Resistance to chemotherapy

Breast cancer is initially high responsive to chemotherapy, but unfortunately relapses and resistances are critically limiting the success of therapy and affect patient's survival. This plays a decisive role in the in the (neo)-adjuvant setting and has been estimated to cause treatment failure in >90% of patients with metastatic disease. It could be of intrinsic nature or acquired during the course of treatment. In this case, tumors may become resistant to drugs other than those initiating the resistance, despite the fact that these drugs may have different mechanisms of action.

Drug resistance may be caused by multiple factors at different levels of chemotherapeutical action (e.g. decreased drug accumulation due to decreased drug influx or increased drug efflux; altered intracellular drug transport; decreased drug activation or increased drug inactivation). The second important mechanism could be an enhanced repair or tolerance to damage of DNA or protein structures, which are targets of most drugs. As logical consequence on the other side these targets or their co-factors can be altered (e.g. metabolite levels, downstream effectors of cytotoxicity or signal pathways). Changes in whole genome or deletion/amplification of single genes as well as modifications (by transcription, processing or translation) of their protein products can be also critical variables of response or resistance respectively to cytotoxic therapy. The interactions between tumor and normal tissue come in the fore of discussion more and more [1].

Data from several studies support the superiority of polychemotherapy over single agent regimens (particularly anthracycline-taxane combinations) [2]. The choice of agents for combination regimens is based on the assumption of non-cross-resistant drugs. They were investigated by pre-clinical studies, which were mostly of unicellular and in-vitro character. It has leaded subsequently to the design of several approaches like using dose escalation in unselected cohorts of patients with advanced breast cancer aiming to improve clinical outcome, particularly by use of non-cross-resistant agents or sequence of drugs using alternative transport mechanisms.

Findings of modern studies, underlying the importance of heterogeneity of tumors, their microenvironment (e.g. intracellular pH, hypoxia, blood flow etc.), stroma-tumor interactions and possible presence of tumor [3] and normal tissue stem cells find only a marginal way into clinical practice [4], although there is a growing evidence, that these factors are crucial for response of chemotherapy.

Increase drug efflux is one of the best investigated mechanisms of chemotherapy resistance.

ATP binding cassette (ABC)-containing drug efflux transporters play essential role in regulating intracellular drug concentrations that determine cell sensitivity to chemotherapy. P-glycoprotein (PgP) encoded by multidrug resistance 1 gene, multidrug resistance protein (MRP), and breast cancer resistance protein (BCRP) are particularly important for response to chemotherapy [5]. More than 80% of currently used antitumor agents can be transported by these three transporters. They reduce intracellular concentration of drugs and possibly slow up their ability to diffuse in the cell.

Cold shock Y-box protein, which acts as a global marker for chemo resistance plays the crucial role in regulation of MDR gene [6]. Increased levels of YB-1 in primary human breast cancer tissue are associated with intrinsic MDR1 gene expression, inducing up-regulation of its product, PgP. Activation of MDR1 gene expression by YB-1 may occur in response to genotoxic stress provoked by exposure to drugs such as cisplatin or etoposide or e.g. UV-light [7]. It means that resistance to chemotherapy e.g. by PgP is not only of genetic nature but also rapidly inducing during agent administration. Neoadjuvant studies support this hypothesis by increased nuclear YB-1 localisation associated by PgP up-regulation after course of paclitaxel therapy [8]. This could also be an explanation for decreasing response rates in metastatic breast cancer.

The network of MDR regulation seems to be controlled by several genes known as oncogenes and the interactions of nuclear factor(NF)-kB or phosphoinositol-3-kinase-AKT-NF-kB pathways with YB-1 expression have been described and arrive clinical relevance [9].

Our group has recently demonstrated the significant superiority of rapidly cycled tandem high dose chemotherapy over conventional dose dense regimens in high-risk breast cancer patients only with YB-1 expressing tumors. In both study arms similar doses of anthracyclines and no taxanes, which are known substrates of PgP were used [10]. It underlines the impact of MDR phenotype in response to chemotherapy through multiple other factors, like cell cycle, DNA repair and other for instance by interaction with p53 oncogene [11].

New findings on tumor stem cells provide insights in possible mechanisms of drug resistance. There are several studies showing primary chemo resistance of stem cells in various cancers. Both normal stem cells and CSCs commonly express drug pumps such as (ATP)-binding cassette transporters, including multidrug resistance transporter 1 (MDR1) and breast cancer resistance protein (BCRP) [12]. BCRP has been shown to mediate resistance to mitoxantrone, topotecan and doxorubicin as well MTX [13]. Although the immunhistochemical measurement of BCRP should be still established our preliminary results indicate more pronounced effects from dose-intensification of chemotherapy in BCRP expressing tumors, significantly associated with basal-like tumor characteristics. Beside expression of efflux mechanisms stem cells also express metabolites like aldehyde dehydrogenase 1 (ALDH1), mediating resistance to cyclophopshamide e.g. in leukemia and breast cancer cells [14].

However the clinical relevance of stem cells in breast cancer remains unclear, the recently published study has shown for the first time the growing fraction of tumorgenic cells during course of chemotherapy also in partial responding tumors [15], could be a possible explanation for worse prognosis of patients, who do not completely respond to chemotherapy.

The next important mechanism is a decreased drug uptake. Nucleoside analogues are taken up into cells by equilibrative (ENT) and concentrative nucleoside transporters (CNT). hENT 3 and 4 have been identified to transport chemotherapeutic nucleoside analogues, which are essential components of chemotherapy (e.g. gemcitabine, 5-deoxy-5-fluoruracil). Similar function has been described for hCNT1–3. Whether the hENT1 and hCNT2 mRNA expression or their transcription modulation play the decisive role in chemo resistance remain unclear [16].

Drug efficacy can be decisively changed through metabolism form prodrug to active form through metabolism for instance by phosphorylation of nucleoside analogues or glutamation of the antifolates. The glutathione transferases (GST) are also important enzymes and are known to inactive toxins to confer resistance to a wide range of chemotherapeutic agents like doxorubicin, vincristine and others [17]. On the other side variations of activity of essential enzymes, which are necessary for transformation to an active drug (e.g. carboxylesterase 2, cytidine deaminase, and thymidine phosphorylase for capecitabine) and accordingly of activity of related metabolism enzymes like thymidylate synthase and dihydropyrimidine dehydrogenase contribute critically to efficacy of capecitabine chemotherapy [18]. The clinical impact of the metabolism is a controversial point of discussion. Recent data does not support e.g. variability of high-dose cyclophosphamide pharmacokinetics in depend of the wide polymorphisms spectrum of metabolizing enzymes (diverse CYP's, ALDH1, GST) within a randomized trial on HD chemotherapy in breast cancer, on the other side similarly to polymorphisms in drug transporters, metabolizing polymorphisms have been reported to influence significantly survival of patients with various malignancies (for review see [19])

Variability of drug targets is discussed as a powerful predictive marker particularly in breast cancer. Topoisomerase (Topo) II alpha is a validated target of anthracyclines, frequently used in a majority of breast cancer patients. Topo II gene amplification or deletion are possible mechanisms of resistance or sensitivity to anthracyclines. Particularly its neighbourhood to Her-2/neu gene on chromosome 7 and their frequent co-amplification make this protein very interesting for validation within prospective trials. The retrospective analysis from randomized trials suggests the assumption of predictive effect of anthracycline-containing therapies only in patients with Her-2/neu tumors with Topo II co-amplification [20]. Similarly variability of beta-tubulin expression (e.g. different isotopes, gene mutation) are considered as possible mechanism of resistance to taxanes [21].

Many chemotherapeutical agents like cyclophosphamide and platinum analogues cause direct damage of DNA and resistance to these compounds results from activation of repair mechanisms. Nucleotide (NER) and base excision repair (BER), homologues recombination as well as mismatch repair are the most important DNA repair pathways. For example BRCA1 and 2 genes causing the majority of familiar breast cancers are multifunctional proteins and are playing role in transcriptional activation, repair and homologues recombination of DNA as well as working as checkpoint of cell-cycle and apoptosis. There are several pre-clinical data, that alkylating chemotherapy should be more effective in BRCA 1 deficient tumors due to absent DNA repair following exposure to chemotherapy [22]. Our results from WSG AM 01 study underline particularly this hypothesis, because cyclophosphamide and thiotepa were used for dose intensification within the high dose arm and had more effect in triple-negative tumors [23]. But typical for BRCA1 phenotype triple-negative tumors seem to be generally more chemo sensitive to polychemotherapy containing also taxanes and anthracyclines [24]. BRCA1 abnormal transport between cytoplasm and nucleus, e.g. by KPNA-alpha protein seems also to contribute to global chemo resistance against several agents and their dosage [25].

There are several networks implicated in DNA repair and interacting with cell cycle and apoptosis influencing chemo resistance. p53 as a central tumor suppressor gene is considered as a very important mechanism in drug resistance. Loss of p53 should theoretically limit the ability of chemotherapy to induce apoptosis and intact p53 should cause programmed cell death through bcl-2 and caspases cascade. But chameleonic characteristics of p53 and other tumor suppressor and oncogenes make this network more complex [26]. Various other regulators of cell cycle like e.g. cyclins D1 and E contribute also to chemo resistance and Her-2/neu oncogene is associated with poorer response to e.g. CMF through altered cell-cycle control and apoptosis checkpoints [27].

Use of small molecules or immunotherapy, reported decreasing the numbers of possible tumor stem cells, novel agents non-resistant to known mechanisms and use of agents conferring resistance will be considered as future directions to overcome chemo resistance in breast cancer. In this context resentizing of cells to apoptosis or re-introducing of tumor suppressor genes, like wild-type p53 are promising strategies for suppressing resistance mechanisms.

References:

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