Peptides and biocomplexes in anticancer therapy

1.1 Cancer and metastasis origins and global burden

Hippocrates (460 B.C.) first established the word carcinos from the Greek (which means crab) to point at what we now know as a tumour. This term referred to the morphology of the tumour Hippocrates saw, the irregular shape and the hanging blood vessels reminded him of crab claws and limbs. This animal symbol was preserved, and the term “cancer” was employed afterwards by Aurelius Cornelius Celsus (from the Latin “Canker”, which also means crab). The first cases of cancer were reported on papyrus by Egyptians (1600 B.C.).

Joseph Claude Anthelme Récamier, a French gynaecologist was the first to coin the word “metastasis” in 1829 as being a spread cancer found in the bloodstream. Sixty years after, Stephen Paget strengthened knowledge by proposing the “seed and soil” theory stating that “the distribution of the secondary growth is not a matter of chance” but the cancer cell’s (the seed) ability to invade an organ (the soil) strongly depends on the properties of this organ [1] (reviewed in [2]). Without describing any molecular aspect, he however pointed out one of the most important features of metastases. These notions were deepened later by James Ewing, in 1928, in his “Treatise on tumours”.

Nowadays, cancer is the leading cause of death in economically developed countries and has emerged to be the second one in developing countries [3]. The increasing population ageing and lifestyles imposed by the emerging consumption society like sedentary behaviour, inadequate nutrition, and smoking mainly account for this curse. In 2008, global cancer statistics estimated 12.7 million new cancer cases and 7.6 million cancer-associated deaths. Lung cancer so far is the deadliest cancer in males representing 22.5% of cancer deaths, and the second deadliest cancer in females after breast cancer (13.7%). (Figure. 1).

Figure. 1

Estimation of cancer-related deaths in 2008

1.2 Genomic instability and the other side of the Darwinian coin

Mutations in the genome affecting the function of proteins that regulate homeostasis of the cell account for cancer development. Every form of cancer displays high genomic instability. Genomic instability refers to the high rate by which changes rise in chromosome structures and numbers, and therefore in genomic functions [4]. Genomic instability favours the continuous acquisition of DNA aberrations which, as a result of the selection pressure, prompt the cancer cells to adapt, resist, and become continuously more aggressive. Cancer is therefore the result of Darwinian natural selection, where the fittest cell would have the opportunity to grow and divide. Paradoxically, the other side of the coin is the eventual death of the “host” and therefore the cancer extinction.

How genomic instability arises is still a subject of intense debate. There are two main opposing theories concerning this matter. The mutator hypothesis supports that an original early mutation renders the precancerous cell genetically unstable (e.g. by disabling “caretaker” genes affecting the DNA repair machinery) and leads to an increase in the rate of spontaneous mutations [5]. This allows the cancer cell to transform, and according to the selective pressure (e.g. anticancer therapy, hypoxia, etc.) to adapt to or escape an unfavourable environment. This theory however predicts that mutations affecting the fitness of a cell are also occurring that would lead to cancer extinction, obviously this does not reflect the reality.

The oncogene-induced DNA replication stress model states that mutations in sporadic cancer can arise and affect oncogenes or tumour suppressor genes (by opposition to “caretaker” genes). In the absence of a functional tumour suppressor called p53, which prompts the cell to undergo senescence or alternatively apoptosis when DNA is not intact, these oncogenic mutations would lead to the collapse of the DNA replication forks and ultimately to DNA strand breaks and genomic instability [4]. High throughput genetic data obtained on breast and colon cancer favour the oncogenic stress model as the majority of the DNA mutations that were found lie within non-caretaker genes [6, 7]. Certain non-caretaker genes appear almost always mutated in the analysed samples like those encoding for K-Ras (Kirsten rat sarcoma viral oncogene homolog), APC (adenomatous polyposis coli), and PI3K (phosphoinositide 3-kinase). In contrast, mathematical predictions shift the balance in favour of the mutator hypothesis [5]. It is not strange that the reality is more complex and surely imbricates elements of these two models.

1.3 The hallmarks of cancer

Neoplastic diseases are highly complex and heterogeneous. However, these diseases have shared common traits. Douglas Hanahan and Robert Weinberg published in 2000 “The hallmarks of cancer”, a report of principles that rationalise the complexity displayed by malignancies [8]. Six distinctive and complementary hallmarks that eventually favour tumour growth and metastatic dissemination have been proposed at this time and updated 10 years after [9]. They are briefly explained here (Figure. 2).

Figure. 2

The hallmarks of cancer.

The accumulation of mutations due to genomic instability favours the appearance of the following hallmarks:

1. Sustained proliferative signalling. Cancer cells have the ability to maintain constant proliferation. In contrast, normal cells control their proliferation which confers on them the maintenance of homeostasis and tissue architecture. Cancer cells reprogram the release of growth factors and the expression of their associated receptors in order to increase their proliferation rate. Every pathway that controls positively or negatively the mitogenic activity is a target of choice for carcinogenesis-inducing mutations. Cancer cells may even also reprogram and induce neighbouring normal cells to emit growth factors [10].

2. Evasion from growth suppressors. Sustained constant proliferation occurs through the first-mentioned hallmark but cannot be completed unless the negative regulators of proliferation are neutralised. Some proteins, the so-called tumour suppressors, are responsible to sense and further decide if the cell should undergo division, senescence, or apoptosis. Rb (retinoblastoma protein) [11] and p53 (commonly called the “guardian of the genome”) [12] are such proteins and are unsurprisingly common targets of inactivating mutations.

3. Resistance to cell death. Normal cells that undergo replication failure or those that encounter excessive stress normally undergo a form of cell suicide called apoptosis. The regulation of apoptosis is finely tuned at physiological levels, in particular by showing an equilibrated balance between anti- and pro-apoptotic protein activities [13]. Apoptosis can be triggered by extrinsic factors (for example by engagement of the Fas receptor) or from within the cell (intrinsic mitochondrial cascade), typically after DNA damage. Both situations lead to the eventual activation of the proteases called caspases, which cleave various proteins essential for cell homeostasis maintenance, and therefore leads to progressive cell disassembly. Genomic instability allows the cancer cells to generate an unbalanced anti-apoptotic signature by activating anti-apoptotic signalling and repressing the pro-apoptotic ones [13]. The diversity of strategies that cancer cells use to counteract apoptosis reflects the multiplicity of signalling leading to cell death. Other characterised kinds of cell death like autophagy [14] and necrosis [15] are also bypassed by cancer cells.

4. Enabled replicative immortality. Normal cells are only able to pass through a limited number of division cycles, after which they enter into a viable but non-replicative senescence phase. They alternatively enter in a phase called crisis and die by apoptosis. This fate is mainly attributed to the telomere (chromosome extremities) shortening [16]. Telomeres of dividing cells shorten at every cell cycle, which is thought to eradicate their protective effect on chromosomal ends. The telomerase is a specialised DNA polymerase that adds DNA segments to the telomeres. In normal cells, telomerase is almost always absent, while more than 90% of the reported immortalised cells display high levels of telomerase [16]. Acquisition of the enhanced telomerase activity confers a strategy for unlimited replication of cancer cells.

5. Induction of angiogenesis. The fast growth and division of cancer cells necessitates constant support in nutrients and oxygen. These latter are supplied by the blood. The generation of new vessels is called angiogenesis and is a physiological process during embryogenesis and female reproductive cycling. It is however mainly switched off at adult stages and physiological conditions. To alleviate the tumour needs of nutrients and oxygen, tumour cells have evolved strategies to create their own vasculature [17]. The angiogenic switch triggered by cancer cells is heterogeneous but almost always results in production of a released factor called vascular growth factor-A (VEGF) that prompts endothelial cells to from a neovasculature [18].

6. Activated invasion and metastasis. The ability of cancer cells to disseminate through the body is undoubtedly their most harmful property, which almost always leads to death. It is also the least understood hallmark of all. In addition to providing the nutrients and oxygen, the newly created vessels offer to cancer cells the possibility to disseminate across the body. Only a few cancer cells will have the ability to complete the long metastatic journey. Succeeding in initiating metastasis involves several critical steps discussed in detail below.

7. Other biological processes. In addition to the six hallmarks set out in this section, studies conducted over the last decades identified other biological processes as emerging hallmarks of cancer. Among those is the ability of cancer cells to escape from immune surveillance and the reprogramming of energy metabolism, a process that is also called the “Warburg effect”. In addition to the fact that the cancer cells can escape from immune surveillance, recent data indicate that immune cells could also provide growth, survival, and pro-angiogenic factors to the tumour cells (reviewed in [19]). To fuel their increasing need of energy, the cancer cells are prone to reprogram their energy metabolism by opting for an aerobic glycolytic metabolism, which is a mechanism allowing to produce lactate starting from glucose in presence of oxygen [20]. This glycolytic switch acidifies the tumour microenvironment which results in several selective advantages for the cancer cells (reviewed in [20]). Another important observation is the increasing consideration for the protective role of the stroma associated with cancer cells.

1.4 Molecular basis of the metastatic cascade

The metastatic cascade is highly complex and involves sequential multistep processes [21]; the consequence of this is that only a few pre-metastatic cells will survive until the end of the process to eventually form metastases [22]. There are still debates on which mode is utilised by the cell to form a metastasis [23]. The linear progression model states that all the mutations that are necessary to disseminate are acquired within the primary tumour while the parallel progression model favours the hypothesis that mutations are acquired sequentially in a spatial manner, and therefore that cells which leave the primary tumour are not fully metastatic [24].

Regardless of the dissemination mode, metastatic cells should be able to undergo a physiological process called epithelial-mesenchymal transition (EMT). EMT is a reversible reprogramming allowing epithelial cells to dedifferentiate and acquire de-adhesive and pro-migratory properties [25]. Concretely, pre-metastatic cells have first to detach from the primary tumour mass (Figure. 3). This phenomenon is now widely accepted to be a consequence of the loss of the E-cadherin adhesion receptors that mediate cell-to-cell interactions [26]. Modulation of the interaction with the extracellular environment is also a crucial event and is mainly achieved by activating or repressing a set of adhesion receptors called integrins. The activation of proteases such as the matrix metalloproteinases (MMPs) permits cancer cells to find a path through the extracellular matrix (ECM), which is a network of proteoglycans, polysaccharides, and fibers mainly constituted by collagen, fibronectin, laminin, and vitronectin [27]. This is however not sufficient for the invasion process. In order to weave a path, the cells must increase their motility machinery by modulating the cytoskeleton (discussed in detail below).

Figure. 3

The multistep metastatic cascade

Once the pre-metastatic cell has reached the lymphatic or hematogenous systems, it has to cross the endothelial barrier, a process called intravasation [28]. The dissemination is then driven by the flow of the lymph or blood, but cells that are not able to survive in an anchorage-independent fashion will die at this step. Additionally, surviving cells should escape from the immune system surveillance. The succeeding ones are then helped by platelets and leukocytes to attach to the vessels in a selectindependent manner and will then cross the endothelial barrier back, a process called extravasation [28]. Finally, cells have to invade the distant organ parenchyma, which is often different from the primary tumour microenvironment in terms of ECM components [28]. At this moment, the metastatic cells reverse the mesenchymal phenotype in order to acquire a proliferative and structured state back; this program is called mesenchymal-epithelial transition (MET) and allows the cells to colonise and form micrometastasis and then eventually macrometastases [29].

Despite the impressive obstacle-associated journey to form a metastasis, the metastatic cell proteome only slightly differs from the primary tumour cell as exemplified in the breast-to-bone malignant dissemination [30]. Overexpressed proteins involved in this breast-to-bone dissemination signature are proteins involved in bone-specific functions like bone homing and degradation, but also proteins involved in more general aspects of dissemination like angiogenesis (FGF5 and CTGF), extracellular matrix degradation (MMP-1 and proteoglycan-1). Metastatic markers that target the motility machinery were also found; for example RhoC (Ras homologous C), a small GTPase involved in actin cytoskeleton remodelling [31].

Any failure to complete one single step of the metastatic cascade would lead to the arrest of the entire process. This therefore raises hope for finding drugs that prevent the metastatic cascade by targeting isolated and specific features of metastatic cells.

2.1 Therapy against cancer and metastasis

Genomic instability makes cancer a moving target. The most commonly used treatment against cancer remains surgery when applicable, in addition to radiotherapy and systemic therapies including chemo-, immune-, and hormonal therapy. Surgery aims to physically remove the tumour mass; it is however not feasible in every organ as well as in many invasive cancer, the shape of which is not well delimitated. Radiotherapy uses ionizing radiation to kill malignant cells. Ionizing radiation induces DNA simple and double strand breaks that lead the cell to undergo apoptosis or mitotic catastrophe. Fifty percent of the cancer-diagnosed patients are treated with radiotherapy with possible adjuvants of anticancer drugs.

Chemotherapies are organised in several groups:

1. The alkylating agents that cause DNA cross-linking and strand break, leading to replicative failure and apoptosis.

2. The antimetabolites are analogues of nucleic acids that inhibit enzymes involved in DNA synthesis.

3. The alkaloids that bind and disrupt the microtubule cytoskeleton, this latter being crucial for the mitotic spindle and therefore for the cell cycle.

4. The antibiotics that also cross-link the DNA.

5. The inhibitors of topoisomerase, an enzyme required during DNA replication.

Immunotherapy generally aims to target or treat tumours with immuno-enhancers. These latter prompt the immune system to attack the tumour cells. Hormonal therapy is also suited for treating certain cancers that strictly depend on the presence of a hormone. In this case, the therapy aims to block the hormone-induced signalling.

Side-effects and resistance to most of these therapies remain however a major concern. The genomic instability favours heterogeneity and therefore the possibility to render a few cells resistant to the drug used, which is the main issue in cancer therapy. Nowadays, plenty of small molecule inhibitors have come out and appear promising as well as target more specifically some hallmarks of cancer (Figure. 4). An attractive strategy to reduce resistance to therapy is to prevent cells from having the possibility to use an “emergency exit” by creating a cocktail of drugs that target different hallmarks of the cancer cells (Figure. 4). The emergence of personalised therapy will help to define the Achilles heel of each tumour and therefore to use the appropriate cocktail of drugs.

Figure. 4

Therapeutic targeting of the hallmarks of cancer

As mentioned earlier, patients die from metastases. Unfortunately, only a few drugs target the metastatic cascade per se[32]. The others are rather hampering the proliferation of pre-established metastases. An illustration of this is the B-Raf inhibitors for malignant melanoma treatment. B-Raf, an effector of Ras, was often found mutated and thereby constitutively activated in malignant melanoma [33]. B-Raf inhibitors development substantially helped in treating these cancers, they however target the proliferating capacity of the metastasis and not their dissemination capacities. Drugs that are used to counteract the metastatic cascade are mainly anti-angiogenic agents, inhibitors of matrix metalloproteinases (MMPs) and those that target the tumour microenvironment of the secondary site [34]. An anti-angiogenic agent such as Bevacizumab, a monoclonal antibody directed against VEGF, is now used for treating metastatic colorectal cancer. Such drugs prevent vessel development and have the double effect of depriving the cells of nutrients and of a transport mean. An example of targeting the secondary site microenvironment is provided by Denosumab, an antibody targeted against RANKL (receptor activator of nuclear factor kappa-B (NF-κB) ligand). The release of RANKL leads to bone resorption, which is a facilitating event during bone invasion. Neutralizing RANKL with Denosumab is used against osteoporosis and for bone metastasis prevention. The invasion process is also the subject of intense therapeutic development. Melanomas and glioblastomas are currently treated with inhibitors of α_V-integrin, an adhesion protein involved in cell-ECM interaction. α_V-integrin inhibitors comprise neutralising antibodies (such as etaracizumab) or cyclic peptides derived from the RGD tripeptide integrin ligand (such as cilengitide). Anti-MMPs were also developed and are currently under clinical trials. They prevent invading cells from digesting the extracellular matrix. A novel class of inhibitors, the inhibitors of c-MET, a tyrosine kinase that controls drug resistance, stemness, invasion, and angiogenesis, seem promising as they simultaneously target several cancer hallmarks at the same time. Despite all that, cancer therapy still lacks effective anti-metastasis drugs.

3.1 Photodynamic therapy (PDT)

Newly emerged therapies of cancer include the combination of multiple facets of approaches; one of the promising applications is photodynamic therapy (PDT). This type of therapy has emerged as an alternative approach to chemotherapy and radiotherapy for cancer treatment. Such therapy is based on a photosensitizer (PS), which is perhaps the most critical component of PDT, and this newly introduced therapy is a continuously interesting area of intense scientific research. Classically, the photosensitizer’s traditional use was achieved by molecules like porphyrins, which have dominated the field of photosensitizer therapy (Figure. 5). However, photosensitizer agents, major disadvantages are low water solubility, poor light absorption, and reduced selectivity for targets. In order to overcome these disadvantages, experts in the field have introduced polysilsesquioxane (PSilQ) nanoparticles that are crosslinked homopolymers formed by the condensation of functionalised trialkoxysilanes or bis(trialkoxysilanes). PSilQ particles provide an interesting platform for developing PS nanocarriers. Tackling the major problems of introducing the therapeutic components, the PSilQ nanoparticles provide the reliability to carry a large payload of PS molecules; their surface and composition can be tailored to be more target-specific based on the load. In addition to their small size, nanoparticles can penetrate deep into tissues and be readily internalised by cells. The authors describe the PSilQ nanoparticles with a high payload of photosensitizers that were synthesised, characterised, and applied in vitro. The network of this nanomaterial is formed by “protoporphyrin IX (PpIX) molecules chemically connected via a redox-responsive linker”. Under reducing environment such as the one found in cancer cells, the nanoparticles can be degraded to efficiently release single photosensitizers in the cytoplasm. In fact, recent advances have shown that the phototoxicity of this porphyrin-based PSilQ nanomaterial was successfully demonstrated in vitro using human cervical (HeLa) cancer cells. Future research will build on this finding in order to improve the platform so that the nanoparticles hopefully can be further functionalised with other components including for example polyethylene glycol (PEG) and target-specific ligands to improve its biocompatibility and target specificity.

Figure. 5

Photodynamic therapy in targeting cancer. Non toxic luminescent probe of Rhenium that can be activated by a specific wavelength in order to induce an anti-cancer compound

3.2 Metal complexes as platforms for cancer therapy

Metals are essential cellular components that play a major role in the function of several indispensable biochemical processes for living organisms, mainly being enzymes cofactors. Metals are endowed with unique characteristics that include redox activity, variable coordination modes, and reactivity towards organic substrates. Metals are tightly regulated under normal conditions due to their high reactivity, and therefore aberrant metal ion concentrations are associated with various pathological disorders, including cancer. For the above-mentioned reasons, coordination complexes, either as drugs or prodrugs, become very attractive probes as potential anticancer agents. After the discovery of cisplatin, cis-[PtII(NH₃)₂Cl₂], which led to the interest in platinum(II) and other metal-containing complexes as potential novel anticancer drugs. However the interests in this field are concerned with uptake, toxicity, and resistance to metallodrugs.