Hallmarks of cancer and therapy resistance

Cancer is a group of diseases consisting of over 100 different conditions. The primary distinction of cancers is based on the tissue of origin (e.g. lung cancer, breast cancer) and the cell type (e.g. carcinomas, sarcomas, adenocarcinomas) [12]. Despite the complexity of the many types of cancers, Hanahan and Weinberg defined the following 6 hallmarks that characterize all cancer types: (1) acquiring autonomous growth signals, (2) evasion of growth inhibitory signals, (3) evasion of apoptotic cell death, (4) unlimited replicative potential, (5) capability to form new blood vessels – angiogenesis – and (6) invasion and metastasis [13]. The original list of hallmarks were later completed with two enabling characteristics. Genome instability and tumor-promoting inflammation provide a permissive environment to acquire the above hallmarks. Reprogramming energy metabolism and avoiding immune destruction were defined as emerging hallmarks, as their importance in carcinogenesis is still under debate [14].

1.1.1. Role of mutations and plasticity of the tumor cells in drug resistance

Although it is not considered as a hallmark, intrinsic or acquired anticancer therapy resistance is frequently observed in all cancer types. The phenomenon of drug resistance occurs when tumor cells become insensitive to treatment. The development of cancer drug resistance is linked to DNA mutations or other metabolic changes including epigenetic events due to the plasticity of cancer cells, since these alterations serve as sources of new phenotypes during tumor progression [15] [16]. The new phenotypes emerge as a consequence of the multiple environmental pressures that compel the malignant cells to continuously change and adapt. In agreement with the Darwinian evolution model, cells, which obtained genetic mutations (or epigenetic changes) that result in growth advantage become predominant compared to neighboring cells. Clones with higher proliferation rate then expand within the tumor. Successive advantageous mutations/alterations lead to waves of clonal expansion, resulting in tumor heterogeneity [17] [18].

The consecutive mutations that accumulate and contribute to carcinogenesis are affecting

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mainly proto-oncogenes, tumor supressor genes and DNA repair genes, sometimes called

“drivers” of cancer [12]. According to the Cancer Genome Atlas Pan-Cancer effort, where mutation patterns of 3281 tumors from 12 cancer types were thoroughly analyzed, 2 to 6 such driver mutations are sufficient for oncogenesis [19]. The analysis also identified oncogenic mutations that can occur in any type of cancer, such as the mutations of histone modifiers, but reported several tissue specific genetic alterations as well, which affect mostly transcriptional factors and transcriptional regulators. Taken together, mutations and epigenetic plasticity of the malignant cells create a heterogeneous tumor where subclones can have different DNA alterations ranging from point mutations to large chromosomal aberrations (e.g. chromosomal translocations) [20] [21] [22] [23] and abnormal DNA methylation pattern variations [24] [25]. Tumor heterogeneity has a profound clinical impact. Therapy acts as a selective pressure, and in response, the fittest – most resistant – subclones are selected to survive, which manifests in tumor relapse [26]

[27].

As an example, 5-fluorouracil (5-FU) resistance of colorectal cancer appears with the recurrence of the tumor that was initially responding well to this chemotherapeutic agent.

When mRNAs were extracted from the tumor that already became resistant to 5-FU treatment, high levels of dihydropyrimidine dehydrogenase (DPD) transcripts were found. DPD is responsible for the inactivation of 5-FU before it is converted to its active form (to fluorodeoxyuridine by thymidine phosphorylase) [28]. Moreover, amplification of the TYMS locus (thymidylate synthetase, target of the activated 5-FU) was correlated with poorer survival among patients with advanced, metastatic colorectal cancer, who were treated with 5-FU previously [29] [30] [31].

Resistance derived from tumor heterogeneity is more pronounced when targeted drugs are used, due to the highly specific targeting of certain malignant alterations. As an example, when patients with chronic myelogenous leukemia (CML) are treated with imatinib, kinase-domain mutations commonly emerge, leading to clinical drug resistance and relapse [32]. A study proved that these mutations can already be present in a tumor subpopulation at the time of diagnosis, before imatinib treatment is initiated [33]. Another example is when patients with colorectal cancer are treated with cetuximab and irinotecan, a subpopulation with p.K57T missense mutation in the MAP2K1 gene will likely outgrow in the tumor, which contributes to poor response to therapy [34].

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1.1.2. Mechanisms of drug resistance and multidrug resistance (MDR)

The most common mechanisms that confer drug resistance to tumor cells are the (a) increased metabolic degradation of drugs, (b) keeping molecular targets at a low level, (c) increased DNA damage repair, (d) changed apoptotic pathways, (e) mutation in the target protein, (f) trapping drugs into acidic compartments, (g) alterations in the cell cycle and checkpoints and (h) the increased efflux by energy dependent transporters [15] [35].

Besides the above conventional mechanisms, cells can ‘escape’ from the treatment by epithelial-mesenchymal transition (EMT) [15], and certain micro-RNAs seem to contribute to the emergence of drug resistance as well [36].

Certain mechanisms from the above list can confer resistance to a single agent (the agent with which the treatment was conducted), while other forms provide cross-resistance to additional drugs. If cross-resistance is efficient against structurally unrelated and functionally distinct drugs, the phenotype is termed “multidrug resistance” (MDR) [37].

Overexpression of the ATP-binding cassette efflux transporters (ABC-transporters) in the plasma membrane of the malignant cells is one of the most common and effective mechanisms of MDR. The main ABC-transporters related to multidrug resistance are ABCB1 (P-glycoprotein, P-gp) [38], ABCG2 (BCRP) [39] [40] [41] and ABCC1 (MRP1) [42]. These transporters have broad substrate specificity, they can recognize and extrude a wide range of xenobiotics from the (tumor) cells, keeping low intracellular drug concentrations. Many anticancer therapeutics, either conventional (e.g. vinca-alkaloids) or targeted drugs (e.g. EGFR inhibitors) that are currently in use are subjects of transport by the ABC pumps [37]. The substrate specificity of the 3 main MDR ABC-transporters are partly overlapping, nevertheless when cultured cancer cells are treated with chemotherapeutics, the major mechanism of multidrug resistance is mediated by P-glycoprotein [35]. In addition, when mice bearing Brca1- and p53-deficient mammary tumors were treated with doxorubicin (a P-gp substrate), resistance frequently occurred and was linked to the overexpression of the P-gp ortholog Mdr1a and/or Mdr1b [43] [44].

P-gp is widely expressed also in many human cancers, including cancers of the gastrointestinal tract (small and large intestine, liver cancer, and pancreatic cancer), cancers of the hematopoietic system (myeloma, lymphoma, leukemia), cancers of the genitourinary system (kidney, ovary, testicle), and childhood cancers (neuroblastoma, fibrosarcoma) [45]. In hematological malignancies, sarcomas, breast cancer and other

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solid cancers, the contribution of P-gp to poor chemotherapy response was demonstrated [37]. P-gp mediated (and in general ABC-transporter mediated ) MDR is still a serious obstacle in cancer therapy, as during treatment, if the fittest clones, which arise due to the selective pressures are overexpressing P-gp, the tumor will most probably stop responding to P-gp substrate chemotherapeutics.

1.1.3. Physiological role and function of ABCB1

ABC-transporters are transmembrane proteins controlling the passage of their substrates across biological membranes and barriers under normal physiological conditions. Thus, ABC-pumps play a crucial role in the distribution of their endogenous substrates (reviewed in [37]). Moreover, ABC transporters make up a complex cellular defense system responsible for the recognition and removal of environmental toxic agents [46].

Accordingly, ABCB1 [47] and ABCG2 [48] [49] proteins are expressed e.g. in the blood-brain barrier, where they protect the blood-brain from xenobiotics. P-gp is expressed also in the liver, intestine and kidney [45] [50].

P-glycoprotein is a 170,000-dalton molecular weight phosphoglycoprotein comprised of two transmembrane domains (TMD), each containing six transmembrane helices, and two nucleotide binding domains (NBD) [51]. Extrusion of xenobiotics by P-gp from the cell is an energy-dependent action fueled by ATP hydrolysis [52].

Figure 3. Structure of P-gp, based on molecular dynamics simulation [54]. Green: lipid bilayer, red: aqueous phase. A) Inward-facing state of P-gp. B) outward-facing structure of P-gp.