Biomarkers of Therapeutic Resistance in Breast Cancer

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BIOMARKERS (S DAWOOD, SECTION EDITOR) Biomarkers of Therapeutic Resistance in Breast Cancer Sudeep Gupta & Ashish Singh & Bharat Singh Bhosale & Bhawna Sirohi Published online: 23 October 2013 # Springer Science+Business Media New York 2013 Abstract Despite improvements in early detection and adjuvant treatment, a significant fraction of women with early stage breast cancer will eventually develop a recurrence. While improvement in outcomes in metastatic disease has been achieved with new therapeutic options almost all patients eventually develop tumor progression due to drug resistance. We review the mechanisms of resistance to commonly used endocrine, chemotherapeutic, and targeted agents in breast cancer and the interplay of the biomarkers therein. Keywords Resistance . Biomarkers . Breast cancer . Systemic therapy Introduction Approximately 30 % of women with early-stage breast cancer develop recurrent disease despite advances in early detection and multimodality treatment [1]. The choice of therapy in primary and recurrent settings requires careful consideration of the clinical characteristics and molecular/pathologic features of the tumor to maximize the benefit/risk ratio. Systemic treatment of breast cancer includes cytotoxic, hormonal, and targeted agents, which are used in the (neo) adjuvant and recurrent/metastatic settings. Overall, systemic agents are active at the commencement of therapy in 90 % of patients with primary cancers and 50 % with metastatic disease. However, after a period of time, resistance to therapy occurs in a fraction of early cancers and is almost universal in metastatic ones. Here we review the mechanisms of therapeutic resistance to commonly used systemic therapies in breast cancer and the biomarkers that may be potentially useful in this context. Endocrine Resistance in Breast Cancer Clinical studies over the last 23 decades have identified 2 forms of resistance to tamoxifen therapy: intrinsic (de novo) resistance, wherein ER-negative and some ER-positive tumors do not respond to treatment from the very outset, and acquired resistance, wherein ER-positive tumors that initially responded to treatment subsequently exploit the tamoxifen/ ER complex as a stimulatory rather than inhibitory growth signal [2]. De novo Resistance to Endocrine Therapies A number of factors may contribute to intrinsic tamoxifen resistance including [3]: & Variable expression of the alpha and beta isoforms of estrogen receptor and of progesterone receptor (PgR). & Interference with binding of coactivators and corepressors of estrogen receptor. & Alternatively spliced ER mRNA variants. & Modulation of ER activity through increased expression of growth factors (eg, type 1 epidermal growth factor (EGF) receptor [EGFR1], and the type 2 EGFR, also called HER2). & Inherited drug metabolic (CYP2D6) genotypes leading to variable drug metabolism. & Mutations in the BRCA1 gene, which appears to modulate expression of ER-alpha. & Altered expression of specific micro RNAs. Differential expression of ER and PgR may play a role in determining endocrine responsiveness. Clinical results show that women with tumors that are ER-positive/PgR negative are less responsive to hormonal treatment than women whose tumors are ER-positive/PgR- positive [4]. Moreover, it has now been shown that the quantitative levels of expression of S. Gupta (*) : A. Singh : B. S. Bhosale : B. Sirohi Tata Memorial Centre, Mumbai, India e-mail: [email protected] Curr Breast Cancer Rep (2013) 5:275283 DOI 10.1007/s12609-013-0127-7

Transcript of Biomarkers of Therapeutic Resistance in Breast Cancer

Page 1: Biomarkers of Therapeutic Resistance in Breast Cancer

BIOMARKERS (S DAWOOD, SECTION EDITOR)

Biomarkers of Therapeutic Resistance in Breast Cancer

Sudeep Gupta & Ashish Singh & Bharat Singh Bhosale &

Bhawna Sirohi

Published online: 23 October 2013# Springer Science+Business Media New York 2013

Abstract Despite improvements in early detection andadjuvant treatment, a significant fraction of women with earlystage breast cancer will eventually develop a recurrence.While improvement in outcomes in metastatic disease hasbeen achievedwith new therapeutic options almost all patientseventually develop tumor progression due to drug resistance.We review the mechanisms of resistance to commonly usedendocrine, chemotherapeutic, and targeted agents in breastcancer and the interplay of the biomarkers therein.

Keywords Resistance . Biomarkers . Breast cancer .

Systemic therapy

Introduction

Approximately 30 % of women with early-stage breast cancerdevelop recurrent disease despite advances in early detectionand multimodality treatment [1]. The choice of therapy inprimary and recurrent settings requires careful considerationof the clinical characteristics and molecular/pathologicfeatures of the tumor to maximize the benefit/risk ratio.Systemic treatment of breast cancer includes cytotoxic,hormonal, and targeted agents, which are used in the (neo)adjuvant and recurrent/metastatic settings. Overall, systemicagents are active at the commencement of therapy in 90 % ofpatients with primary cancers and 50 % with metastaticdisease. However, after a period of time, resistance to therapyoccurs in a fraction of early cancers and is almost universal inmetastatic ones. Here we review the mechanisms oftherapeutic resistance to commonly used systemic therapiesin breast cancer and the biomarkers that may be potentiallyuseful in this context.

Endocrine Resistance in Breast Cancer

Clinical studies over the last 2–3 decades have identified 2forms of resistance to tamoxifen therapy: intrinsic (de novo)resistance, wherein ER-negative and some ER-positivetumors do not respond to treatment from the very outset, andacquired resistance, wherein ER-positive tumors that initiallyresponded to treatment subsequently exploit the tamoxifen/ER complex as a stimulatory rather than inhibitory growthsignal [2].

De novo Resistance to Endocrine Therapies

A number of factors may contribute to intrinsic tamoxifenresistance including [3•]:

& Variable expression of the alpha and beta isoforms ofestrogen receptor and of progesterone receptor (PgR).

& Interference with binding of coactivators and corepressorsof estrogen receptor.

& Alternatively spliced ER mRNAvariants.& Modulation of ER activity through increased expression

of growth factors (eg, type 1 epidermal growth factor(EGF) receptor [EGFR1], and the type 2 EGFR, alsocalled HER2).

& Inherited drug metabolic (CYP2D6) genotypes leading tovariable drug metabolism.

& Mutations in the BRCA1 gene, which appears to modulateexpression of ER-alpha.

& Altered expression of specific micro RNAs.

Differential expression of ER and PgR may play a role indetermining endocrine responsiveness. Clinical results showthat women with tumors that are ER-positive/PgR negativeare less responsive to hormonal treatment than women whosetumors are ER-positive/PgR- positive [4]. Moreover, it hasnow been shown that the quantitative levels of expression of

S. Gupta (*) :A. Singh : B. S. Bhosale :B. SirohiTata Memorial Centre, Mumbai, Indiae-mail: [email protected]

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both ER and PgR are associated with prognosis such thattumors that have stronger expression are associatedwith betteroutcome [5, 6].

ERαmutations have been described in recent years, whichmay lead to the synthesis of a nonfunctional receptor thatcould lead to resistance to hormonal manipulation. A lysine-arginine substitution at 303 (K303R) of ERα is recognized asa factor associated with tamoxifen resistance andhypersensitivity to estrogen [7, 8]. Polymorphisms of ESR1gene (encoding ERα) and 29 other genes of interest werestudied in a cohort of 1001 patients as part of the ProspectiveOutcomes in Sporadic vs Hereditary breast cancer (POSH)study. In univariate analysis, 2 ESR1 polymorphisms (rs3020410 and rs 889312) were significantly associated with apoorer prognosis in patients treated with adjuvant hormonetherapy. However, the prognostic impact could not beconfirmed in multivariate analysis [9, 10]. Recently, a splicevariant leading to a truncated form called ERα36 has beendescribed. This transcript is initiated by a promoter containedin the first intron and differs from the full form of ER (ERα66)by the loss of transcriptional activation (AF1 and AF2) areas,while preserving the binding domains DNA and receptordimerization. ERα36 expression was significantly andindependently correlated with a reduction in relapse-freesurvival and disease-specific survival in 2 cohorts of patientstreated with tamoxifen, with or without chemotherapy [11,12]. Phosphorylation, sumoylation, and methylation of ER isalso associated with extranuclear ER function by inducing itsinteraction with SRC and PI3KCA and activating the Aktpathway [13]. Hypermethylation of ER has been describedin some breast cancers but its predictive or prognosticsignificance is not yet established [14].

The response to hormone therapy in relation topolymorphisms of cytochrome 2D6, which may modify themetabolism of tamoxifen, has been studied in great detail inrecent years. For a long time, 4-OH-tamoxifen was consideredthe main active metabolite of tamoxifen, with a 100 timesgreater affinity for ER than tamoxifen [15]. Recent studieshave described endoxifen (N-desmethyl tamoxifen 4-OH)whose affinity for ER is similar to that of 4-OH tamoxifen,but with higher (an average of 10 times) plasma concentrationthan tamoxifen. However, the concentration of endoxifen isparticularly variable from 1 patient to another depending onpolymorphisms of the gene encoding for CYP2D6. Based onthese polymorphisms, patients can be classified into rapid andpoor metabolizers, and this has been studied as a potentialpredictive factor for tamoxifen response [16]. Despite greatinitial promise, 2 recent analyses from large prospectiverandomized trials have failed to confirm the predictive roleof CYP2D6 status [17, 18].

Indices of proliferation have also been used as biomarker ofresponse/resistance to endocrine therapy. In the IMPACT trial,which included 330 patients, patients were randomized to

receive anastrozole, tamoxifen, or their combination. The studydesign included estimation of Ki67 as a pharmacodynamicintermediate endpoint, at baseline and after 2weeks of endocrinetherapy. Anastrozole showed a significantly greater suppressionof Ki-67 compared with tamoxifen, but there was no significantdifference between tamoxifen and the combination. Importantly,recurrence free survival could be predicted based on the degreeof suppression of Ki67 during 2 weeks of endocrine therapy[19]. However, the value of this kind of analysis remainsto be proven in large prospective studies. The clinicalutility of measuring the 2-week suppression of Ki67 isunder evaluation in the 4000-patient Peri-OperativeEndocrine Therapy for Individualizing Care (POETIC)window-of-opportunity study [20].

Acquired Resistance during Endocrine Treatment

Loss of ER expression over time, accounts for tamoxifenresistance in only a minority of patients [21]. PgR loss occursmore frequently, and when this occurs, the tumor takes a moreaggressive course [22].

Growth factor pathways appear to play a central role inacquired resistance to tamoxifen. Signaling through EGFRand the HER2 receptor appears to bypass the estrogenrequirement for breast cancer cell growth and may driveinitially ER+cells into an endocrine therapy-resistant state[23, 24]. In 1 study tumors with higher IGF1 ligand andERα expression took longer to develop resistance, whiletamoxifen resistant tumors had lower IGF1 and ERαexpression compared with tamoxifen sensitive tumors [25].

Emerging studies suggest that high levels of tumor EGFRexpression may serve to identify those women for whomadjuvant tamoxifen provides little benefit [26]. However,EGFR expression might be associated with a generally poorerprognosis [27], and whether alternative adjuvant strategies,either other forms of hormone therapy or chemotherapy,would alter the clinical course in these patients is uncertain.Prospective trials are required to address these issues andresults from ongoing phase II trial of panitumumab, nab-paclitaxel, and carboplatin for patients with primaryinflammatory breast cancer without Her 2 over expressionwould be interesting in this context [28].

The PI3K–Akt–mTOR pathway modulates growththrough estrogen receptor signaling and EGF receptor familyof receptor tyrosine kinases and has been implicated inresistance to endocrine therapy in breast cancer. There are 3major classes of PI3Ks, but only class I PI3Ks have beenheavily implicated in oncogenesis [29]. Signaling throughthe PI3K–Akt–mTOR pathway promotes the tumorigenicphenotype and drug resistance through effects on multiplecellular processes such as apoptosis, proliferation, motility,cell transformation, metabolism, and DNA repair [30]. ER

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can form a complex with the p85 subunit of PI3K at the cellmembrane to initiate PI3K signaling. Furthermore, AKT andsome downstream effectors (eg, GSK3b and p70S6 kinase)can activate ER transcriptional activity. PI3K and RAS canalso modulate the transcription of ER target genes throughJNK and subsequent formation of the AP-1 transcriptionfactor complex [31]. There is clinical evidence to suggest thatinactivation of PI3K via either overexpression of HER2 orfibroblast growth factor receptor (FGFR)1, or loss of inositolpolyphosphate-4-phosphatase type II (INPP4B) is a cause ofanti-estrogen resistance [32]. Mutations in the PI3K alphacatalytic subunit (PI3KCA) are the commonest geneticabnormality identified in ER positive breast cancer (~30 %)and such mutations generally correlate with good long-termprognosis. However, there are conflicting reports regardingthe prognostic implications of PI3KCA mutation in breastcancer which would necessitate further studies and analysis[33]. It is also been reported that either altered activity of thePI3K/AKT pathway or high expression of AKT couldpredispose to endocrine resistance [34]. The benefit of mTORinhibitor everolimus plus the steroidal aromatase inhibitorexemestane was shown in the BOLERO-2 trial, whichenrolled 724 women who had progressed on anastrazole.The combination resulted in an improvement in PFS (median,6.9 vs 2.8 months; P <0.001 and response rate (9.5 % vs0.4 %) [35]. The results of this trial indicate that addition ofm TOR inhibition is a valid strategy to reverse endocrineresistance.

Micro RNAs (miRs) are noncoding regulatory RNAmolecules of 19–24 nucleotides which modulate theexpression level of certain proteins based on sequencecomplementarities with target mRNA molecules. Differencein expression of certain miRs has been reported to correlatewith tamoxifen resistance, therapeutic response, andprognosis in breast cancer [36].

Her 2 Expression and Endocrine Resistance

Amplification and/or overexpression of HER2 may beassociated with primary resistance to endocrine therapy.Preclinical studies suggest physiologic "cross-talk" betweenthe HER2 and ER signal transduction pathways [37]. HER2expression in human breast cancer cells is downregulated byestrogens. Conversely, transfection and overexpression ofHER2 promotes estrogen independent growth and tamoxifenresistance in ER positive human breast cancer cells [38].Among the hypothesized mechanisms whereby overexpressionof HER2 renders cells hormonally independent arephosphorylation of the ER, ligand-independent ER activation,and regulation of hormone receptor expression [38, 39].Although these data provide a rational explanation for the lowerresponse of HER2-overexpressing tumors to endocrine therapy

seen in several clinical studies, most were retrospective andnonrandomized. Randomized trials have not providedconsistent and confirmatory evidence of endocrine resistancein HER2-overexpressing tumors, either in the adjuvant ormetastatic disease setting.

It has been hypothesized that the adverse influence of HER2overexpression is limited to therapies that are based upon aligand binding agent (eg, selective estrogen receptormodulators such as tamoxifen) as opposed to ligand-depletingtherapy (eg, ovarian ablation, aromatase inhibitors). Thishypothesis was based on an analysis of a trial in which 250postmenopausal women with ER-positive breast cancer whowere ineligible for breast conserving treatment were randomlyassigned to 4 months of preoperative letrozole (2.5 mg daily) ortamoxifen (20 mg daily) [40]. Women with HER2-positivetumors had significantly lower response rates to neoadjuvanttamoxifen compared with those who were HER2-negative(17 % vs 40 %). In contrast, no difference was noted in theresponse to letrozole (69 % vs 53 % for HER2 positive andnegative tumors, respectively). However, most recent evidencesuggests that the interaction of HER2 overexpression andrelative resistance to endocrine treatment is regardless of thetype of endocrine therapy [5, 41].

In metastatic disease, multiple studies have assessedresponse rates to endocrine therapy among women withHER2-positive vs negative tumors. Although the results aremixed, the bulk of evidence favors an adverse influence ofHER2 overexpression on response to treatment withtamoxifen [42, 43]. Table 1 illustrates the range of findingsin women with hormone receptor-positive tumors.

In the TAnDEM study, 207 postmenopausal patients wererandomly assigned to treatment with the combination ofanastrozole plus trastuzumab or anastrozole alone [44].Combination therapy resulted in a significant improvementin PFS (median, 4.8 vs 2.4 months, P=0.001). Of note, 70 %of patients treated with anastrozole crossed over atprogression. Another randomized trial tested the combination

Table 1 Relationship between response to tamoxifen and HER2 status

Study Number Response % inHer 2 negative

Response % inHer 2 positive

Wright 1992[43] 72 37 7

Leitzel 1995[78] 300 41 21

Berns 1995[42] 259 56 17

Archer 1995[79] 92 80 19

Newby 1997[80] 155 56 0

Yamauchi 1997[81] 94 56 9

Elledge 1998[82] 205 57 54

Houston 1999[83] 241 56 38

Hayes 2001[84] 242 34 32

Data modified from Ellis et al. [77]

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of lapatinib plus letrozole in 219 postmenopausal women[45]. Dual blockade resulted in improved PFS comparedwith single agent letrozole (median, 8.2 vs 3 months;P =0.019) and a higher rate of clinical benefit (ORR plusstable disease >6 months, 48 % vs 29 %; P=0.003). Theseresults suggest that the addition of HER2 targeted therapy toendocrine manipulation can reverse resistance to the latter, atleast partially and transiently.

Trastuzumab Resistance in Breast Cancer

Not all women with HER2 overexpression respond totrastuzumab. Theoretically, resistance to trastuzumab mightbe caused by (1) altered receptor antibody interaction, (2)activating of the downstream pathways by increased signalingfrom either other members of the HER family or otherreceptors, or (3) constitutive activation of downstreamelements. These include downregulation of HER2 andalterations in or upregulated expression of genes involved inmodifying biological pathways (eg, PTEN, insulin-likegrowth factor receptor I, and c-myc). One possibility is thata subgroup of HER2-positive tumors also expressesp95HER2, a truncated form of the HER2 receptor that haskinase activity but lacks the extracellular trastuzumab-bindingdomain [46, 47]. In a study of 46 patients with metastaticbreast cancer, only 1 of 9 patients expressing p95HER2responded to trastuzumab, compared with 19 of 37 of thoseexpressing full-length HER2 [47].

Preclinical data from this study also suggested that tumorsexpressing the truncated receptor remain sensitive to othertherapies targeting HER2 (eg, lapatinib). Significant level ofcrosstalk occurs among receptors within the HER family.Although trastuzumab reduces HER2-mediated signaling, itmight not reduce signaling from other HER receptors. HER1/HER3 heterodimers or HER1/HER1 homodimers mightinitiate downstream signaling even in the presence of HER2blockade.

Ligands of the HER family including heregulin andbetacellulin have been shown to mediate acquired resistanceto trastuzumab [48•]. In HER2-positive breast cancer, geneticalterations leading to upregulation of the PI3K/AKT/ mTORpathway most frequently occur through loss of PTEN proteinexpression (30 %–40 % of tumors) or activating mutations inPIK3CA (E545K or H1047R; 20 %–25 % of tumors) [49].Results from the Phase III, randomized, double-blindstudy of everolimus plus trastuzumab and vinorelbinein women with HER2-overexpressing locally advancedor metastatic breast cancer previously treated with ataxane and resistant to trastuzumab (BOLERO-3) werepresented at the Annual ASCO Meeting 2013 andshowed only a modest improvement in PFS but noimprovement in OS [50].

In a recent study it was shown that in ER-positive tumorsthe response to neoadjuvant treatment with trastuzumab plusanthracyline-taxane chemotherapy is driven by the degree ofHER2 mRNA expression [51•]. This phenomenon could notbe observed in the ER-negative subset. Also For ESR1-negative/HER2-positive tumors the amount of HER2 mRNAis not further relevant for response once a tumor is in theHER2-positive group. An interesting STEPP analysis fromthe adjuvant trastuzumab NSABP B-31 trial examined thedegree of HER2 mRNA expression and correspondingtrastuzumab benefit separately for patients with estrogenreceptor-positive and estrogen receptor-negative disease. Thestriking finding was that among patients with estrogenreceptor positive disease, trastuzumab benefit in terms of 8-year disease-free survival was entirely confined to those withthe higher levels of HER2 mRNA expression [52].

Multiple studies have addressed whether circulating HER2protein ECD levels (c-ECD) predicts response to HER2-directed treatment. Baseline c-ECD levels do not appear topredict an increased odds of response, but in a pooled analysisof 7 trials of first-line trastuzumab with or withoutchemotherapy, patients with a 20 % or greater decline in c-ECD levels over baseline had significantly higher responserates to trastuzumab (57 % vs 28 %) as well as significantlylonger TTP and overall survival compared with those with alesser degree of decline [53, 54]. However, a separate reviewof 63 studies concluded that concentrations of HER2 ECD arenot consistently related to patient outcomes [55]. Thus theclinical utility of assessing or following c-ECD levels duringHER2-directed therapy is not established. The ASCO expertpanel on tumor markers in breast cancer recommended againstthe routine use of serum c-ECD in any clinical setting [56].

Since multiple trastuzumab resistance mechanisms produce achange in signaling networks and protein phosphorylationpatterns, mapping phosphotyrosine signaling networks usingquantitative proteomics can be utilized for identifying novelmechanisms of trastuzumab resistance. This could potentiallybe used to identify, quantify, and functionally screen proteinsthat might be involved in trastuzumab resistance [57]. In thiscontext, quantitative phosphoproteomics-siRNA screeningstrategy has identified proteins related to the Src kinase pathway,including CDCP1/Trask, epiplakin, embryonal Fyn-associatedsubstrate, focal adhesion kinase, and Paxillin as being relevant.Src is a promising therapeutic target for overcomingtrastuzumab resistance [58].

TKI Resistance in Breast cancer

Lapatinib and other approved TKIs targets receptors anddownstream members of the ERBB/RAS pathway. Lapatinibinhibits the tyrosine kinase domain of the epidermal growthfactor receptor (EGFR) and human epidermal growth factor

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receptor 2 (HER2). It is approved for the treatment ofmetastatic breast cancer, where the overall response rate tothis treatment is 24 % in first line use [59]. Because ofmoderate response rate observed with oral TKIs there is anincreasing need to identify biomarkers which are drivers orpotential markers of resistance to targeted therapy.

The factors that confer primary or acquired resistance tolapatinib are not well characterized. One report indicated that,similar to trastuzumab, loss of PTEN or activation of PIK3CAthrough hotspot mutations (E545K or H1047R) conferredresistance to lapatinib in HER2-overexpressing breast cancercells [60]. In another report, the mechanism of resistance tolapatinib was proposed to be independent of PTEN loss andthe H1047R PIK3CA-activating mutation, but dependent onthe E545K mutation within the PIK3CA gene [61]. LowPTEN expression or PIK3CA mutations were not predictiveof resistance to 6 weeks of lapatinib monotherapy in womenwith locally advanced breast cancer [62].

AXL is a membrane-bound receptor tyrosine kinasecontaining a kinase domain closely related to MET and anextracellular domain resembling that of neural cell adhesionmolecules [63]. Overexpressing cDNA for AXL inducestransforming ability through tyrosine phosphorylated140 kDa protein having oncogenic properties. IncreasedAXL expression has been identified as a novel mechanismof resistance to lapatinib and trastuzumab in lapatinib-resistantclones from BT474 human breast cancer cells, which are ER,PgR, and HER2 positive. AXL kinase inhibition by foretinib,a multikinase inhibitor targeting AXL, MET, and vascularendothelial growth factor receptor (VEGFR), restoreslapatinib and trastuzumab sensitivity in these cells [64].

HER2 somatic mutation has been identified in cancerslacking HER2 gene amplification. In a recent study [65] 13HER2 somatic mutations from 25 patients (identified from atotal of 1499 patients for an overall HER2 mutation rate ofapproximately 1.6 %) were functionally characterized. Themutationswere grouped into several different phenotypes. Sevenof these 13 were activating mutations, including G309A,D769H,D769Y,V777L, P780ins, V842I, and R896C. Neratinibwas effective in all the identified mutations. Lapatinib was lesspotent than neratinib and was not effective in reducing thegrowth of cells bearing L755S or P780ins mutation.

Resistance to Cytotoxic Therapy

After an initial period of response to cytotoxic therapy,progression occurs in the vast majority of patients withmetastatic breast cancer. The use of combination regimenshas been adopted to overcome resistance to single agents.But very often, tumors develop resistance to drugcombinations. Due to the multifactorial causation ofcarcinogenesis, attributing treatment failure to a single factor

is inappropriate. Research on biological basis of molecularlevel treatment failure has the potential to explain thisphenomenon and provide appropriate solutions.

The common mechanisms of chemotherapeutic drugresistance include decreased intracellular drug concentration(drug transporters, metabolic enzymes), disturbances affectingthe cell cycle arrest, apoptosis and DNA repair, activation ofsignaling pathways, epigenetic modifications, and alterationsin the availability of drug targets [66].

Taxane Resistance in Breast Cancer

Classically, taxanes exert their action through binding to betatubulin, components of microtubules, resulting in the formationof stable microtubules. Subsequent arrest at the mitoticcheckpoint results in apoptosis presumably through G2/M arrestand apoptosis through themitochondrial pathway. Paclitaxel canalso cause disruption of microtubules during interphase, therebydisrupting growth and metabolism [67••]. Activation of thespindle assembly checkpoint is required for paclitaxel-inducedcell death. Cyclin-dependent kinase (CDK) 1 activity andCDK2 activity in cancer cells, which reflect the activation stateof the spindle assembly checkpoint and the growth state,respectively, could predict sensitivity to paclitaxel [68]. Twomechanisms of resistance to taxanes have been reported in cellsexposed to low concentration of taxanes for a prolongedduration. One involves changes in the expression of β-tubulinisotopes, mainly β-III, whereas the other is a part of the MDRsystem. Another suggested mechanism is upregulation ofcaveolin-1, a membrane component which is involved inintracellular signaling and small molecule transport [69].

Breast cancer cell lines with low levels of BRCA1 areassociated with taxane resistance [70]. Various mechanismsof resistance have been postulated including a differentialapoptotic response by BRCA1. BRCA1-induces increase inc-Jun N-terminal kinase (JNK)/stress-activated protein kinasephosphorylation leading to apoptosis. BRCA1 is important fortranscriptional upregulation of spindle assembly checkpointproteins and loss of its function inhibits their critical disruptionby taxanes. On the other hand higher response rates werereported in BRCA1 or BRCA2 mutations when treated withDNA-damaging regimens like anthracycline or platinumagents [71••]. If confirmed in larger trials it can change theway we treat patients with inherited germline mutations inthese genes and a subset of women with triple-negativedisease, whose tumors exhibit true BRCAness [72].

Anthracycline Resistance in Breast Cancer

The main mechanism of resistance to anthracyclines isincreased expression of the P-170 glycoprotein related to the

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enhancement of drug efflux. The evidence supporting thisincludes correlation between this protein and resistance,transfer of the cloned MDR1 gene, and reversal of resistanceby agents that block P-170 [66]. Expression of the MDR1gene may be transcriptionally modulated by doxorubicinalong with inhibitors of protein kinase C and calmodulin.Resistance is more complicated in vivo, with many normaltissues and significant number of cancers showing increasedexpression of MDR gene [73]. The expression of a 190-kDaprotein, which is a member of the ATP-binding cassettetransmembrane transporter superfamily, also correlates withresistance. In the absence of alterations in MDR1 ortopoisomerase II expression, multidrug resistance protein(MRP) expression in isolation can also produce resistance toanthracyclines [74].

Proteomics studies using Antibody Microarray (AbMA)have identified various differentially expressed proteinsimplicated in resistance to chemotherapy. The 14-3-3 theta/tau isoform, which plays a role in signal transduction, haspreviously been implicated in resistance to anthracycline ortaxane therapy in breast cancer cells. High expression of 14-3-3 isoform theta/tau is associated with chemotherapy resistancein breast cancers [75]. Bcl-2-like apoptosis related proteinsBID (pro-apoptotic) and Bcl-xL (anti-apoptotic) are involvedin the apoptotic pathway of resistant cell lines. Also there is adecreased expression of BID in its active cleaved form (tBID)in chemoresistant tumors [75].

Recent years have witnessed large scale genomiccharacterization of breast tumors in an attempt to understandthe exquisite heterogeneity in this disease. Novel subtypeshave been discovered and characterized. While thesetechnologies are not yet ready for routine implementation asbiomarkers in clinical practice, they are beginning to explainthe clinical behavior of many patients. For example, a recentstudy that used integrated sequencing and copy numberanalysis uncovered novel subgroups within ER positivetumors that are associated with relative resistance to hormonalmanipulation [76].

Conclusions

Multiple studies, the majority in vitro, have identified severalmechanisms of drug resistance and biomarkers of drugresistance in breast cancer. How these processes operatein vivo and their clinical impact has to be further studied incontrolled prospective trials involving patient tumorspecimens correlated with therapeutic responses todifferent agents. The use of newer technologies suchas genomics and proteomics will continue to expand this fieldof study. These discoveries will help in designing clinicaltrials to study drug resistance and personalize therapy forwomen with breast cancer.

Compliance with Ethics Guidelines

Conflict of interest Sudeep Gupta declares that he has no conflict ofinterest. Ashish Singh declares that he has no conflict of interest. BharatSingh Bhosale declares that he has no conflict of interest. Bhawna Sirohideclares that she has no conflict of interest.

Human and Animal Rights and Informed Consent This article doesnot contain any studies with human or animal subjects performed by anyof the authors.

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