Clinicopathologicalsignificance of β-tubulin isotypeIII gene expression in breast cancer patients


Qingqing Hea,1,?, Bo Pengb,1, Dayong Zhuanga, Lei Xiaoa, Luming Zhenga, Ziyi Fana, Jian Zhua, Benmei Xub, Cheng Xub,Jiangman Zhaob, Liming Wub, Peng Zhoua, Lei Houa, Fang Yua and GuoweiZhaoa aDepartment of Thyroid andBreast Surgery,Jinan Military GeneralHospital of PLA, Jinan,Shandong,China bZhangjiang Center for Translational Medicine, Shanghai, China


BACKGROUND: The molecular classi? cationof breast cancer mainly focuse sone strogenreceptor(ER), Progesteronereceptor (PgR), and Human Epidermal Growth Factor Receptor 2(HER2/Neu) status detected by immunohistochemistry (IHC) analysis. The β-tubulin isotype III (TUBB3) gene was thought tobe amarker of taxane resistance or cancer aggressiveness.

METHODS: To evaluate the clinicopathological signi?cance of TUBB3 expression in breast cancer patients, we measured TUBB3 mRNA levels in 92 breast cancer patients by Quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and examined their correlation withER, PgR, and HER2status detected by IHC.

RESULTS:We observe dasigni? cant positive correlation between the TUBB3mRNA expression and the immuno histo chemical positivity of both PgR (p = 0.000) and HER2 (p = 0.001). In addition, TUBB3 mRNA expression was associated with lymph nodes status(P = 0.008) andtumor stages(0.029), but nocorrelation wasfound withother clinicopathological features, suchas age, pathohistological grades and tumor size.

CONCLUSIONS: In conclusion, TUBB3 expression correlated signi?cantly with molecular markers of breast cancer, such as PgR and HER2, suggesting that TUBB3 mRNA level facilitate the identi?cation of a subset of patients who respond to Taxane treatment in addition to hormonal therapy and trastuzumab.

Keywords: Breast cancer, ER,PgR, HER2, β-tubulin isotype III

1. Introduction

Breast cancer is the most frequentlydiagnosed cancer in women in western countries, and it is the second most common cause of cancer-related deaths [1]. Despite recent improvements in the diagnosis and management of early disease, approximately 50% of women with breast cancer will develop distant metastases [2].

Breastcancer is a heterogeneousdisease thatcan be classi?ed into subgroups on the basis of hormone receptor status, HER2 expression levels, and gene expression pro?ling [3–5]. Patients of some breast cancer subgroups may have high response rates to speci?c chemotherapeutic drugs, whereas others may derive a relatively small bene?t from drugs. At the same time they have to be exposed to treatment-related toxicity [6–10]. This underscores the need for predictive biomarkers that can facilitate the selection of optimal treatment or alternative regimen for patients of breast cancer of various subclasses. Accordingly, predictive biomarkers offer the potential to improve the bene?t:risk ratio of a giventherapeuticagent.

Among those putative markers is the structural proteinclassIII β-tubulin(TUBB3)[11–13].TheTUBB3 gene was initially thoughtto encode a neuron-speci?c protein[14–16]and to be amarker of taxanere sistance or cancer aggressiveness [17]. High expression of TUBB3 correlates with low response rates in patients treated with taxane- or vinca-alkaloid-containingregimens and with reduced survival in patients with nonsmallcelllungcancer (NSCLC) [18,19],breast[20],or ovarian cancer [21]. Although originally identi?ed as a mechanismof drugresistance to taxanes[22], recent studies have shown that TUBB3 is involved inan adaptive response to low oxygen levels and poor nutrient supply in a growing number of solid tumors [17,23]. This explains the involvement of TUBB3 in drug resistance independent of whether the disease is treated with a regimen that includes a microtubule targeting agentornot[24].

Clinically, the signi?cance of alteration of TUBB3 expression has been clearly established. Nevertheless the relationship between TUBB3 and hormone receptors, HER2 has not been very clear in breast cancer. Currently,ER, PgR, and HER2/neu status are molecular marker suse dintheroutine treatment of breast cancer. The presence or absence of ER and PgR are valuable prognosti cfactors providing predictive valueas to the potential bene?ts from hormonaltherapy. Approximately 70% of meta static breast tumor sare ER and/or PgR positive, and these patients tend to have a greater chance of effective tumor response and longer over all survival than patient swith ER/PgR- negativetumors since they can be targeted with treatments such as tamoxifen, a selective ER modulator [25–27]. Furthermore, patients with ER/PgR-positive early breast cancer have are duced risk of recurrence and death following adjuvant hormonal therapy, whereas patients with ER/PgR-negative disease derive minimal bene?t from these treatments [28]. The epidermalgrowth factor receptor HER2/neu is overexpressed or ampli?ed in approximately 30% of breast cancers, and its presence is associated with a worse prognosis [29]. The human monoclonal antibody trastuzumab was developed to bind the HER2/neurecep to rand block it sactivity [30]. Assessment of HER-2 status also is importantfor predicting response to other speci?c chemotherapy regimens.

In the present study, we studied the relationship between TUBB3 expression and hormone receptors and HER2 status in breastcancer patients.

2. Materialsandmethods

2.1. Patients’ characteristics

All 92 enrolled patients were cases of primary operablebreastcancer. Patients’ clinicalhistorydatawas acquired from the ?les of the Department of Thyroid and Breast Surgery, Jinan Military General Hospital, Jinan, China. The patients’ age ranged from 21 to 74 years. The histological diagnosis of the formalin?xed, paraf?nembedded sections were done and con?rmed by three experienced pathologists. The diagnosis read as 84 invasive ductal carcinomas (91.3%), 1 invasive lobular carcinomas (1.1%), 6 other types carcinoma (6.5%), and 1 unknown histological types (1.1%). Multiple clinicopathological and molecular characteristics were obtained, including age, tumor size, histological type, histological grade, lymph node status, TNM stage, tumor molecular subtype (as de?ned by the immunohistochemical expression of ER, PgR, and HER2).

The experiments were undertaken with the understanding and written consent of each subject, and that the study conforms with The Code of Ethics of the World Medical Association (Declaration of Helsinki), printed in the British Medical Journal (18 July 1964). This study had been carried out inaccordance with the ethical standard of ethics committee of Jinan Military GeneralHospital,Jinan,China.

2.2. Immunohistochemistry (IHC) analysisfor ER, PgRand HER2proteins

Immunohistochemistry (IHC) was performed on 3μm- thick tissue sections prepared from formal in-?xed, paraf?n-embeddedtissue.Sections weres tained by the standard method using antibodies against ER, PgR and HER-2 according to the manufacturers’ instructions.Brie?y,3-μm-thick sections were cut from paraf?n blocks containing representative tumor samples. Paraf?n sections were dewaxed in xylene, rehydrated through a series of graded alcohols, placed in 10 mM citrate buffer and submitted to heatr etrieval using avapor lock for 40 min. After heating, the slides were allowed to cool to room temperatureand brie?y washed with Tris-buffered saline. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 5 min .Normal serumwa susedfor 30min in order to block non-speci?c immunoassaying. Immunohistochemical staining was performed using an avidin-biotin peroxidase system. Following washes in PBS, biotinylated universal secondary antibody were applied for 30 min. The sections were incubated with the avidin-biotin complex reagent for 30 min and developed with 3, 3-diaminobenzidine tetrahydrochloride (DAB) in phosphate-bufferedsaline, pH 7.5, containing 0.036% hydrogen peroxide for 5 min. Light Mayer’s hematoxylin was applied as a counterstain. The slides were then dehydrated in a series of ethanol and mountedwith Permount.Breast carcinomatissues consistently showing ER, PgR or HER2 ampli?cation and strong protein expression by immu nohisto chemical analysis wasuse dasaposi tivecon trolsample.Normal breast tissue wa susedinnegative control samples.

Asemiquanti tativescoring of percentage of positive cells with ER and PgR nuclear staining was used: 0 = no staining, 1+=1–10% staining, 2+=11–50% staining, and 3+=> 50% positive nuclear stain. Nuclear ER and PgR stain intensity was graded as weak, moderate,or strong.HER-2 expressionwas scored according to the degree and the proportionof membrane staining according to HercepTest protocol [31]. HER2 expression was negative with a score of 0 or 1+. A score of 0 was de?ned as no staining or membrane staining in less than 10% of tumor cells. A score 1+ comprisedfaintor partiallystainedmembranein more of 10% of tumortissue. Over expression of HER-2 was scored as 2+ when weak to moderate complete membrane staining was present in more than 10% of tumor cells. A score of 3+ was interpreted as strong, complete membrane staining in more than 10% of the tumor. The pathologic reviews with IHC staining for all the surgical specimens were done prospective lyand comprehensively by two experienced pathologists in ourhospital.

2.3. qRT-PCRfor the expression of TUBB3

Fresh frozen specimens of tumor and adjacent tissues were obtained from 92 patients. Specimens were microscopically examined to assess quality and to verify the histopathology. Specimens were pulverized by pulp re?ner under Trizol reagent (Life Technologies, California, USA). Total RNA was extracted with TaKaRa RNAiso Reagent (Takara Bio, Inc., Otsu, Shiga, Japan). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was applied for the assessment of TUBB3 gene. Total RNA were reverse transcribed with RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, Thermo Scienti?c, Massachusetts, USA) for generation of cDNA with ABI 9700 PCR (Applied Biosystems, Carlsbad, California, USA). Forty cycles of nucleic acid ampli?cation were applied using ABI 7500 Real-Time PCR (Applied Biosystems, Carlsbad, California, USA), and the cycle threshold (CT) value of the target gene was identi?ed. CT values were normalised by subtracting the CT value of the housekeeping gene GAPDH from the CT value of the target gene (CT). RNA results were then reported as 40-CT values, which would correlate proportionally to the mRNA expression level of the target gene [32]. Expression of the TUBB3 gene, as well as the reference gene GAPDH, was assessed in triplicate by PCR using the SYBR green System in an ABI PRISM 7900HT (Applied Biosystems, Darmstadt, Germany). The Primer sets used for ampli?cation of TUBB3 and GAPDH were as follows (5’-3’):

TUBB3 ForwardPrimer:
Reverse Primer:
GAPDH ForwardPrimer:

In the absence of a reliable gold standard and following distributional studies, we used the 25th percentile of observed TUBB3 mRNA expression levels asthresholds for categori zation of tumor stop ositive or negativecases [32].The chosencut -off swere foundto be close to the natural cut-off sin distributional studies. Moreover, cut-offs on the quartiles offer easily interpretable,
reproducible and objective results.

2.4. Statistical analysis

In the absence of a reliable gold standard and following distributional studies, we used the 25th percentile of observed TUBB3 mRNA expression levels as thre sholds for categorization of tumors top ositiveor negativecases [32]. Thechosencut -off swere found to becloseto the natural cut-off sindi stributional studies. Moreover, cut-offs on the quartiles offer easily inter pretable,reproducible and objective results.

2.4. Statistical analysis

Descriptive statistics comparingTUBB3 expression with the clinicopathological characteristics were analyzed by the chi-square test or, when necessary, by Fisher’s exact test. A computer program package StataTM (Version 10.0, StataCorp, College Station, TX, USA) was used for all statistical testing and management of the database, and a signi?cant level of 5% was consideredstatistically signi?cant.

3. Results

3.1. Patient sand tumour characteristics

Themain clinic opath ologic character is ticsof the 92 patients were shown in Table1. 92 patients,all women (agerange21–74years,meanage 50.6years)were included in the study. The immunohistochemical staining for ER, PgR and HER2 proteins were shown in Fig.1.Tumors of 60(65.2%) women were ER positive and 30(32.6%) ER negative, with the remaining two patients’ ER status of tumor were not identi?ed. Tumorsof 53 (57.6%)patients were PgR positive and 38 (41.3%)were Pg Rnegative.Tumor PgR status was not determine dinone patient.HER-2 positives taining was observe dintumors of 48(52.2%) patients and negative staining in tumors of 41 (44.6%) patients, HER-2 status could not be determined in there maining 3 patients. Tumors of 16 patients showed both ER and HER-2 negative staining, tumors of else 22 patients were ER negativebutHER-2 positive. Tumorsin 44 patients expressed both ER and HER-2 positive, tumors of additiona l38 patient srevealed ER positive but HER -2 negative.


From 92 patients, 90 (97.8%) were treated by endocrine and/or chemotherapy: 1 (1.1%) patients were treatedonlyby endocrinetherapy;48 (53.3%)patients received only chemotherapy; and 41 (44.6%) patients were treated by both end ocrine therapy and chemotherapy. 1 (1.1%) patients were not treated by endocrine therapy neither by chemotherapy. Treatment data regarding endocrine and chemotherapy were not available for1 (1.1%)patients.

3.2. Correlationbetween TUBB3 expression and clinicalfeatures

The distribution of TUBB3 mRNA values is shown in Fig. 2. The correlations of TUBB3 mRNA status (positive or negative) with standard clinicopathological factors are shown in Table 2. In general, negative TUBB3 mRNA expression was signi?cantly associated with tumor higher malignancy state, such as advanced stage (p = 0.029), positive nodes (p = 0.008).Speci?cally,TNMstagesofII–IIIwereseenin 84% TUBB3 mRNA-negative versus 59.3% TUBB3 mRNA -positive tumors, while positive nodes were observed in 45.6% vs. 76.9% of TUBB3 mRNA positive versus negative tumors, respectively. But the expression of TUBB3 was not signi? cant lyrelate dtoage (P = 0.397), tumor size (P = 0.446) or menopause condition(p = 0.474).

3.3. Associationbetween TUBB3expression andER, PgR,HER2 status

The correlations of TUBB3 mRNA status (positive or negative) with ER, PgR and HER2 are shown in Table 3. In general, positive-mRNA expression of TUBB3 gene was signi?cantly associated with PgR positivity (P = 0.000) and HER2 positivity (P = 0.001). There was no correlation between the expression of TUBB3 and ER status (P = 0.246). Speci?cally, HER2 positive tumors were observed in 66.7% TUBB3 mRNA-positive tumors versus 26.9% in TUBB3 mRNA-negative tumors while positive expression of PgR was observed in 72.1% TUBB3 mRNA-positive tumors versus 26.9% in TUBB3 mRNA-negativetumors.



4. Discussion

Our study analyzed the association between clinic opathological features and the expression of TUBB3 in aseries of breast cancer patients.TUBB3 mRNA positive expression was associated with lymphnodes status (P =0.008)and tumorstages(p =0.029).ThemRNA expression of TUBB3 in breast tumors was also observed to be associated with the presence of PgR(P = 0.000) and HER2 (P = 0.001) proteins, but there was no correlation between the expression of TUBB3 and ERstatus (P = 0.246).

In vitro studies or in clinicalinvestigations,TUBB3 seems to play a crucial role in the development of chemoresistance to antimicrotubule agents and be a prognosticmarkerofdisease evolution.TUBB3 tumor expression could thus be considered as a predictive marker of paclitaxel resistance in breast cancer [33].Furthermore, high expression levels of TUBB3 have been shown to be associated with poor prognosis in ovarian cancer patients treated by paclitaxel-based chemotherapy[21] and in carcinomas of unknown primarysite [19].



Here,themostinteresting? nding in this study is that the high expression of TUBB3 geneissigni?cantlyrelated with PgR positive breast tumors. Progesterone is well known for its abilities to modula tedirectly the expression ofgrowth factorrecep to rpa thway sand downstream cell cycle regulatory genes known as nuclear proto-oncogenes [34]. Progesterone can direct mammary epithelial growth, differentiation, and survival. PgR is a gene regulatory protein that is dimeric. PgR status has an established role in the tumor response to hormonal therapy. Based on the ?nding that high expression of βIII-tubulinwas associated with paclitaxel resistance in advanced breast cancer, our results indicated that Pg Rpositive breast cancer patients mayhave more likelihood in paclitaxelresistance because of the co-relationshipof PgRand TUBB3 expression.

It is noteworthy that information in the literature regarding a thorough evaluation of both HER2 and TUBB3 is very limited. Jun M [35] et al., found that overexpression of TUBB3 and ampli?ed HER2 genes predicted good response and favorable progression free survival in HER2-positive breast cancer patients treated with paclitaxel and trastuzuamab. Our results also showed that positive HER2 was significantly related with the presence of TUBB3 mRNA which was consistent with George’ s? ndin g[32]. However, some studies reported that there was no signi?cantassociation found between tubulin expression and HER2/neu overexpression[36]. Possible reason of the discrepancy could be due to diverse retrospective designs, sample size, or heterogeneity of the eligible patients ( with different numbers of previous lines of treatment). In this study, we did not analyze the correlations of TUBB3 gene expression and overall survival (OS), disease-free survival (DFS), we could not evaluate the prognostic and predictive utility of TUBB3 gene in breast cancer patients. However, determination of TUBB3 status in breast cancer with HER2 expression would help identify asubse to fpatient swhomight havea responseto Taxane in additionto trastuzumab.

Our results showed that TUBB3 correlate swith PgR s tatus, butdoesnotcorrelatewith ERstatus. Thisdoes not contradict with the fact that PR is a highly ERdependent gene, as previous researches have not only shown the evidence of discordant expression of PR and ER, but also providedat least four reasonsfor this discordance. First, ER does not always activate PR. ER functions via two separated ways, nuclear initiated steroid signaling (NISS) and membrane initiated steroidsignaling(MISS).The forme risk nown as classical and genomic ER activity,causing transcription of PR,while the latte risk nownasnon - classical and nongenomic ER activity, causing PR down regulation. In a classical ER-PR pathway, ER locates inside the nucleus, binds to estrogen, recruits a coregulator complex, and regulates transcription of PR. However, in a non-classical ER-PR pathway, ER is located outside the nucleus, binds to estrogen on the plasma membrane, and activates growth factor receptors (EGFR or HER2) and their downstream signaling pathways, such as phosphoinositide 3-kinase (PI3K) /Akt/ mammalian target of rapamycinm (mTOR). This leads to downregulation of PR [37–40]. Factors which potentiate MISS include hyperactive growth factor signaling, phosporylation of ER, low levels of nuclear ER, and so on [41,42]. Second, activation of PR by ER depends on estrogen level. Previous studies have reported that low circulating level of estrogen in postmenopausal women is not suf?cient to induce PR expression [43,44]. In the present study, > 40% subjects are postmenopausal women, thus the ER-PR pathway maybeinactive.Third, other proteins and growing factors can also regulate PR expression directly. For example,Insulin-likegrowthfactor-1(IGF-1)andEGFR have been proved to regulate expression of PR independent of ER levels or activity in two different studies [45,46]. Horwitz et al., also observed that some breast cance rcell lines continuous lyexpres shigh level of PR independentof estrogens or ER [47]. Forth, the limitation of testing technique smay lead to false determination of ER and PR status. Immunohistochemistry (IHC) is routinely used for hormone receptor analysis currently, the limitations of which have been mentioned by series of publications[48–53]. Factors, such as pre-analytic issues storage, ?xation method, intensity of anti genretrieval,type of anti body,lack of apositive internal control signal, variability in system control samples, may have effects on IHC results [52,53]. Therefore, it is reasonable that expression of TUBB3 correlates with PR status but does not correlates with ER status.

In summary, our work has manifested the signi?cance of the correlation of TUBB3 mRNA level with PgR, HER2 status. Still, their function and relation to other biomolecules should be further investigated, while their prognostic and potentially predictive value remainsto be estimated.


The authors thank all staffs of Shanghai Biotecan Pharmaceuticals Co., Ltd. for their technical assistance of TUBB3 gene expression analysis. This work was supported by Shandong Provincial Natural ScienceFoundation(China)(No.ZR2012HM072)andthe President Funding of Jinan Military General Hospital (No.2011M 03).


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