76 SCIENCE AND CULTURE, MARCH-APRIL, 2014

CANCER STEM CELLS AND MULTIPLE DRUG RESISTANCEIN BREAST CANCER*

A. MUKHOPADHYAY**, A. CHAKRABORTY, S. MUKHOPADHYAY AND J BASAK

Million of new cancer patients are diagnosed each year and over half of these patients die fromthis disease. As the second leading cause of cancer deaths, breast cancer in estimated to bediagnosed in over one million people worldwide and to cause more than 400,000 deaths eachyear. Chemotherapy is part of a successful treatment to many cases; however, the development ofmultidrug resistance (MDR) to it becomes a major obstacle so as to as few as half of the breatcancer patients treated benefit from chemotherapy. MDR is a term used to describe the phenomenoncharacterized by the ability of drug resistant tumors to exhibit simultaneous resistnace to a numberof structurally and functionally unrelated chemotherapeutic agents. At present, many mechanismshave been found to be responsible for it, including over expression of the members of the adenosinetriphosphate (ATP)-binding cassette (ABC) membrane transporter family, changes of apoptosis-related genes, the alteration of DNA repair gene, cancer stem cells and so on. And up to date,many methods were adopted to overcome MDR, for example natural drugs, chemical drugs andgenetic therapy. Advanced cases of breast cancer with 2nd line of treatment failure are chemo-resistant and high in BCSCs. Herein, we will use an aggressive approach to test for best chemo-and/or targeted therapy on BCSCs.

ARTICLE

* Second Ila Roy Memorial Lecture Organised by ISNA deliveredon 30th September, 2013.

** Netaji Subhas Chandra Bose Cancer Research Institute, Kolkatae-mail : hmcwt@dataone.in

Introduction

Breast cancer is the most common form of cancerdiagnosed in women worldwide. Although the rateof mortality as a result of breast cancer has

decreased in western countries due to early detection, it isnow the leading cause of cancer death (about 60%) amongfemales in economically developing countries. Thirtypercent of the breast cancers in India are in the second orthird decade of life and they are resistant to conventionalmode of therapies. The concept of cancer stem cellsresponsible for tumor origin, maintenance, and resistanceto treatment has gained prominence in the field of breastcancer research. The therapeutic targeting of these cellshas the potential to eliminate residual disease and may

become an important component of a multimodalitytreatment. In the past two decades, more than thirty newanticancer drugs have been introduced, but survival rateshave improved only marginally for many forms of cancer.In contrast to most cancer cells, cancer stem cells are slow-dividing and have a lowered ability to undergo apoptosisand a higher ability of DNA repair, making them moreresistant to traditional methods of cancer treatment.

Breast cancer, the most common form of canceramongst women, also has the second highest morbidity ratein the world (10.9% of all cancers). With an estimated 1.38million new cancer cases diagnosed in 2008, it is also themost common cancer both in developed and developingregions (Globocan cancer fact sheet, 2008). In spite ofmuch advancement in breast cancer treatments over theyears, relapse of this disease with time (approximately 40%of all breast cancer patients with 60-70% relapse cases withmetastasis) serves as a major roadblock in complete cure;

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the only established reason behind this, being the underlyingpresence of a small population of stem-like cells calledcancer stem cells i.e. the breast cancer stem cells.

Chemotherapy plays a major role in the treatment ofpatients with breast cancer, which in most countries is stillthe leading cause of cancer deaths among women. Theefficacy of chemotherapy is limited by the intrinsic presenceof development of drug-resistant tumor cells. Knowledgeof the clinically active mechanisms of drug resistance mightresult in new treatment strategies either by overcoming drugresistance or by drug-resistant patient for particulartreatment modalities.

Advanced cases of breast cancer with 2nd line oftreatment failure are chemo-resistant and high in BCSCs.This proposal uses an aggressive approach to test for bestchemo-and/or targeted therapy on BCSCs. The idea is thatablation of BCSCs will lead to improved therapeutics, lessertoxicity in patients and improved mortality and morbidityrates. Novelty of the project is in utilizing the informationobtained from patient specific stem cells and using themin a clinical setting.

Estimation of number of BCSCs in breast cancertissue and bone marrow for advanced cases with 2nd lineof treatment failure by identifying cell surface markers withCD44+/CD24-/ALDH1+ phenotype; also we will estimateother breast cancer stem cell markers like CD133, CD326,CD184 etc. Culture of whole tumor cells for ex vivo drugsensitivity towards patient-specific personalizedchemotherapy followed by statistical analysis of failure rateof patients even after chemo-predictive assays. This isfollowed by chemo-predictive assays on BCSC but that willnot be used in the clinical scenario. Comparison of chemo-

predictive assays of whole tumor cells and BCSC intreatment failure cases to predict if BCSC targeted therapycould have been an alternative approach.

Mechanisms of MDR

The Adenosine Triphosphate (ATP)-bindingCassette (ABC) Membrane Transporter Family :Elevated expression of ATP-binding cassette (ABC)transporters is considered to be the main cause of MDR inbreast cancer. ATP-binding cassette (ABC) transporters area family of transporter proteins that contribute to drugresistance via adenosine triphosphate (ATP)-dependent drugefflux pumps, which can result in an increased efflux ofthe cytotoxic drugs from the cancer cells, thus loweringtheir intracellular concentrations. Up to date, more than100 ABC transporters from prokaryotes to humans and 48human ABC genes have been identified that share sequenceand structural homology3. The proteins which are relatedto the MDR in breast cancer are mainly including p-glycoprotein (p-gp), multidrug resistance-related protein(MRP) and breast cancer resistance protein (BCRP).

P-glycoprotein : The glycoprotein was namedPglycoprotein (P-gp) because the protein can modulatemembrane permeability with respect to a number ofapparently unrelated drugs including actinomycin D,methotrexate, daunomycin, and colchicine7. The MDRmediated by P-gp is also called “classical MDR”.

Multidrug Resistance-related Protein 1 (MRP) :In 1992, Susan Cole and Roger Deeley observedamplification and increased expression of a novel gene innon-P-gp expressing small cell lung cancer DOX resistantcell lines and this is the MRP1 (ABCC1) (MDR relatedprotein) gene. The protein encoded by this gene is also a

member of ABC transporters.

Apoptosis and MDR : P53-As a tumor suppressor, p53 playsa pivotal role in inducing apoptosisin response to cellular damage,including DNA damage. However,mutant p53 plays an opposite rolein the regulation of apoptosis, thatis mutant p53 is an anti-apoptosisfactor.

Other apoptosis relatedgenes and MDR- Mitochondrial(intrinsic pathway) and cell surfacereceptor (Fas) mediated (extrinsicpathway) apoptosis are the twomain routes leading to programmedFig 1. Flowchart of P-glycoprotein modification

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cell death. MCF-7 cells can undergo apoptosis by thesequential activation of caspases-9 (associated withmitochondrial mediated apoptosis), -7, and -6. Recently, asplice variant form of caspase-3 has been shown to beoverexpressed in chemoresistant, locally advanced breastcancers, and is particularly associated with response tocyclophosphamide.

MDR-related Enzyme : Glutathione S-transferase(GST)-GST is a member of phase II detoxification enzymesthat catalyses the conjugation of glutathione (GSH) to awide variety of endogenous and exogenous electrophiliccompounds. Because of their capacity to react withelectrophiles, radicals and reactive oxygen species, GSTs,together with GSH, have a major role in the protectionagainst oxidative stress.

DNA Topoisomerase II (tope II) – DNAtopoisomerase II (topo II) is a nuclear phosphoproteininvolved in DNA replication and chromosome dynamics.These enzymes catalyse the ATP-dependent passage of oneDNA duplex (the transport or T-segment) through atransient, double-stranded break in another (the gate or G-segment), navigating DNA through the protein using a setof dissociable internal interfaces, or ‘gates’ [65,66]. Thefamily of DNA topoisomerase II includes two related butgenetically distinct isoforms isforms TOPII_ and II_ inmammalian cells.

Glucosylceramide Synthase – Recent years, manystudies have shown that sphingolipids are not only structuraland insert components of cell membranes but alsoassociated with myriadprocess of cells including theproliferation, survival and death of cells. As an importantmember of sphingolipid metabolism, ceramide have beenproven to be a second messager of apoptosis [79, 80].Cellular stress is known to increase ceramide levels in cells.So it is easily to understand that increased ceramide hasbeen oberserved in response to many anti-cancer drugs,such as doxorubicin, vincristine, paclitaxel, etoposide, PSC833 and fenretinide. Many enzymes have been confirmedto be responsible for the regulation of ceramide levels, such

as ceramide synthase and sphingomyelinase which areresponsible for the ceramide generation, and sphingomyelinsynthase and ceramidase which take part in the ceramidemetabolization [81]. Glucosylceramide synthase (GCS) isone of them. As an enyzme which catalyzes the first stepin glycosphingolipid synthesis, GCS transfers UDP–glucoseto ceramide to form glucosylceramide, which have beenfound to involve in many cellular processes such as cellproliferation, oncogenic transformation, differentiation, andtumor metastasis.

Cancer Stem Cells and MDR : Stem cells aredefined as cells with both self-renewal capacity and theability to produce multiple distinct differentiated cell typesto form all the cell types that are found in the mature tissue.Thus, these two characteristics of stem cells confer theunique property of asymmetric division. Stem cells arequiescent or slowly cycling cells maintained in anundifferentiated state until normal functioning of theorganism needs their participation. Stem cells are classifiedinto two principal types : embryonic and adult stem cells.Recent studies have revealed that they play important rolein cancer biology. Cancer stem cells (CSC) have beendetected in many tumors, such as retinoblastoma andmelanoma [94,95]. In breast cancer, a CD44+/CD24-or low/Lin-cell population was first identified as CSC. Later,aldehyde dehydrogenase (ALDH) 1 activity was reportedto be associated with stem/progenitor properties in breastcancer.

Sex Hormones and MDR : ER- Estrogen hormonesinduce expression of c-myc and c-fos protooncogenessufficient for cell division and breast cancer progression

PR- Like estrogens, the physiological effects ofprogesterone are mediated by interaction of the hormonewith the progesterone receptor. Transcriptional regulationby E2 and progesterone (P4) likely contributes to themodulation of P-gp levels.

EMT and MDR : An epithelial–mesenchymaltransition (EMT) is a biologic process that allows apolarized epithelial cell, which normally interacts with

Fig 2. The relationship between EMT and progression of tumours

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basement membrane via its basal surface, to undergomultiple biochemical changes that enable it to assume amesenchymal cell phenotype, which includes enhancedmigratory capacity, invasiveness, elevated resistance toapoptosis, and greatly increased production of ECMcomponents.

Methylation and MDR : Epigenetic alterations incancer often serve as potent surrogates for geneticmutations. Methylation of CpG dinucleotides is animportant pattern of epigenetics. Methylation can directlyinterface with the binding of transcription factors to inhibitreplication.

Strategies to Reverse MDR

Since MDR phenomena have been recognised, the warfighting against it has been continuing. Many strategies havebeen devised to overcome it and mainly divided into threetypes : modulators, immunotherapy and genetic therapy.

Modulators of P-gp : Numerous compounds havebeen shown to inhibit the drug efflux function of Pgp andtherefore, reverse cellular resistance. The process ofchemosensitization involves the co-administration of aMDR modulator with an anticancer drug in order to causeenhanced intracellular accumulation via impairing the P-gp function.

TABLE 1. Selected list of P-gp modulators

Immunosuppressant Anti-arrhythmic agent

Cyclosporin A Quinidine

Valspodar (PSC833) Antifungal agent

HIV protease inhibitors Ketoconazole

Ritonavir Sedative

Saquinavir Midazolam

Nelfi navir Acridone carboxamide

Calcium channel blocker LY 335979 (zosoquidar)

Verapamil GG918 (GF120918)

Bepridil Peptide chemosensitiser

Diltiazam Reversin 121

Flunarizine Reversin 205

Progesterone antagonist Anti-oestrogen

Mifepristone (RU486) Tamoxifen

Immunotherapy of MDR : The use of monoclonalantibodies, several of which can inhibit P-gp mediated drugefflux in vitro. The monoclonal antibody (mAb) MRK 16is the first antibody used for reversing MDR.

Genetic Therapy of MDR : The genetic therapy of

MDR mainly includes two methods. In the first methodmultidrug resistant bone marrow cells were produced bytransfescting them with vectors carrying the MDR1 cDNAand this process allowed bone marrow cells to apply achemotherapeutic regimen at otherwise unacceptable doses,and thus overcoming MDR.

The other method is inhibiting MDR proteinsincluding transcriptional translational inhibition through theintroduction of antisense oligonucleotides or ribozymes orRNA interference.

Present Research Works in this Field

National : Dr. Rangarajan (2009) and his group atDepartment of Molecular Reproduction, Development and

TABLE 2. Selected list of modulator targeting otherMDR-related genes.

Name Inhibitors

MRP1[3] MS-209

XR-9576 (tariquidar)

VX-710 (biricodar)

Isothiocyanates

tRA 98006

Agosterol A

Rifampicin

NSAIDs

BCRP(ABCG2)[3] GF-120918 (elacridar)

tRA 98006

Flavonoids

Phytoestrogens

Imatinib mesylate

Fumitremorgin C

TAG-139

GST-pi[150] Clofibrate

Ethacrynic acid

GSH analogs

Gossypol

Indomethacin

Misonidazole

Piriprost

Quinones

Quereetin

Sulfasalazine

GCS[91] PDMP

PPMP

Miglustat

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Genetic, Indian Institute of Science, Bangalore are studyingphenotypic and functional characterization of humanmammary stem/progenitor cells in long term culture. Cancerstem cells exhibit close resemblance to normal stem cellsin phenotype as well as function. It has recently been shownthat human breast stem cells can be enriched in suspensioncultures as mammospheres. However, little is known aboutthe behavior of these cells in long-term cultures. Sinceextensive self-renewal potential is the hallmark of stemcells, they undertook a detailed phenotypic and functionalcharacterization of human mammospheres over long-termpassages. Professor M Radhakrishna Pillai and his groupat Rajiv Gandhi Centre for Biotechnology,Thiruvananthapuram are studying tumor stem cell profilesin breast cancer. Chemo-predictive assay is till date nottried widely on Indian population. Only report was fromTata Memorial Hospital, Mumbai by Pathak1. They studiedin vitro chemo-sensitivity profile of oral squamous cellcancer and correlate the results with clinical response tochemotherapy.

International

The hypothesis that tumors may originate from a rarepopulation of cancer stem cells (CSCs) has gainedtremendous popularity in recent years and is supportedextensively by several pioneering works. Cancer therapiestargeting CSCs have unlimited potential for relapse freesurvival of cancer patients2. Magnus3 has established thatthis small proportion of tumor cells rather than transitamplifying cells or differentiated cells must be targeted toachieve longterm cure. The ability to therapeutically attack

Fig. 3. Schematic presentation of Immunotherapy in MDR

stem cells hinges upon identifyingunique targets like cell surfacemarkers. Al-Hajj and colleagues4

showed that human breast CSCs,identified on the basis of CD44+,CD24–/low, Lin- expression, couldform tumors when as few as 100cells were injected into nonobesediabetic/severe combinedimmunodeficiency disease (NOD/SCID) mice. These cells have someof the key characteristics of stemcells. When 20,000 cells withoutthis phenotype were used, they wereunable to form a tumor. Theseeperiments indicate that tumorinitiation could be driven by rareBCSCs. It is reported that a smallpopulation of CD44+/CD24-/Lin-human breast cancer cells wereenriched for tumorigenic potential.

Specifically, the CD44+ phenotype is correlated positivelywith breast cancer initiator cells. There are reports showingthat all CD44+/CD24– cells do not show stem cellproperties. Additional marker aldehyde dehydrogenasepositive cells subtype the stem cells from the CD44+/CD24-cells. Moreover, there was a high prevalence of ALDH1expression among breast carcinomas5. Ginestier6 reportedthat expression of ALDH1 was considered to be a markerof malignant stem cells and putative cancer stem cellsexpressing the combined CD44+/CD24-/low/ALDH1p+phenotype showed an especially high tumorigenic capacity,being able to form tumors from as few as 20 cells.

It has long been known that chemotherapeutic drugsinitiate self-destruction in the drug-sensitive tumor cells.In this era of personalized medicine, chemo-predictiveassays like ChemoFX, MICK assay, EDR help to determinehow chemotherapy will impact cancer cells before itimpacts cancer patients and thus allows oncologists toprescribe chemotherapy that would be the most effectiveagainst the tumor cells of that patient. In 2007, Cree7

reported a potential randomized clinical trial of tumorchemo-sensitivity assay with recurrent platinum-resistantovarian cancer cases and documented a trend towardsimproved response and progression-free survival for assay-directed treatment8 predicted that it is feasible to assessthe chemo-responsiveness of small breast lesions using theChemoFx assay to assist in choosing neo-adjuvantchemotherapy for breast cancer patients. In 2011, Schink9

and Copeland reported that chemo-sensitivity assays havea role in the management of recurrent ovarian cancer casesyielding a 0.70 correlation with response and accuratelypredicting progression-free and and overall survival.

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However, Agiostratidou10 drew a correlation between invitro cytotoxicity and clinical response to chemotherapy inovarian and breast cancer patients and concluded thatalthough in vitro chemo-sensitivity/resistance is a valuableassay, further analysis and implications of other factors arerequired for a general evaluation of cytotoxic treatment forpatients with ovarian and breast cancer.

Netaji Subhas Chandra Bose Cancer Research Instituteis a renowned tertiary care cancer hospital in Eastern India.The proposed investigating group has already come acrossa larger number of breast cancer patients with their diseaseprogression profile and is well experiened in selection ofpatients. The members have already collected samples frombreast cancer tissue and bone marrow of breast cancerpatients and done a preliminary study with CD44+/CD24-cells. A total of 10 ex vivo chemo-naive patients withbiopsy proven triple-negative metastatic breast cancer wereselected randomly and tested for CD44/CD24 cell surfacemarkers following immunosorting using magnetic cell sorterand immunophenotyping by flowcytometric analysis.isolated BCSCs were cultured for drug sensitivity towardsplatinum, anthracycline and docetaxel. Correlation wasdrawn between cell differentiation, % of stem cells anddrug response. Accordingly chemotherapy was designed fora particular patient. Percentage of BCSCs in pre- and post-chemotherapeutic condition was further compared. We havedetected BCSCs in 90% of cases. Among positive samples,89% patients showed platinum sensitivity and rest werefound to be anthracycline sensitive. No sensitivity todocetaxel was observed. In lieu of this, cisplatin wasapplied in vivo and % of BCSCs came down to 6.58%from initial 11.16% (for a representative case).

Sl. No. Percentage of BCSCsPre-chemoterapy Post-chemotherapy

1. 11.16 6.58

2 2.30 0.20

3. 4.59 0.27

4. 0.90 0.86

5. 5.62 13.47

A secondary cell homing assay for predicting chemo-sensitivity was published by Co-PI in Molecular CancerTherapeutics in 2010. Here it has been demonstrated usingHEY ovarian cancer cells the response to paclitaxel in anex vivo situation.

Objectives

Estimation of number of BCSCs in breast cancertissue and bone marrow for advanced cases with2nd line of treatment failure by identifying cellsurface markers with CD44+/CD24-/ALDH1+phenotype; Also we will estimate other breastcancer stem cell markers like CD133, CD326,CD184 etc.

Culture of whole tumor cells for ex vivo drugsensitivity towards patient-specific personalizedchemotherapy followed by statistical analysis offailure rate of patients even after chemo-predictiveassays. This is followed by chemo-predictive assayson BCSC but that will not be used in the clinicalscenario.

Fig. 4. Representative FACS data of BCSCs isolated from paticuts pre- and post-chemotherapy

Comparison of chemo-predictive assays of wholetumor cells and BCSC intreatment failure cases topredict if BCSC targetedtherapy could have been analternative approach.

Methodology

Acquisition of breastcancer patients fromchemotherapy.

Identification of BCSCs byidentifying different cellsurface markers usingFACS.

Isolation of breast cancercells from whole tumor of

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patients to perform drug sensitivity test ex vivowith standard chemotherapeutic drugs like 5-Fluorouracil, Epirubicin, Cyclophosphamide etc.and some specific target drugs like Trastuzumab,Imatinib etc.

Designing predictive chemotherapy for a particularpatient based on the results.

Isolation of BCSCs from whole tumor samplesexpressing specific surface markers and culturethem for ex vivo drug sensitivity with the sameset of drugs.

Outcomes

1. Detection of up-front chemo-resistant patients.

2. Define best chemotherapy for treatment failurepatients.

3. Define correlation between BCSC with TTR, PFS,OS, and chemo-resistance.

4. Define stem cell markers that would best definepre and post chemo BCSC load.

5. Determine a therapeutic paradigm by personalizingtreatment for individual patient.

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