University of Groningen Towards new personalized treatment options for patients with genomically unstable tumors van Gijn, Stephanie Elise

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University of Groningen

Towards new personalized treatment options for patients with genomically unstable tumors

van Gijn, Stephanie Elise

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Publication date: 2019

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van Gijn, S. E. (2019). Towards new personalized treatment options for patients with genomically unstable tumors. Rijksuniversiteit Groningen.

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531746-L-bw-van Gijn 531746-L-bw-van Gijn 531746-L-bw-van Gijn 531746-L-bw-van Gijn Processed on: 6-6-2019 Processed on: 6-6-2019 Processed on: 6-6-2019

Processed on: 6-6-2019 PDF page: 49PDF page: 49PDF page: 49PDF page: 49

Elevated protein expression of

TPX2 is positively associated with

genomic instability in breast

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ABSTRACT

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype for which therapeutic treatment options are currently scarce. TNBCs are characterized by a high degree of genomic instability. Since normal cells do not tolerate high levels of genomic instability, it is thought that tumor cells are ‘rewired’ to survive high levels of genomic instability. Previously, we found that cancer cells with a DNA repair defect due to BRCA2 inactivation have enhanced sensitivity to inactivation of TPX2. This study sought to determine whether TNBCs express high levels of TPX2 protein and whether TPX2 protein expression is associated with genomic instability. In a set of 449 invasive breast carcinoma samples TPX2 expression was immunohistochemically analyzed and associated with protein expression of markers of genomic instability (phospho-RPA32-Ser33 and _{γ-H2AX) as well as an oncogenic driver} of genomic instability (Cyclin E). Among the different breast cancer subtypes, TNBCs showed highest levels of TPX2 protein expression. Furthermore, TPX2 expression was positively associated with tumor grade, and expression of pRPA and Cyclin E. These data indicate a potential dependency of TNBCs on the TPX2/Aurora-A signaling pathway, and point to actionable therapeutic targets for these tumors.

INTRODUCTION

Breast cancer is the second most common type of cancer worldwide and the second most common cause of death in women in developing countries1,2_{. Breast cancers are classified as triple negative}

breast cancer (TNBC) when they lack expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression. TNBCs are typically very heterogeneous and behave aggressively, with early (visceral) metastatic spread, and are associated with poor prognosis2_{. Very little progress has been made in their systemic treatment with conventional}

chemotherapies. In addition, TNBCs often lack clearly defined oncogenic drivers, which excludes patients with TNBC from treatment with currently available targeted therapeutic approaches2_{. The}

development of new treatment options for patients with TNBC is therefore a profound unmet need. A key property of TNBCs is their high degree of genomic instability through defects in DNA repair, which is a central process in their carcinogenesis3_{. Genomic instability refers to the progressive}

accumulation of genomic alterations, such as amplifications and deletions (i.e. somatic copy number alterations; SCNAs). This process of genomic instability contributes to the acquisition of the cancer hallmarks (e.g. sustained proliferative signaling, invasion and metastasis). Normal cells do not tolerate high levels of genomic instability4-6_{. TNBC cells, however, have apparently adapted}

to cope with high levels of genomic instability. Better understanding of how tumor cells are molecularly ‘rewired’ to survive genomic instability could reveal new therapeutic targets or strategies.

To gain more insight into this rewiring process, we previously used functional genomic mRNA (FGmRNA) profiling to determine the degree of genomic instability for 16,172 cancer samples. Utilizing these samples in a transcriptome-wide association study (TWAS) enabled us to rank genes based on the association between their expression levels and the degree of genomic instability7_{. One of the top}

hits in this TWAS analysis was the microtubule nucleation factor TPX2, which together with its partner Aurora kinase A (Aurora-A), is essential for the assembly of a functional mitotic spindle and for faithful chromosome segregation8_{. Pre-clinical validation experiments revealed that cancer cells with a DNA}

repair defect due to BRCA2 inactivation have enhanced sensitivity to inactivation of TPX2 or Aurora-A9_.

In line with the findings, elevated expression of TPX2 has been observed in a variety of cancers and is an indicator of worse patient prognosis8,10-14_{. Collectively, these observations suggest that genomically}

unstable tumors become dependent on the TPX2/Aurora-A signaling pathway for their survival. In this study, we investigated whether genomically unstable TNBCs express higher levels of TPX2 protein compared to the other less genomically unstable breast cancer subtypes. Furthermore, we explored whether TPX2 expression positively associated with expression levels of markers of genomic instability. To this end, we performed immunohistochemical analyses in a large cohort of breast cancer samples. This enabled us to assess the level of TPX2 protein

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expression in hormone-receptor based breast cancer subtypes (including TNBC) and determine its association with clinicopathological parameters and two markers of genomic instability (phospho-RPA32-Ser33 and _{γ-H2AX) and an oncogenic driver of genomic instability (Cyclin E)}15,16_.

RESULTS

Breast cancers display elevated protein expression of TPX2

Representative examples of TPX2 staining patterns containing negative, weak and strong staining are depicted in Figure 1A. Staining of TPX2 showed a strong nuclear staining pattern with occasional weak cytoplasmic staining. Examples of TPX2 staining on representative breast cancer samples and normal breast samples are depicted in Figure 1B. The TPX2 H-score for breast cancers ranged from 46 to 196 with a median H-score of 156. Normal breast samples had a median TPX2 H-score of 75 ranging from 55 to 95 (Fig. 2).

TPX2 expression shows highest expression in TNBCs

Next, we investigated protein expression levels of TPX2 across breast cancer subtypes (Figure 3A). A significantly higher TPX2 protein expression was observed in TNBCs, when compared to the ER+_PR+_HER2-_{subtype (p = 0.01) (Figure 3A). Since expression of the androgen receptor}

(AR) has previously been linked to biological and clinical behavior of TNBCs17_{, we analyzed}

AR-positive and AR-negative TNBCs separately. However, no significant difference in TPX2 expression in AR-positive compared to AR-negative TNBCs was found (p ≥ 0.05) (Fig. 3B).

negative staining weak staining strong staining Figure 1

A

B

breast cancer tissue staining

norrmal breast tissue staining

Figure 1: Representative immunohistochemical stainings of TPX2. (A) Example im-ages of negative, weak and strong TPX2 staining in breast cancer tissues. (B) Magni-fied example images of TPX2 staining in breast cancer tissues and normal breast tissues.

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TPX2 expression is associated with tumor grade and expression of pRPA and Cyclin E

The observed elevation of TPX2 expression in TNBCs, led us to hypothesize that the extent of TPX2 expression could act as an indicator of the degree of genomic instability in breast tumors. For this reason, we associated expression of TPX2 with clinicopathological parameters and with expression of two markers that are linked to genomic instability, namely pRPA and _γ-H2AX, and expression of Cyclin E, an oncogenic driver of genomic instability. Using univariate analyses, we found that tumor grade (Beta=0.161, p=0.000), γ-H2AX (Beta=-0.098, p=0.008), pRPA (Beta=0.247, p=0.000) and Cyclin E cytoplasmic (Beta=0.181, p=0.000) and Cyclin E nuclear staining (Beta=0.119, p=0.000) showed a statistically significant association with TPX2 expression (Table 2). Subsequently, we selected these statistically significant variables for multivariate analyses. Multivariate analyses showed that tumor grade (Beta=0.162, p=0.001), pRPA (Beta=0.247, p=0.000) and cytoplasmic Cyclin E staining (Beta=0.176, p=0.000) are predictors of TPX2 expression.

Figure 2

A

average H -sc or e T PX 2 individual samples 0 100 50 150 200

breast cancer samples (n=475) normal breast samples (n=9)

0 100

50

Figure 2: Waterfall plot of average TPX2 H-scores of two or three cores. Each bar on the horizon-tal axis represents an individual sample. Breast carcinoma tissue samples are depicted in yellow and normal breast tissue samples in green.

ER- PR - HER2- AR-ER- P R - H ER2- AR+ 0 50 100 150 200 250 Figure 3 A B ER+ P R+ H ER 2-ER+ P R+ H ER2+ ER- PR - HER2- ER- P R- H ER2+ 0 50 100 150 200 250 H-score TPX2

Breast cancer subtype

H-score TPX2

Breast cancer subtype

* * ns

Figure 3: Distribution of TPX2 H-scores among the breast cancer subtypes. (A) A Kruskal-Wallis with Dunn’s Multiple Comparison test was used for testing for significance. A significant difference was only found between ER+_PR+_HER2-_{and ER}-_PR-_HER2-_{(p=0.01). (B) Division of TNBCs into AR}-_{and AR}+

groups did not result in a significant difference in TPX2 H-score using a Mann-Whitney U test (p=0.87).

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DISCUSSION

In this study, we examined protein levels of TPX2 in breast cancer subtypes. Previously, TPX2 expression was shown to be positively associated with the degree of genomic instability9,18_{. Since TNBCs present}

with the highest degree of genomic instability across the breast cancer subtypes, we hypothesized that TNBCs would show the highest levels of TPX2 protein expression3_{. Indeed, our analysis confirmed}

that TPX2 expression was most elevated in TNBCs compared to the other breast cancer subtypes. In addition, we determined whether high protein expression levels of TPX2 were associated with clinicopathological parameters. Our results showed that protein expression of TPX2 associated positively to the clinicopathological parameter tumor grade in multivariate analysis. The significant positive association between tumor grade and expression of TPX2 indicates that breast cancers with a high tumor grade have elevated levels of TPX2 protein expression and that TPX2 expression is an independent predictor of tumor grade. Since high tumor grade is linked to a poor prognosis, elevated levels of TPX2 in breast cancers could predict a poor prognosis. Although TPX2 expression has been investigated considerably in other cancer types12,19-23_{, this study for the first time investigated}

protein levels of TPX2 using immunohistochemistry in a large cohort of breast carcinoma samples. To determine whether elevated TPX2 expression was associated with features of genomic instability, we assessed the association of TPX2 expression with two markers of genomic instability, pRPA and _{γ-H2AX, and to the expression of a key driver of genomic instability, Cyclin E}15,16_{. We found}

that TPX2 expression associated positively with protein expression of cytoplasmic Cyclin E and pRPA in multivariate analyses. Expression of TPX2, pRPA and Cyclin E, are cell cycle-dependent, however, it is unlikely that TPX2, Cyclin E and pRPA are associated due to their oscillating patterns24,25_{, since}

phosphorylation of RPA and elevation of Cyclin E occur at the G1-S phase transition, whereas TPX2 predominantly accumulates during the G2-M phase transition15,26,27_{. Our multivariate analyses}

showed that cytoplasmic Cyclin E expression -but not nuclear Cyclin E expression- was associated with TPX2 expression. Cytoplasmic Cyclin E staining has been shown to strongly correlate to reduced overall survival unlike nuclear Cyclin E staining28_{. The high affinity of cytoplasmic Cyclin}

E to cyclin-dependent kinase 2 (CDK2) can force cells to progress from the G1-S phase of the cell cycle, which can cause replication stress28_{. Collectively, these results suggest that elevated expression}

of TPX2 could act as a marker indicative of replication stress, a source of genomic instability. Furthermore, we found that overall expression levels of TPX2 were higher in the breast cancer tissues compared to normal breast tissue. Of the breast cancer subtypes, TNBCs expressed the highest protein levels of TPX2, which could be a requirement for their survival. A recent study showed that beyond the mitotic function of TPX2/Aurora-A, this complex also regulates DNA double-stranded break repair and protects stalled replication forks in conditions of replication stress29_{. So}

Table 2: Univariate and multivariate linear regression analysis between expression of TPX2 and other parameters.

Univariate linear regression analysis Multivariate linear regression analysis Hazard

Ratio 95%-CI P Hazard Ratio 95%-CI P

Age 0.037 -1.64 3.65 0.198 Tumor size (mm) 0.020 -2.79 2.93 0.277 Tumor grade 0.161 1.43 7.23 0.000 0.162 1.65 6.91 0.001 g-H2AX staining -0.098 -6.64 0.50 0.008 -0.093 -6.44 0.53 0.153 pRPA staining 0.247 4.53 10.27 0.000 0.247 4.59 10.27 0.000 CyclinE (cytoplasmic) 0.181 2.93 8.23 0.000 0.176 2.87 8.10 0.000 CyclinE (nuclear) 0.119 0.12 8.05 0.000 0.120 -0.24 8.05 0.064 R2_{=0.181 (univariate), R}2_{=0.183 (multivariate)}

Table 2: Univariate and multivariate linear regression analysis between expression of TPX2 and other parameters.

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far, it was known that Fanconi anemia/BRCA proteins are able to protect stalled replication forks from MRE11-dependent degradation30,31_{. This recent study showed that also the TPX2/Aurora-A}

complex is able to protect newly replicated DNA from MRE11-dependent degradation, albeit via a distinct pathway. Specifically, binding of TPX2 to 53BP1, a promoter of non-homologous end joining (NHEJ), counteracts 53BP1 function and thereby protects newly replicated DNA from MRE11-dependent degradation through a mechanism dependent on Aurora-A activity29_{. These}

findings, together with our earlier previous data, explain why genomically unstable cancer cells harboring defects in Fanconi anemia/BRCA proteins would depend on the TPX2/Aurora-A pathway for their survival9,29_{. As a consequence, inactivation of the TPX2/Aurora-A signaling pathway in}

genomically unstable cancer cells could lead to synthetic lethality, since replication forks cannot be stabilized, causing replication fork collapse and toxic DNA double-stranded breaks16,32,33_.

Currently, no drugs exist that target TPX2, however, multiple Aurora-A inhibitors have been developed which are currently evaluated in clinical trials34-36_{. Thus far, clinical activity of Aurora-A}

inhibitors in combination with cytostatic drugs have been observed in a range of tumor types37_.

At this moment, metastatic triple negative breast cancer patients are recruited for a clinical trial investigating clinical activity of an Aurora-A inhibitor together with a dual TORC1/2 inhibitor (NCT02719691, clinicaltrials.gov).

In conclusion, this study shows that among breast cancer subtypes, highest levels of TPX2 expression are found in TNBCs, which are highly genomically unstable. Future research will need to explore whether depletion of the TPX2/Aurora-A signaling pathway could be a new potential target in the treatment of genomically unstable TNBCs.

METHODS and MATERIALS

Patients

Resection specimens of 449 consecutive invasive breast carcinomas diagnosed in the University Medical Center Groningen (UMCG, The Netherlands) between 1996 and 2005 were collected and obtained from the archive of the Department of Pathology and Medical Biology (UMCG, The Netherlands). The patient cohort was combined from two series which were previously described38,39_.

Patient selection was based on the availability of sufficient paraffin-embedded tumor tissue. Additionally, resection specimens of 108 consecutive primary invasive triple negative breast carcinomas diagnosed in the UMCG (The Netherlands) between 2006 and 2017 were collected. In total, tumor samples of 58 patients (10.4%) were excluded due to insufficient tumor material on the tissue microarray (TMA) or lack of one of the hormone receptor stainings, making it unable to determine breast cancer subtype. This resulted in a total study cohort of 499 patients (Table 1). Healthy control tissue was obtained from resection specimens of 9 patients who underwent reduction mammoplasty in the UMCG in 2018, which allowed to determine TPX2 protein expression levels in normal tissue. Clinicopathological characteristics were retrospectively gathered from electronic patient records. This study was conducted in agreement with the principles of the Declaration of Helsinki and within the rules and regulations posed by the Institutional Review Board (IRB) of the UMCG.

Tissue Microarray

TMAs were constructed from formalin-fixed, paraffin-embedded tumor blocks. For orientation, the most representative invasive tumor area was marked on the original haematoxylin and eosin (H&E)-stained slides. From this area, three 0.6 mm diameter punches were taken from the donor blocks and transferred into a recipient paraffin block using a manual tissue arrayer (Beecher Instruments, Sun Prairie, USA). In total, nine TMAs were constructed each containing between 141-216 samples.

Immunohistochemistry

Serial sections of 3µm were cut from the TMA blocks with a standard microtome. All sections were stained for TPX2 (Novus Biologicals, NB500-183) and androgen receptor (AR) (SP107, Roche). In addition, TNBC sections were stained for ER (SP-1, Ventana), PR (1E2, Ventana) and HER2 (SP-3,

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Thermo Fisher Scientific) to confirm receptor status. ER, PR, HER2 and AR antibodies were pre-diluted and sections were stained on a Ventana Benchmark Ultra immunostainer (Ventana Medical Systems) according to the manufacturer’s protocol. For TPX2 staining, sections were deparaffinised in xylene, rehydrated in decreasing concentrations of alcohol and washed with demineralised water. Heat-induced antigen retrieval was performed by cooking the sections for 15 minutes in 10 mM citrate buffer (pH 6.0). Incubating the sections in 3% H₂O₂ in methanol for 15 minutes blocked endogenous peroxidase reaction. To permeabilize the tissue, sections were washed in 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) with 0.4% Triton X-100 (PBS-T). Subsequently, non-specific binding was blocked by incubating the tissue sections with 5% BSA in PBS-T for 30 minutes. The

Table 1: Patient and tumor characteristics

N (%) No. of patients 499 (100) Sex Male 0 (0) Female 499 (100) Age Median 57 Range 26-90 Breast cancer subtype

ER(+) PR(+) HER2(-) 211 (42.3) ER(+) PR(+) HER2(+) 120 (24.0) ER(-) PR(-) HER2(-)AR(-) 101 (20.2) ER(-) PR(-) HER2(-) AR(+) 39 (7.8) ER(-) PR(-) HER2(+) 28 (5.6) Tumor grade I 103 (20.6) II 185 (37.0) III 203 (40.6) Unknown 9 (1.8) T-stage T1 (tumor size is ≤ 20mm) 282 (56.5) T2 (tumor size is >20mm but ≤

50mm) 168 (33.7) T3 (tumor size is > 50mm) 36 (7.2) Unknown 13 (2.6) Therapy CTX 116 (23.3) RTX 97 (19.4) CTX + RTX 281 (56.3) Unknown 5 (1.0) Relapse during follow-up

Yes 412 (82.6) No 83 (16.6) Unknown 4 (0.8) ER=estrogen receptor, PR=progesterone receptor, HER2=human epidermal growth factor receptor 2, AR=androgen receptor, CTX=chemotherapy, RTX=radiotherapy

Table 1: Patient and tumor characteristics.

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primary monoclonal antibody against TPX2 was diluted in PBS-T (1:100) containing 1% BSA and incubated overnight at 4°C. Sections were washed in 1% BSA in PBS-T for 10 minutes and incubated with anti-rabbit secondary antibody (Dako, 1:50) for 1 hour at room temperature (RT). Visualization was performed with the diaminobenzidine peroxidase reaction and sections were counterstained with haematoxylin. Sections were dehydrated in increasing concentrations of alcohol and mounted. Immunohistochemical staining of Cyclin E, γ-H2AX and phospho-RPA32-Ser33 (hereafter referred to as pRPA) was performed by deparaffinising slides in xylene, incubating slides for 30 minutes with 0.3% H₂O₂ and incubating with primary antibodies against Cyclin E (Santa Cruz, #sc-198, 1:1000, 1 hour at RT), γ-H2AX (Millipore, #05-636, 1:300, 1 hour at RT) and pRPA (Bethyl, #A300-246A, 1:6400, half an hour at RT). After washing and incubation with a secondary antibody, visualization was performed with the diaminobenzidine peroxidase reaction and sections were counterstained with haematoxylin.

Evaluation of immunohistochemistry

Scoring of ER, PR and AR was based on the percentage of cells with positive nuclear staining, with a score of >1% considered as positive40_{. HER2 expression was scored as 0, 1+, 2+ or 3+ according to the ASCO/}

CAP testing guideline41_{. For TPX2, scoring was based on the percentage of cells with negative, weak and}

strong nuclear staining (Fig. 1). Scoring of TPX2 was performed by two independent observers (SvG and MZ). Disagreements were reassessed and resolved by consensus. When no consensus was achieved, a final score was given by an experienced pathologist (BvdV). Histoscores (H-scores) could range between 0-200 and were calculated as staining intensities (0=negative, 1=weak and 2=strong) and multiplied by the percentage of positive tumor cells (0-100%). Quantitative staining of Cyclin E (cytoplasmic and nuclear), _{γ-H2AX and pRPA was scored as described}42_{. Expression of Cyclin E staining was separated}

into cytoplasmic Cyclin E and nuclear Cyclin E staining, as previous studies showed that the isoforms do not only differ in localization and molecular weight but also in their association to overall survival28_. Statistical analysis

All statistical analyses were performed with IBM SPSS Statistics version 25. A Kruskal-Wallis test and Dunn’s Multiple Comparison test were performed to explore differences in TPX2 protein expression distribution across breast cancer subtypes. Correlations between TPX2 expression and clinicopathological parameters and tumor expression of Cyclin E, γ-H2AX and pRPA were assessed using univariate linear regression analyses. Significant results from univariate analyses were then selected for multivariate linear regression analyses. P-values ≤ 0.05 were considered statistically significant.

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