy treatment and also due to earlier diagnosis resulting from the widespread use of mammography (Siegel et al., 2012).  In the case of localized breast cancer, the 5-year relative survival rate is 98.6%; which declined to 83.8% for regional stage and 23.3% for distant stage. Other factors that influence survival include tumor grade, hormone receptor status, and human epidermal growth factor receptor 2 (HER2) status.

There are differences between African American women and white women on the basis of stage and survival. African American women are less likely than white women to be diagnosed with local stage breast cancer (51% vs 61%) and have lower survival rates than white women within each stage of disease. Though difficult to explain this reasons, it may be explained in large part by a combination of socioeconomic factors, less access to care among African American women, and biological differences in cancers (Siegel et al., 2012).

Figure 1. 8:  Estimated Numbers of US Cancer Survivors by Site. (Siegel  et al., 2012)

1.2.5 Biomarkers and chemotherapy in Node-negative breast cancer

The most prevalent form of breast cancer worldwide is node-negative breast cancer (Fig. 1.9) and in regions or countries with widespread breast cancer screening and disease awareness among women, it is likely to be rated between 65%–70% of breast cancer patients (Harbeck and Thomssen, 2011). Most patients have no or only a few (1-3) axillary lymph nodes involved and therefore have a good chance of being cured. There is no substantial difference in the underlying tumor biology between node-negative and node-positive disease, and the question that remains in adjuvant chemotherapy today is in proper patient selection. Node-negative breast cancer does not automatically suggest a good prognosis, or the lack of a need for chemotherapy.

Data from Adjuvant Online show that the mortality risk may even be higher in patients with node-negative grade 3 tumors than the risk demonstrated in some patients with node-positive disease, suggesting a high risk among these patients to indicate adjuvant chemotherapy. But then, there is still a good degree of uncertainty in determining whether patients with node-negative disease actually benefit from chemotherapy, which lead many clinicians to hesitate before indicating chemotherapy in many node-negative patients. (Harbeck and Thomssen, 2011). With node-positive disease, it  is associated with an overall mortality rate of approximately 20%, and oncologists do not hesitate to prescribe chemotherapy for these patients.

Adjuvant systemic therapy in women with early-stage disease is guided by prognostic and predictive factors, including stage, grade, estrogen receptor (ER) and progesterone receptor (PR) status, Ki-67 status and HER2 amplification. These parameters help physicians to select adjuvant systemic therapy. However, these remain imperfect tools, in that some patients receive systemic chemotherapy even though they can be cured by surgery alone. If these parameters are used alone in selecting treatment as recommended by 1998 and 2001 St Gallen consensus statement, up to 90% of nodenegative breast cancer patients will be candidate for adjuvant chemotherapy, although only about 30% of them will relapse and thus need adjuvant chemotherapy. In terms of tumor grade, it is certainly important in that it is predictive of risk over time, but it lacks standardization. In case of undifferentiated cancers (grade 3), patients are truly at highrisk and may benefit from chemotherapy, whereas in case of well-differentiated grade 1 cancers, the risk of recurrence may be rather low. However, in the heterogeneous group of grade 2 tumors, it is essential to know for which patients the benefits of chemotherapy will outweigh its potential side effects.

Therefore, new prognostic factors are still required to optimize treatments among these patients. However, most patients are offered chemotherapy according to current guidelines, leading to over-treatment of a large proportion, since there is no means of clearly identifying those patients who will not relapse and hence do not need adjuvant chemotherapy. (Goldhirsch A et al., 2005).  One of the major clinical questions is how to identify those patients who may be able to avoid adjuvant chemotherapy because of their low risk of recurrence.

 

Figure 1.9: Approximate percentages of patients with node-negative disease at time of diagnosis in different parts of the world. (Harbeck and Thomssen, 2011).

1.2.6 Node status and relapse Rate

Another clinical question is how to identify individuals with high risk who may benefit from adjuvant chemotherapy. One important risk factor for disease relapse is nodal stays. Most patients with node-negative breast cancer have a fairly good ten-year overall survival with loco-regional treatment alone, (Fig 1.10) however, about 30% relapse developing distant metastasis. In fact, approximately 70% of node-negative patients respond sufficiently to surgery, radiotherapy, and endocrine therapy, and do not require additional chemotherapy. The problem faced by most clinicians now face is that approximately 30% of node-negative patients will need chemotherapy because of their risk for recurrence, but there are limited tools currently available to identify this subset of patients. Data from Adjuvant! Online have shown that even patients with grade 1, estrogen receptor– negative, node-negative tumors may have a high relapse rate of almost 20% over 10 years. Indicating that the relapse rate is even higher in patients with grade 2 and grade 3 tumors  (Harbeck and Thomssen, 2011).

Therefore, markers to predict individual risk who may benefit from adjuvant chemotherapy and also to identify patients not requiring aggressive adjuvant therapy are urgently needed, so as to avoid unnecessary exposure of women to the potential toxicity and side-effects of such treatment, and also to reduce the overall cost of breast cancer management as well as preventing under-treatment of node-negative breast cancer are needed.

The main cause of morbidity and mortality in patients with cancer is the formation of distant metastases, which is a multistep event involving local invasion, degradation of extracellular matrix, angiogenesis, intravasation, evasion of apoptosis and survival in circulation, extravasation and growth at secondary site. Key mediators of this process include certain proteinases such as uPA, PAI-1, MMPs and ADAMs which has caused increase attention to be drawn on these factors as potential prognostic markers for risk assessment in node-negative breast cancer. However, unlike MMPs, uPA , and PAI-1, little work has been done on ADAMs role as a prognostic factor.

Figure 1.10: Lymph node status and relapse rate in primary breast cancer (Harbeck and Thomssen, 2011).

1.3 Expression and functions of ADAMs

The ADAMs (A Disintegrin And Metalloproteases) family, are modular type I transmembrane proteins which belongs to the zinc protease superfamily. Although they are frequently referred to as a family, they are made up of a subfamily of the M10 family which, in turn, belongs to the metzincin clan of metalloproteases (Duffy MJ et al., 2009; Arribas J et al., 2006).   Forty gene members have been identified so far, but then it is believed that about 21 functional in humans. Members of the family have a modular design, characterized by the presence of metalloprotease and integrin receptor-binding activities, and a cytoplasmic domain that in many family members specifies binding sites for various signal transducing proteins (Seals and Courtneidge, 2003). Based on their primary structure analysis, it has been indicated that, in addition to the disintegrin and metalloprotease, all ADAMs contain the following domains: signal peptide, prodomain, cysteine-rich, epidermal growth factor (EGF)-like, transmembrane and cytoplasmic (Fig. 1.11).

ADAMs expression profile can vary considerably and are mostly not expreesed in Escherichia coli, Saccharomyces cerevisiae, or plants but are rather  found in vertebrates,

Caenorhabditis elegans, Drosophila, and Xenopus. The fission yeast Schizosaccharomyces pombe has what may be an early progenitor of the ADAMs family, although its properties have not been studied (Seals and Courtneidge, 2003). Transcription of ADAM mRNA is modulated both positionally and temporally. Although some ADAMs are testis specific, others are found in variety of tissues and the transcription of ADAMs 1-6 in the testis is developmentally regulated. For example, in mammals many of them (including ADAMs 2, 7, 18, 20, 21, 29, and 30) are exclusively or predominantly expressed in the testis and/or associated structures. Other members (ADAMs 8, 9, 10, 11, 12, 15, 17, 19, 22, 23, 28, and 33) show a more broad somatic distribution.  Originally, ADAMs 9, 12, and 19 were cloned from myoblasts, but have been shown subsequently to be more broadly expressed. (Seals and Courtneidge, 2003)

Many ADAMs have been reported to be expressed in multiple splice form. For instance, ADAM22, ADAM29 and ADAM30 with two to three forms that vary in the lengths of their cytoplasmic tails, but then no functional differences in their isoforms have been reported. On the other hand others have produced proteins with markedly different activity as in the case of ADAM12 with two splice forms: L,which produces a membrane-bound protein, and S, diverges just upstream of the transmembrane domain, which results in a shorter form that is secreted fromthe cell (Seals and Courtneidge, 2003). Studies by Shi et al (Shi et al., 2000) indicated that ADAM12-S has functional IGFBP-3 and IGFBP-5 protein cleavage activity and due to its overexpression during pregnancy, it is possible that ADAM12-S is responsible for increasing the pool of IGF in the bloodstream during pregnancy through IGFBP proteolysis.

Another protein to consider is ADAM28. It produces isoforms with different subcellular localization patterns and tissue expression. There are three isoforms in Murine ADAM28 , two larger ones for encoding membrane-anchored proteins and expressed in the epididymis and lung, and smaller one predicted to encode a secreted protein with testis-specific expression. However,in human  there are only two forms: the secreted form which is  preferentially expressed in the spleen, and the membrane-bound form with specificity to lymph node (Seals and Courtneidge, 2003). Other ADAMs with documented evidence of alternativel splicing are ADAM9,  ADAM10, ADAM11 and ADAM3.

The ADAMs have been implicated in processes such as the activation of the proforms of certain growth factors and cytokines as well as the shedding of the extracellular domains of growth factor receptors and adhesion proteins, control of membrane fusion, and cell migration, as well as physiological processes such as muscle development, fertilization, neurogenesis, adipogenesis, myogenesis and cell fate determination.  Another function  is the activation of NOTCH signalling by Notch ligand Delta shedding from the cell surface by ADAM-10 (Rocks et al., 2008). Accumulating evidence demonstrates ADAMs as proteins that  support both proteolytic activity and cell adhesion, making  them candidates to mediate both the remodelling of the extracellular matrix (ECM) and the changes in cell adhesion that characterize certain pathological processes such as tumor development, bacterial infection, cardiac hypertrophy, and asthma (Murphy, 2008). Of these different diseases, it is in cancer where most research has been carried out .

Key features of malignant tumours are their abilities to invade surrounding tissues, to have access to the vascular and lymphatic systems, and to disseminate to distant organs by metastatic spreading (Butler et al.,  2006). Major ADAMs shown to play a role in cancer include ADAM8, -9, -10, -12, -15, -17, -19, -28 and ADAMTS1, -4 and -5. The related group of ADAMTS are secreted soluble proteins that contain a variable number of thrombospondin-like repeats (Fig. 1.11). Consistent with a causative role in cancer, several ADAMs are emerging as potential cancer biomarkers for aiding cancer diagnosis and predicting patient outcome. Furthermore, a number of selective ADAM inhibitors, especially against ADAM10 and ADAM17, have been shown to have anti-cancer effects. Collectively these results have led to the proposal of these metalloproteases as putative targets of anti-tumor therapy.

 

Figure 1.11: The topography of the ADAMs and related metalloproteases. Comparison of domain structures of ADAMs, SVMP P-II, SVMP P-III, SVMP-IV, ADAM-TS and MMPs. ADAM protein contains an N-terminal signal peptide (S.P.), a pro-peptide domain, a metalloprotease domain, a disintegrin-like domain, a cysteine-rich region, an EGF-like domain, a transmembrane domain(TM) and a cytoplasmic domain (Cyt. Tail). (Lu et al., 2007).

 

Table 1.1 Potential functions of human ADAMs

 

ADAM	Potential functions and features	Localizatio n
Proteolytically inactive
ADAM2	Sperm-egg binding and fusion	Sperm
ADAM7	Sperm maturation	Testis
ADAM11	Integrin ligand, neural adhesion, tumor supressor	Brain
ADAM18	Oocyte recognition	Testis
ADAM22	Adhesion	Brain
ADAM23	Tumour suppressor, Cell adhesion, neural development	Brain, Heart

 

ADAM29	Unknown	Testis
Proteolytically active
ADAM8	Shedding of adhesion molecules, leukocyte receptors, neutrophil infiltration, osteoclast stimulation	Macrophag e, neutrophil
ADAM9	Alpha-secretase activity, cellular adhesion, Shedding of HB-EGF, TNF-p75 receptor, cleavage of APP, digestion of fibronectin and gelatin,	Various tissues
ADAM10	Alpha-secretase activity, shedding of TNF alpha, betacellulin, HER2, Notch, and collagen IV,cellular adhesion, digestion of gelatin and myelin, basic protein, cleavage of delta, APP, L1, and CD44, shedding of HB-EGF, presence of RRKR sequence.	Kidney, Brain, Chondrocyt e
ADAM12	Cellular     adhesion,     shedding     of     HB- EGF,muscle formation, presence of RRKR sequence, digestion of IGFBP-3 and 5,  digestion of collagen IV, gelatin and fibronectin	Osteoblast, muscle, chondrocyt e, placenta
ADAM15	Cellular           adhesion,         expression             in arteriosclerosis, digestion of collagen IV and gelatin	Smooth muscle cell, chondrocyt e, endothelial cell, osteoclast
ADAM17	Release of several growth factor ligands, e.g., TNF-alpha,TGF beta,and specific  EGFR/HER ligands, cellular adhesion, Shedding  of TRANCE and HB-EGF, presence of RRKR sequence, cleavage of APP, Notch, L-selectin and CD44	Macrophag e, Various tissues
ADAM19	Formation of neuron, digestion of neuregulin, sheddase, dendritic cell development.	Testis
ADAM28	Shedding of IGFBP3, immune surveillance, Digestion of myelin basic protein.	Testis, lung, lymphocyte , pancreas, uterus
ADAM33	Mutation in bronchial asthma patients, cleavage of APP, KL-1 and      insulin B         chain, involved             in pathogenesis of gastric cancer via IL-18 secretion	Lung (flbroblast, smooth muscle)

 

(Duffy et al., 2011)

1.3.1 Subcellular location of ADAMs

ADAMs have been proposed to probably synthesized in the rough endoplasmic reticulum and later mature in a Golgi compartment  and this is done by the removal of the prodomain from the ADAM precursor protein . Studies have proved that, bulk of the protein resides in a region near the nucleus, where they colocalize with Golgi markers (Seals and Courtneidge, 2003). However, certain ADAMs such as ADAMs 9, 10, 15, 17, and 28 can also be detected on the cell surface. Hougaard et al (Hougaard et al  ., 2000) reported that there was a regulated transition of the alternatively spliced short form of ADAM12 (ADAM12-S) from intracellular compartments to the cell surface, whiles the large form was retained in the trans-Golgi network.

However, several ADAMs family members may be active intracellularly. For instance, it has been reported that, most of the mature form of ADAM15 is resistant to trypsinization treatment, indicating a predominantly intracellular pool. Also the metalloprotease activity of ADAMs 10, 17, and 19 can also occur within intracellular compartments. Nevertheless, the cell type, the ADAM, and the substrate involved may determine such differences in subcellular localization and activity.

Figure 1.12: A schematic  overview of ADAMs synthesis, processing, and function.  (Seals and Courtneidge, 2003).

 

1.3.2 Structure and Domain activity of ADAM Proteins

The generalised structure of an ADAM protein contains 8 distinct domains or regions. In the typical ADAM protein, these domains are a signal domain, a prodomain, a metalloproteinase domain, a disintegrin or integrin-binding domain, a cysteine rich region, an EGF (epidermal growth factor)-like domain, a transmembrane sequence and an intracellular C-terminal end  (McGowan et al., 2007). Like most proteases, the ADAMs are initially synthesised as enzymatically-inactive precursor proteins. 

1.3.2.1 The prodomain

At the N terminus of ADAMs recides a signal sequence that directs ADAMs into the secretory pathway and a prodomain that lead to ADAMs maturation. (Seals and Courtneidge, 2003). The metalloprotease site of ADAMs are kept inactive, through a cysteine switch by the prodomain .The conserved cysteine residue located within the prodomain preferentially coordinates the required active site zinc atom, and thereby sequesters the metalloprotease domain in an inactive conformation  (Edwards et al., 2008; Becker et al., 1995) . For protease activation, this prodomain is removed by a furinlike convertase or by autocatalysis, depending on the specific ADAM (Murphy, 2008). The mechanism employed is the cleavage of the prodomain from the rest of the protein  by proprotein convertases (PCs) at a conserved Rx(R/K)R motif, thereby effectively releasing the prodomain and switching the zinc coordination to the metalloprotease domain, enabling it to undertake its catalytic activity. Several studies support this mechanism (Seals and Courtneidge, 2003).

Aside this mechanisms, there are cases in which ADAMs may undergo autocatalytic activation. Example is when, activity-blocking mutations in the metalloprotease domains of  ADAM8 and ADAM28, produces only the precursor form of the protein in transfected cells (Schlomann et al., 2002). Another functional aspect of the prodomain is to chaperone the proper folding of the metalloprotease domain of ADAMs. It has been suggested by studies that the removal of the prodomain of ADAM17 generates a protease-inactive protein (Milla et al., 1999). Similarly, an ADAM10 construct lacking its prodomain is catalytically inactive in vivo. Hence, it can be concluded from this evidence that, the prodomain appears to be necessary  in assisting  in the proper folding of ADAMs, in the structuring of the catalytic active site, and in the proper transit of ADAMs throughout the secretory pathway (Seals and Courtneidge, 2003).

1.3.2.2 The metalloprotease domain

This domains are well conserved, but then, only 25 out of a known total of 40 members of the family (ADAMs 1, 8-10, 12, 13, 15, 16, 17, 19-21, 24-26, 28, 30, and 3340), have the zinc binding catalytic site consensus sequence of HEXXHXXGXXHD (single letter amino acid code) where X is stands for any other amino acid; the three H residues bind zinc, the G allows a turn and E constitutes the catalytic residue, comprised of a water molecule tetrahedrally coordinated to the zinc, and the E residue acting as a catalytic base (Lu et al., 2007).

 

Metalloprotease domain is located next to prodomain and only about 60% of ADAMs exhibit nonprotease activity, although they all posses this domain. On the basis of their structural definition, of the 21 human ADAMs identified, only 13 are proteolytically active. ADAMs shown to exhibit protease activity include ADAM8, 9, 10, 12, 15, 17, 19, 28 and 33 (Kawaguchi et al., 2002). The mechanism of  the proteolytic activity, which is the best defined function of ADAMs currently, has been more accurately evaluated through crystallization of the metalloprotease domain.  At the  active site is zinc and water atoms necessary for the hydrolytic processing of protein substrates, and which are coordinated by three conserved histidine residues and a downstream methionine. The methionine lies in a Met-turn motif that loops around to face the consensus HExxHxxGxxH site. Although individual proteins among the various metzincins exhibit certain distinguishable structural features that may impart specificity for substrates and protease inhibitors, there exist remarkable conservation within this catalytic site (Seals and Courtneidge, 2003).

1.3.2.3 The disintegrin domain

The disintegrin-like domain located at the downstream of the metalloprotease domain, consists of 60 to 90 amino acid long with 6 to 15 Cys residues with sequence similarity to that  of the snake venom disintegrins (Marcinkiewicz C, 2005). This sequence  is found in all ADAMs and they binds to integrins, which are a group of adhesion proteins implicated in cell adhesion, migration and cell signalling (Stupack, 2007) . Snake venom disintegrins , which confers the ability of these molecules to interact with integrins in different cell systems have been characterized as potent inhibitors of the function of various integrins. The disulfide bridge pattern of RGD-containing disintegrins, which may be important to their biological activity, especially their potency and selectivity has been determined by means of chemical methods, NMR spectroscopy and crystallography. Not all ADAMs have the RGD sequence and in place of that, others contain sequences such as KGD, MVD, MLD, VGD, ECD, or MDG (single letter amino acid code). (Lu et al., 2007)

ADAMs distegrin derived its name for its presence in the snake venom metalloproteases (SVMPs), involved in binding of platelet integrin receptors. Consequently,the binding to this receptors, prevents the association of platelets with their natural ligands such as fibrinogen, and results in a block in platelet aggregation at the wound site. This disintegrin-mediated interaction of SVMPs along with the breakdown of basement membrane components by their metalloprotease activity leads to the severe hemorrhaging caused by bites from snakes harboring these toxins (Seals and

Courtneidge, 2003). Although, the disintegrin domain has been widely described as being able to interact with integrin molecules and therefore mediating cell-cell and cell-matrix interactions (Reiss et al., 2006), it  has been shown not to be available for protein binding due to protein folding (Takeda et al., 2006). It may be therefore  considered as a structural feature rather than an integrin ligand.

1.3.2.4 The cysteine-rich and EGF-like domains

The Cys-rich and EGF-like domains, though not much is known about them may play very important role for interactions of ADAMs with other proteins such as chaperones involved in biosynthesis and or with other partners on the cell surface. Structurally, the two domain consist of about 160 amino acid with 10 to 14 Cys residues and about 40 amino acid  with 6 Cys residues, respectively. EGF domains have been indicated to be found in proteins that are either completely secreted or have transmembrane regions that tether the protein to the cell surface (Lu et al., 2007). On the other hand, the Cys-rich domain is located at the carboxy terminal end of ADAMs, which has been considered likely to complements the binding capacity of the disintegrin-like domain and  imparts specificity to disintegrin domain-mediated interactions (Emi et al., 1993). The Cys-rich domain of TACE/ADAM-17 may play a role in the release of the pro-domain and may be required for the shedding of interleukin 1 receptor type II as well aid in as well the recruitment of accessory proteins involved in targeting TACE to some substrates (Reddy et al., 2000) Several other fuctions have been reported for Cystein-rich domain as follows:

It has been indicated to be involved in cell-cell fusion  (Huovila et al., 1996), regulating protease activity and controlling substrate specificity (Reiss et al., 2006), function as a ligand for the cell-adhesion molecule syndecan, especially syndecan-4  (Iba et al., 1999, 2000).

 

1.3.2.5 Transmembrane domain

Mostly, ADAMs belong to the  type I membrane proteins, anchored through a transmembrane (TM) domain near the C-terminus and  also have an alternatively spliced form that diverges before the TM domain, leading to the production of a soluble, secreted form. All of the ADAMTSs lack a TM domain and are therefore termed as secreted proteases. Examples of  ADAMs with transmembarne domain include 11, 12, 17, and 28 (Cerretti et al., 1999; Lu et al., 2007).

The ability to posses both soluble and membrane-anchored forms, allows ADAMs to regulate events not only on or near the cell surface, but also at a distance from cells.

However, very little is known as to whether all the membrane-anchored ADAMs have a soluble counterpart generated through either alternative splicing or shedding from the cell surface. (Cerretti et al., 1999; Lu et al., 2007)

 

1.3.2.6 The cytoplasmic tail

ADAMs have unusual cytoplasm tail, with many rich in proline, serine, glutamic acid and or lysine. They are highly variable both in length ( between 40 to 250 amino acid) and in sequence and contains a phosphorylation sites and SH3 binding domains .This domain contains noticeable and specialized motifs that have been postulated to be involved in the inside-out regulation of metalloprotease activity, the outside-in regulation of cell signaling, and or the control of maturation and subcellular localization (Lu et al., 2007; Seals and Courtneidge, 2003).  PxxP binding sites for SH3 domain-containing proteins are the most notable motifs which can be found in human ADAMs 7, 8, 9, 10, 12, 15, 17, 19, 22, 29, and 33. Several ADAMs also have potential phosphorylation sites for serinethreonine and/or tyrosine kinases. Not only might this regulate ADAM function directly, but the resulting phosphotyrosine residues could also provide ligands for SH2 domaincontaining proteins (Seals and Courtneidge, 2003).

In a study, ADAM9 binds to endophilin I and SH3PX1 (Howard et al., 1999). Given the potential function of endophilin I and SH3PX1 in vesicle sorting, it is speculated that these interactions may play a part in the regulation of ADAM maturation and or subcellular localization. Also it was reported that the membrane proximal region of the tail of ADAM9 associates with the catalytic domain of protein kinase C-alpha (PKC-alpha) (Izumi et al., 1998). The tail of ADAM9 can be phosphorylated by PKC in vitro at one or more serine and threonine residues. It therefore speculated that PKC-alpha helps to recruit ADAM9 to specific sites on the plasma membrane and upon phosphorylation or activation of ADAM9, shedding of HB-EGF occurs (Seals and Courtneidge, 2003).

 

 

1.3.3 Mechanisms by which ADAMs play a role in cancer

There are several different mechanisms through which ADAMs promote cancer formation and progression. Some of these processes includes Cell proliferation, Angiogenesis and Apoptosis.

 

 

 

 

1.3.3.1 Cell Proliferation

Cell proliferation by ADAMs can occur through the following processes:

 

1.3.3.1.1 Activation of positively-stimulating pathways

Several proteolytically active ADAMs regulate cell proliferation by cleaving growth factors or cell surface proteins or by activation of positively-stimulating growth factors. Many of these growth factors are first and foremost synthesised as inactive transmembrane precursor proteins that require ectodomain shedding for activation in order to fuction at its exert maximun capacity. Ligands for several growth factor receptors are processed by ADAM family members  and amongst the best-studied growthstimulating factors that are activated by ADAMs are the EGFR/HER family of ligands (EGF receptor ligands (heparin-binding EGF (HB-EGF), amphiregulin, betacellulin, epiregulin) with primary conversion mediated by either ADAM10 or ADAM17 (Duffy et al., 2011). However, other ADAMs such as ADAM8, 9, 12, 17 and 19  can also activate one or more of these ligands (Horiuchi et al., 2007) .

What actually happens is that, the shed form of the ligands binds to one or more of the EGFR/HER family of receptors. Upon homo- or heterodimerisation, several different pathways including the mitogenactivated protein kinase (MAPK) pathway, the phosphatidylinositol 3-kinase (PI3K) pathway and janus kinase/signal transducer and activator of transcriptional (JAK/STAT) pathway, are activated by downstream signalling which can results in some of the classical hall markers of malignancy such as enhanced cell proliferation, increased cell motility and increased cell survival (Peeters et al., 2009; Duffy et al., 2011).

 

 

Figure 1.13: Mode of action of ADAMs in the activation of EGFR/HER receptor signalling ADAMs  are involved in proteolytic ectodomain shedding of membrane bound ligands. The released ligands (EGF, HB-EGF, TGFa, heregulins) are free to bind to and activate EGFR, HER3 and HER4. Following receptor dimerisation , downstream signalling through many pathways is activated, including MAPK, PI3K and JAK/STAT. (Duffy MJ et al., 2011).

 

In a work reported by Singh et al it was showed that UV irradiation of skin cancer cells activated ADAM9 and 17 which was followed by amphiregulin shedding, EGFR transactivation and increased cell proliferation.  ADAM-10 also contributes to cell proliferation by modulating b-catenin signalling through E-cadherin shedding and increasing gene cyclin D1 levels (Shtutman et al., 1999).

 

 

1.3.3.1.2  Inactivation of growth-inhibitory pathways

Inactivation of growth-inhibitory pathways has been  indicated in TGFb which signals via TGFbR1 and TGFbR.  It has been proposed that, in normal and early malignant cells, TGFb inhibits proliferation where as in progressive malignancy, TGFb promotes proliferation (Ikushima and Miyazono, 2010 ; Duffy et al., 2011). ADAM17 was reported to mediate shedding of the type 1 TGFb receptor, thereby drcreasing TGFb signalling which led to decreased growth inhibition and the reduction in growth inhibition complements the growth stimulation, resulting from increased release of the EGFR/HER ligands (Liu et al., 2009; Duffy et al., 2011).

 

 

1.3.3.1.3. Shedding of adhesion proteins

One of the means of increased cell proliferation is by means of shedding of adhesion proteins by ADAMs. For example ADAM10 appears to be the major sheddase for the release of EGF and betacellulin (Sahin et al., 2004, 2007 ) and also contributes to   E-Cadherin shaddase (Ito et al., 1999) . The sunsequent   release of soluble E-cadherin in the extracellular milieu leads to the abrogation of cell-cell contacts, thereby facilitating cell migration.  ADAM-10 also contributes to cell proliferation by modulating b-catenin signalling through E-cadherin shedding and increasing gene cyclin D1 levels (Shtutman et al., 1999) Najy et al  also reported that ADAM15-mediated shed form of cadherin E bound to and activated HER2 in breast cancer cells. The increased proliferation and migration observed was attributed to the fact, the shed form of cadherin E formed a complex with HER2 and HER3, resulting in  enhanced ERK signalling.

Apart from increased cell proliferation, this shedding might also be expected to weaken cell:cell interaction and thus allow dissociation of potential invasive and metastatic cells in the primary cancer which  could potentially place a malignant cell or group of cells on their pathway to metastais. Shedding of other adhesion proteins such as L-selectin, ICAM-1 or VCAM, on the other hand, might be expected to modulate binding of tumour cells to the vasculature wall and thus play a role in the intravasation (Duffy et al., 2011)

 

 

1.3.3.2 Angiogenesis

Cancer growth and metastasis can be mediated by angiogenesis which consists of the formation of new blood vessels devoted to vascularise the tumour tissue. This process is essential for tumours to grow beyond approximately 2 mm in diameter. (Duffy et al., 2011). Angiogenesis process is under the dependence of a balance of pro- and antiangiogenic factors (Bajou et al., 2004). Proteinases in general, have been initially considered as positive regulators of angiogenesis but recent studies have evidenced complex and sometimes opposite roles of MMPs, ADAMs and ADAMTSs in regulating tumoral angiogenesis (Handsley and Edwards, 2005). However, several evidences have  also proved that ADAMs may promote cancer growth and metastasis through this process. Some of these evidences are: Pulmonary hypovascularisation in mice expressed catalytically inactive ADAM17 (Zhao et al., 2001),  and the deletion of ADAM17 resulted in pathological neovascularisation and reduced growth of injected tumour cells in a mouse model (Weskamp et al., 2010).

 

Some studies carried out has indicated the recombinant disintegrin domain (RDD) of ADAM-15 as a potent inhibitor of angiogenesis. ADAM-15 RDD induces a reduction of MDA-MB-231 tumour growth associated with less tumour vascularization in vivo. Transgenic B16F10 melanoma cells form less metastasis in mouse lungs after turning on RDD expression (Trochon-Joseph et al., 2004). It has been proposed that mechanisms implicating ADAM-15 in the regulation of angiogenesis could be related to the presence of Arg-Gly-Asp (RGD) sequence in the disintegrin domain which binds integrins. In addition, certain ADAM proteinases have been indicated to control cell apoptosis. In a mammary cancer model induced by the expression of polyoma middle T oncoprotein, ADAM-12 has been shown to increase stromal cell apoptosis and decrease tumour cell apoptosis (Kveiborg et al, 2005)  ADAM-10 knock-out (KO) embryos suffer from cell growth arrest and apoptosis associated with an overexpression of full-length E-cadherin (Maretzky et al., 2005).

 

 

 

1.3.4 Evidence of a Role for ADAMs  in Cancer and their potential use as 

Biomarkers

Based on the ability of ADAMs to release ligands which is capable of stimulating cell proliferation as well as migration , several studies from cell lines grown in culture, animal models and human malignancies suggest that a number of ADAMs in cancer formation. The most established ones include ADAM9, ADAM10, ADAM12, ADAM15 and ADAM17 are involved in cancer formation and/or progression of which the strongest evidence for a role in malignancy exists for ADAM17 (Duffy et al., 2009) .

Several studies have shown that increased expression of certain ADAMs enhanced in vitro invasion, proliferation and promoted tumour formation in vivo (McGowan et al., 2007; Borrell-Pages et al., 2003), while decreased expression reduced these processes. It has been shown that deficiency of specific ADAMs such as ADAM9, 15 and 17 resulted in decreased growth of heterotopically injected tumour cells in mice models (Guaiquil et al., 2009).  Biomarkers are potentially useful in cancer detection, prognosis assessment, and predicting therapy outcome or likely resistance to therapy as well as  monitoring ongoing therapy.

 

 

Figure 1.14: An overview of a disintegrin and metalloproteinases (ADAM) in cancer biology. Five different pathways may be involved in ADAM mediated cancer cell proliferation and progression. (1) ProADAMs are activated by furin or matrix metalloproteinases (MMPs). (2) Sheddase activity of ADAMs is stimulated by external factors (e.g. 12-Otetradecanoylphorbol-13acetate [TPA] ), leading to shedding of cell surface ligands such as heparinbinding- epidermal growth factor (HP-EGF) and transforming growth factor (TGF)-α. This process perhaps involves protein kinase C (PKC) and mitogenactivated protein kinase (MAPK) pathways. Then, soluble growth factors such as HP-EGF activate epidermal growth factor receptor on the cells in autocrine and paracrine manners. (3) The interaction of the disintegrin and cysteine-rich domains of ADAM with integrins or syndecans on the cells may help them to cleave the substrates (e.g. extracellular matrix [ECM] ). (4) ADAMs modulate extracellular matrix–integrin interactions, and thus they can indirectly promote proliferation signals through integrins. (5) ADAM may process other undetermined membrane-anchored molecules such as chemokines, cytokines and their receptors, which are related to cancer cell proliferation and progression. CR, cysteine-rich domain; CT, cytosolic tail; Dis, disintegrin domain; E, epidermal growth factor-like domain; MP, metalloproteinase domain; Pro, propeptide domain; TM, transmembrane domain.  ( Mochizuki S and  Okada Y, 2007)

 

1.3.5 Contribution of ADAMs  in different types of cancer as diagnostic marker

 

1.3.5.1 Lung cancer

It has been proved in several studies that dysregulation of the production of several ADAMs leads to  lung cancer. Some of the have been indicated here. Primary bronchial epithelial cells and bronchial cell lines exposed to smoke components showed an increased proliferation rate associated with EGFR phosphorylation with possible mediation by ADAM-17 which can activate several EGFR ligands (Lemjabbar et al., 2003).  Also ADAM17 was reported to be upregulated  in non-small cell lung carcinoma (NSCLC), with possible heregulin3 (HER3) signalling (Zhou et al., 2006).

Another example is ADAM-8, which was strongly expressed in NSCLC by tissue microarray analysis, and correlates with clinical stage of the disease (Ishikawa et al., 2004).  Also, ADAM-9 mRNA and protein expression levels are enhanced in EBC-1 lung cancer cell line displaying a tropism for brain metastasis as compared to parent EBC-1 or EBC-1 cell line with a tropism for bone tissue (Shintani et al., 2004).

ADAM-12 mRNA and protein levels was reported to increase  in NSCLC when compared to non-cancerous tissues (Rocks et al., 2006)  ADAM-15 was reported to be  expressed in both small cell lung carcinoma (SCLC) and NSCLC cell lines with higher expression in tumoral cells than in normal epithelial cells of pulmonary tumours (Schutz et al., 2005).

ADAM28 was one of the first ADAMs shown to be elevated in serum from patients with cancer (Kuroda et al., 2010). ADAM-28, cleaved  insulin-like growth factor binding protein3 (IGFBP- 3),  and was  found to be about 16-fold over-expressed in NSCLC (Mochizuki et al., 2007).

 

 

1.3.5.2 Brain tumours

ADAM-22 , restricted to the brain is  implicated in cell-cell and cell-matrix interactions through their binding to integrins and extracellular matrix and may  be involved in neural development (Sagane et al., 1998). Cytoplasmic variants of ADAM-22 have been indicated to have been  expressed differently in normal human brain tissue and gliomas (Harada et al., 2000), which gives evidence of  ADAM genes expression in brain tumours. Brain tumour cell lines cultured under hypoxic conditions demonstrated an upregulation of ADAM-17 expression levels, and its  activity correlated with increased tumour cell invasion (Zheng et al., 2007). Also, ADAM-8 and ADAM-19 mRNA are upregulated in primary brain tumours and their expression and activity are correlated with invasiveness of glioma cells (Wildeboer et al., 2006). There was an overexpression of the membrane- bound ADAM-12 variant in glioblastomas (Kodama et al., 2004). However, treatment of cultured glioblastoma cells with an ADAM-12 inhibitor decreased the production of the mature HB-EGF indicating its role in HB-EGF signalling pathway  in those cells.

 

 

1.3.5.3 Prostate cancer

There is a possiblity of the existence of both androgen-independent and androgen dependent cases in prostate cancer though early stages involve androgen-dependent (Bertram et al., 2006). Androgen-dependency has been suggested to  interfere with ADAM-related regulation processes since the mRNA expression of several ADAMs is regulated by androgens (McCulloch et al., 2000). Androgen or serum starvation enhances ADAM-9 protein expression in androgen receptor- positive prostate cancer cells (Shigemura et al., 2007).  Peduto et al. reported that well-differentiated prostate cancers developed in ADAM-9–deficient mice compared with poorly differentiated tumors in control mice expressing ADAM-9 . They also showed that overexpression of ADAM-9 in mouse prostate epithelial cells gave rise to epithelial hyperplasia and prostate intraepithelial neoplasia,a putative precursor lesion for prostate cancer  because of its ability to cleave EGFR ligands and the receptor for fibroblast growth factor (Peduto et al., 2005).

In studies on prostate cancer cell lines in culture,overexpression of ADAM-9 was found to be associated with the conversion of LNCaP cells to an androgen-independent and metastatic state (Sung et al., 2008).  In prostate carcinoma, ADAM-9 levels were significantly associated with prostate-specific antigen (PSA)relapse-free survival (Fritzsche et al., 2008). ADAM-8 protein expression has been demonstrated to be significantly associated with higher cancer stages including positive nodal status, and higher Gleason scores (Fritzsche et al., 2006).  Najy et al. also  found that downregulation of ADAM-15 in the prostate cancer cell line PC3 decreased migration and adhesion to specific extracellular protein matrix proteins, such as fibronectin, vitronectin, and laminin.

 

 

1.3.5.4 Liver cancer

In activated hepatic stellate cells, TGF-b1 induces ADAM-12 expression which might also participate in tumour progression (Le Pabic et al., 2005). This suggest that, ADAM-12 might contribute to growth inhibitory signalling in normal epithelial cells which is lost during tumour progression. Several studies have indicated ADAM-17 as a  contributing factor  to EGFR-ligand release and induction of cell proliferation and invasion (Rocks et al., 2008). In a study, it was reported that ADAM-17 mRNA levels were higher in hepatocellular carcinomas than in paired non-cancerous liver tissues suggesting that this proteinase might be implicated in tumour invasiveness by either activating EGFR by amphiregulin (Lemjabbar et al., 2003) or TGFalpha (Borrell-Pages et al., 2003). ADAM-9 in one study has been reported to promote invasiveness of liver metastatic carcinoma cells by degrading basement membrane components such as laminin-1  (Mazzocca et al., 2005).

 

1.3.5.5 Breast cancer

There has been several reports of ADAMs implications in breast cancer patients.

One of the first ADAMs shown to have diagnostic potential was ADAM12 in breast cancer. ADAM- 12, an apoptosis-modulating gene  accelerated the development of tumour by delaying tumour cell apoptosis by  the overexpression of its soluble form lacking the cytoplasmic tail (secreted splice variant of ADAM-12) (Kveiborg et al., 2005; Rocks et al., 2008). In urine, using Western blotting ,ADAM-12 levels were enhanced in breast cancer patients vis-à-vis a healthy control group which suggest a potentially important noninvasive biomarker in breast cancer (Roy et al., 2004).

Using logistic regression analysis, the authors calculated that the predictive probability of the presence of breast cancer was ≥ 80%, when levels of ADAM12 exceeded 40 arbitrary units (Roy et al., 2004). In a follow-up study to above, Pories et al found that urinary ADAM12 levels were also increased in women with putative premalignant lesions of invasive breast cancer such as atypical hyperplasia and lobular carcinoma in situ, compared to levels in healthy controls. This finding, if confirmed, suggests that measurement of ADAM12 in urine could identify women at increase risk of developing breast cancer. In the PyMT mouse model, overexpression of ADAM-12 was found to promote breast cancer progression (Kveiborg et al., 2005). Also, expression of both ADAM12 isoforms was found to be significantly elevated in human malignant breast tissue overexpression which  resulted in increased tumor take, tumor size, and metastasis in vivo. Of the two isoforms, only the secreted isoform, ADAM12-S, enhanced the ability of tumor cells to migrate and invade in vitro and resulted in a higher incidence of local and distant metastasis in vivo (Roy et al., 2004 ).

In vitro studies have shown that overexpression of ADAM- 17 in breast cancer cells increases invasion and proliferation (McGowan et al., 2007) and targeting it, reverts the malignant phenotype by preventing shedding of TGF-a and amphiregulin (Kenny and Bissell, 2007). Active ADAM-28 was found to be overexpressed in breast cancinoma cells ,contributing to  the regulation of cell proliferation through IGFBP-3 cleavage, enhancing the bioavailability of IGF-I (Mitsui et al., 2006). Alternative splicing could also be an important tool used by cancer cells to acquire an invasive phenotype. For example, different isoforms of ADAM-9 proteins and ADAM-15 mRNA have been detected in breast cancer cells (Ortiz et al., 2004) which calls for attention to set up a powerful diagnostic tool by studying the differential production of ADAM-9 or -15 domains. In breast cancer, ADAM-9 expression was significantly higher in node-positive than node-negative primary cancers whereas the active form of ADAM-17 was increased in high-grade versus lowgrade tumors (McGowan et al., 2007) .

In vivo, loss of ADAM-15 decreased metastasis to bone and using breast cancer cell lines,it was reported that ADAM-15 cleaved cadherin E after growth factor deprivation (Najy et al., 2008).  In another study,it was shown  that the human breast cancer cell lines, MCF-7 and MDA-MB453, strongly express ADAM 9, 12, and 17, whereas ADAM 10 and 15 were expressed at a lower level, indicating a putative pathophysiological role of these ADAMs in breast cancer biology (Lendeckel et al., 2005). ADAM17 processed through major histocompatibility complex (MHC) class I molecules was showned to be expressed in breast, ovarian and prostate cancer ,making it a potential immunotherapeutic target in these cancers. (Sinnathamby et al., 2011).

Figure 1.15 Prospective role of ADAMs in breast carcinoma cell proliferation.

ADAM28 is overexpressed as active forms in breast carcinoma cells. ADAM28 cleaves insulin-like growth factor binding protein-3 (IGFBP-3) and releases insulin-like growth factor-I (IGF-I) through the IGF-I–IGFBP-3 complex. IGF-I induces cell proliferation through phosphorylation of the IGF type I receptor (IGF-IR) and extracellular signal-regulated kinase 1/2 (ERK1/2). IGFBP-3 cleavage can be inhibited by treatment with anti-ADAM28 antibody or an ADAM inhibitor, KB-R7785, as well as ADAM28 small interfering RNA (siRNA) (Mochizuki S and  Okada Y, 2007)

1.3.5.6 Gastric and colon carcinoma

In vivo, ADAM-10 and -17 are overexpressed in antral mucosa during H. pylori infection and it has been established that , ADAM-9, -10, -12, -15, and -17 are increased in gastric tumours (Carl-McGrath et al., 2005). ADAM-10, which is found to be overexpressed in vitro after gastric cell infection, could establish a link between Helicobacter pylori-induced inflammation and carcinogenesis in stomach and it acts through EGFR ligand shedding leading to gastric cell proliferation (Joy et al., 2005). In colon carcinomas, ADAM-17 is overexpressed independently of tumour stage or grade and is involved in tumour growth and angiogenesis possibly via an autocrine/paracrine pathway implicating EGFR (Blanchot-Jossic et al., 2005). ADAM-9 is also overexpressed in a colon cell line and is co-localized with E-cadherin suggesting a potential role in ECadherin-mediated metastasis.  It has been showed that a soluble form of ADAM-9 secreted by hepatic stellate cells promoted colon cancer cell invasion in vitro. (Mazzocca et al., 2005).

1.3.5.7 Kidney, bladder carcinoma

Inhibition of ADAM-17 by a dominant negative ADAM-17 mutant prevents pro-HBEGF cleavage, EGFR activation and cell proliferation in kidney carcinoma cells ,indicating the importance of EGFR signalling in the development of kidney cancer since (Schafer et al., 2004). ADAM-12 mRNA was found to be overexpressed in bladder cancer and its levels correlated with disease stage. In another study, the levels of ADAM-12 was also found to be  higher  in the urine from patients with bladder cancer compared with healthy control subjects (Frohlich et al., 2006) and  concentrations tended to be higher in those with the largest invasive tumours. In some cases , urinary ADAM12 levels decreased following surgical removal of the bladder cancer but increased again with recurrent disease (Fröhlich et al., 2006)  which  suggesting the propability of using  the measurement of urinary ADAM12 for monitoring patients with bladder cancer.

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