1 cisplatin hypersensitivity of testicular germ cell - cancer research

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Cisplatin hypersensitivity of testicular germ cell tumors is determined by high

constitutive Noxa levels mediated by Oct4

Matthias Gutekunst1, Thomas Mueller2, Andrea Weilbacher1, Michael A. Dengler1, Jens

Bedke3, Stephan Kruck3, Moshe Oren4, Walter E. Aulitzky5, Heiko van der Kuip1*

1 Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology and University of

Tuebingen, Stuttgart, Germany 2 Department of Internal Medicine IV, Oncology and Hematology, Martin-Luther-University

of Halle-Wittenberg, Halle, Germany 3 Department of Urology, Eberhard Karls University of Tuebingen, Tuebingen, Germany 4 Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel 5 2nd Department of Internal Medicine, Oncology and Hematology, Robert Bosch Hospital,

Stuttgart, Germany

*correspondence

Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology

Auerbachstr. 112

70376 Stuttgart

Germany

Telephone: #49-711-8101-3730; Fax: #49-711-859295

Email: [email protected]

Running title: Oct-4-dependent constitutive Noxa levels determine Cisplatin sensitivity

Key words: Oct-4, Noxa, Testicular Germ Cell Tumors (TGCT), Cisplatin hypersensitivity

Disclosure of Potential Conflicts of Interest: The authors declare no potential conflicts of

interest.

Grant Support: The research leading to these results has received support from the Robert

Bosch Foundation (project 11.5.8000.0094.0 and O2A to MG, AW, MAD, MO, WEA, HK)

and the Federal State of Saxony Anhalt (FKZ 3646A/0907 to TM).

Word counts: abstract: 250 manuscript text: 3912

Research. on January 28, 2019. © 2013 American Association for Cancercancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 9, 2013; DOI: 10.1158/0008-5472.CAN-12-2876

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Abstract:

Testicular germ cell tumors (TGCT) are considered a paradigm of chemosensitive tumors.

Embryonal carcinoma (EC) cells represent the pluripotent entity of TGCT and are

characterized by expression of Oct-4, a key regulator of pluripotency and a determinant of

their inherent hypersensitivity to Cisplatin. However, the mechanisms underlying this Oct-4-

mediated sensitivity are poorly understood. We previously demonstrated that p53 is a major

player in Cisplatin hypersensitivity and therefore investigated whether Oct-4 may directly

affect p53 activity. Despite a significant decrease in sensitivity, depletion of Oct-4 did neither

alter Cisplatin-induced transactivation of p53 target genes nor its subcellular localization.

These data indicate that, rather than directly modulating p53 activity, Oct-4 provides a cellular

context that augments the proapoptotic activity of p53. Since mitochondrial priming by the

Bcl-2 family is a known determinant of chemosensitivity we compared the constitutive levels

of these proteins in Oct-4-positive and Oct-4-depleted cells. We identified Noxa as the only

Bcl-2 family protein to be highly correlated with Oct-4 status and Cisplatin sensitivity.

Compared to differentiated cells, constitutive Noxa levels were significantly higher in Oct-4-

positive cell lines and cancer patient samples. Furthermore, RNAi-mediated knockdown of

Oct-4 resulted in reduced Noxa transcript, in an almost complete loss of constitutive Noxa

protein and decreased Cisplatin hypersensitivity to a similar extent as did Noxa depletion.

In conclusion, our study indicates that Noxa is a central determinant of hypersensitivity to

Cisplatin. Oct-4-dependent high constitutive levels of this BH3-only protein prime EC cells to

undergo rapid and massive apoptosis in response to p53 activation.




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Introduction:

TGCT are the most frequent carcinomas in young male adults with a still increasing incidence

worldwide. Cisplatin-based chemotherapy cures the majority of patients even in advanced

stages due to the extraordinary sensitivity of TGCT to Cisplatin. Thus, TGCT are considered

as the paradigm of a chemosensitive tumor (1). Several mechanisms have been considered to

underlie this hypersensitivity, including the high apoptotic propensity of TGCT cells (2, 3).

The transcription factor Oct-4, a key regulator of pluripotency, is exclusively expressed in

cells of non-malignant pluripotent nature, i.e. embryonic stem cells (ESC) and germ cells, as

well as their malignant counterparts EC and seminoma, where it serves as a specific

malignancy marker (4, 5). Loss of Oct-4 causes a significant reduction of Cisplatin

hypersensitivity and was proposed to account for acquired Cisplatin resistance in refractory

tumors (6, 7). We and others have shown that Cisplatin hypersensitivity is highly dependent

on a functional p53 protein (8–10). The central role of this tumor suppressor was also

demonstrated by the finding that these cells are sensitive not only to Cisplatin but also to

DNA damage-independent p53 activators such as Nutlin-3 (10–12). Together, these results

indicate that hypersensitivity to p53 activation requires an Oct-4-mediated cellular context.

We now report that the low apoptotic threshold of Oct-4-positive TGCT cells is dictated by

the constitutive presence of high Noxa protein levels.




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Materials & Methods:

Cell culture: Cell lines from non-seminomatous germ cell tumors were analyzed: H12.1 (EC),

H12.5 (EC), 2102EP (EC), 1777NRpmet (differentiated state), 1411HP (differentiated state),

NTERA-2D1 (EC), H12.1ODM (in vitro differentiated derivative of H12.1). Origin and

cultivation procedures of these cell lines were described previously (6, 10). The cell lines

833K (EC), kindly provided by Prof. Andrews (University of Sheffield, UK), and GCT-72

(differentiated state), kindly provided by Prof. Pera (University of Southern California, USA),

were cultivated under same conditions. The cell line H12.1RA (in vitro differentiated

derivative of H12.1) was established by treatment of H12.1 with all-trans-retinoid acid during

cultivation in GCT-72-conditioned medium followed by cultivation under same condition as

the other cell lines (6, 10).

H460 (lung) and A2780 (ovary) cells were obtained from the NCI-60 cell line panel and

cultivated in RPMI-1640 with 10 % FCS and glutamine. PBMCs and Fibroblasts were

isolated and maintained as described previously (13, 14).

Reagents: Cisplatin and Nutlin-3 (Sigma) were used at a concentration of 10 µM, Etoposide

was used at 5 µM and Doxorubicin at 100 ng/ml. MG132 was used at a concentration of

1 µM.

Protein expression: Cells were lysed to obtain cellular protein according to standard

protocols. Western blot was performed using a SDS-PAGE Gel Electrophoresis system.

mRNA Expression: Total RNA was extracted and transcribed to cDNA according to standard

protocols. Expression was analyzed using BioMark HD System (Fluidigm) according to

manufacturer’s instructions. TaqMan assays were obtained from Applied Biosystems

(Supplementary Table 1). The relative amount of Noxa mRNA and pre-mRNA was

determined by a SYBR Green-based qPCR assay (7900HT Fast Real-Time PCR System,

Applied Biosystems). Contamination of RNA samples with genomic DNA was excluded by

DNase digestion followed by reverse transcription to cDNA. An intron-spanning primer pair




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was designed to detect Noxa mRNA levels whereas Noxa pre-mRNA was detected using

primers that span an exon-intron junction (for primer sequences see Supplementary Table 2).

Primers for detection of actin expression for normalization were kindly provided by Claudia

Kalla (Institute of Clinical Pharmacology, Stuttgart, Germany).

SRB microculture colorimetric assay: IC50 of Cisplatin and Nutlin-3 were determined as

described (15).

Subcellular fractionation: Cytoplasm and nuclei were separated using Mitochondria Isolation

Kit for Cultured Cells (Pierce) according to manufacturer’s instructions.

Apoptosis assay: Apoptosis was assessed by FITC-conjugated Annexin-V staining (16).

siRNA experiments: To silence Oct-4, Noxa, Puma and p53 we used siGenome SMARTpool

siRNA (Dharmacon, for sequences see Supplementary Table 3). Bim was silenced using Bim

siRNA from Santa Cruz. As a control we used siGenome Non-Targeting siRNA #1

(Dharmacon). Cells were transfected using DharmaFECT#3 (Dharmacon). 48 h after

transfection cells were treated according to requirements. To evaluate the efficacy of siRNA

silencing, protein lysates were prepared and analyzed by Western Blot.

Tissue samples: Tissue samples from TGCT patients were provided by the Department of

Urology, Eberhard Karls University of Tuebingen, Tuebingen, Germany. The local ethics

committee approved the collection of patient samples (315/2012BO2 and 396/2005V) and

informed consent was obtained from the patients. Specimen were frozen in liquid nitrogen

immediately after surgery and stored at -80°C for further use. To obtain protein lysates for

Western Blot, tissue was lysed using FastPrep-24 (MP Biomedicals) and sonication.

Statistics: Data are expressed as standard deviation of the mean (SD). Correlations are

expressed as Spearman rank correlation coefficient.




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Results:

Oct-4 depletion does not compromise the p53 response to Cisplatin

It has previously been demonstrated that Oct-4 protein levels are highly correlated with

Cisplatin hypersensitivity in TGCT cells (6). Therefore, we first tested whether Oct-4 is also

required for the more general sensitivity of TGCT to DNA damage-independent p53

activators (10). This was addressed by assessing the impact of Nutlin-3 on NTERA-2D1 cells

subjected to RNAi-mediated Oct-4 knockdown. As shown in Figure 1A, Oct-4 depletion

reduced Nutlin-3-induced apoptosis to a degree comparable to that observed in cells treated

with Cisplatin. These results suggest that efficient induction of apoptosis by p53 requires the

pluripotent, Oct-4-positive context of EC cells.

Consequently, we investigated possible differences in the accumulation and activation of p53

upon Cisplatin in the presence or absence of Oct-4. In response to cellular stress p53

activation leads to induction of a wide variety of target genes. Hence, we screened a panel of

46 bona fide p53 transcriptional targets ((17, 18); Supplementary Table 1) by qPCR in Oct-4-

positive and Oct-4-deprived NTERA-2D1 cells exposed to Cisplatin. A similar analysis was

also performed with the EC cell line 2102EP that retains a nullipotent phenotype, allowing us

to discriminate between differentiation-dependent and direct effects of Oct-4. Surprisingly,

despite its pronounced impact on apoptosis, Oct-4 depletion did not compromise the p53-

dependent transcriptional response to Cisplatin in both cell lines (Figure 1B). Hence,

induction of a p53-mediated transcriptional program is not the primary determinant of Oct-4-

mediated hypersensitivity.

Beyond its function as a transcription factor, p53 is capable of facilitating apoptosis in the

cytoplasm. Thus, we monitored the accumulation of p53 upon Cisplatin exposure in both

nucleus and cytoplasm. Notably, upon Cisplatin treatment, p53 accumulated to a similar

extent in both compartments (Figure 1C). Nevertheless, neither p53 accumulation nor its

localization was significantly influenced by knockdown of Oct-4 (Figure 1C), which further




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argues against a direct influence of Oct-4 on p53 activity. Together, these results indicate that

rather than directly altering p53 activity, Oct-4 provides an apoptosis-prone cellular context

that augments the proapoptotic efficacy of p53.

Oct-4 determines Cisplatin sensitivity by modulating the Bcl-2 family composition

Recently, it was demonstrated that the response to cytotoxic chemotherapy is dependent on

the relative composition of Bcl-2 family proteins which determines the apoptotic threshold

(19). We therefore investigated a possible correlation of individual Bcl-2 family proteins with

Oct-4 and Cisplatin sensitivity in NTERA-2D1 and 2102EP cells. To that end, we silenced

Oct-4 by RNAi-mediated knockdown and examined the protein levels of the proapoptotic

Bcl-2 family members Bax, Bak, Noxa, Puma, Bim and Bid, as well as the anti-apoptotic

members Bcl-2, Bcl-xL, Bcl-w and Mcl-1. Notably, Noxa was the only Bcl-2 family protein

which could be detected selectively in the presence of Oct-4 (Figure 2A). The fact that this

was observed in both pluripotent NTERA-2D1 and nullipotent 2102EP cells (Figure 2A)

suggests that the reduction of Noxa protein is not just a secondary effect of differentiation but

a direct consequence of Oct-4 depletion. Mcl-1, the major anti-apoptotic binding partner of

Noxa, was also reduced upon Oct-4 knockdown (Figure 2A). Since Noxa levels became

practically undetectable under such conditions (Figure 2A), the Noxa/Mcl-1 ratio was shifted

considerably in favor of Mcl-1, probably establishing a higher apoptotic threshold.

Knockdown of Oct-4 also reduced the levels of the BH3-only proteins Puma and Bim,

especially in 2102EP cells, although not as strongly as observed for Noxa (Figure 2A).

Notably, a role of Puma in hypersensitivity of pluripotent EC cells was previously proposed

(10). The protein levels of all other Bcl-2 family members analyzed did not exhibit

concomitant changes in these cell lines upon Oct-4 depletion (Figure 2A).

Next, we investigated the impact of the Oct-4-dependent Bcl-2 family proteins Noxa, Puma

and Bim on Cisplatin hypersensitivity. Therefore, we transfected NTERA-2D1 and 2102EP




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cells with the corresponding siRNAs and obtained a similar knockdown efficiency for each

siRNA (Figure 2B, right panel). Silencing of Noxa led to a marked decrease in sensitivity to

Cisplatin in both cell lines, comparable to that observed upon Oct-4 knockdown (Figure 2B,

left panel). A similar trend was also seen with Puma, although in NTERA-2D1 cells, silencing

of Puma did not reduce apoptosis as efficiently as did Noxa knockdown. In contrast, Bim

knockdown had only minor effects on Cisplatin hypersensitivity (Figure 2B, left panel). To

investigate possible cross-regulation between these pro-apoptotic Bcl-2 family members,

NTERA-2D1 cells were depleted of each protein and levels of the respective others were

quantified. We found no significant cross-regulation between Noxa, Puma and Bim

(Supplementary Figure 1).

It is of note that knockdown of either Oct-4 or Noxa reduced sensitivity of TGCT not only to

Cisplatin but also to other genotoxic agents such as Etoposide and Doxorubicin as well as to

the DNA damage-independent p53 activator Nutlin-3 (Supplementary Figure 2). These data

suggest that Oct-4 and Noxa are central mediators of the general hypersensitivity of TGCT.

Constitutive Noxa protein levels are closely correlated with Oct-4 and Cisplatin

hypersensitivity in a panel of TGCT cell lines and patient samples

Noxa and Puma are induced in response to several cellular stress stimuli. However, Noxa

protein was also found to be highly expressed in unstressed, undifferentiated EC cells but not

in cell lines derived from other tumor entities (10). We therefore investigated if constitutive

Noxa and Puma levels are maintained via transcriptional mechanisms in EC cells. In contrast

to Puma, Noxa transcript was significantly higher expressed in Oct-4-positive NTERA-2D1

and 2102EP cells when compared to Oct-4-negative lymphocytes, fibroblasts or cancer cell

lines (Figure 3A, left panels). A direct effect of Oct-4 on Noxa mRNA expression was

confirmed by Oct-4 knockdown experiments. Oct-4 depletion reduced Noxa transcript in

NTERA-2D1 and 2102EP cells to a level comparable to that observed in differentiated cells




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(Figure 3A, left panels). To investigate the possibility that Oct-4 may regulate mRNA stability

by post-transcriptional mechanisms, expression of Noxa pre-mRNA was examined and found

to be similarly dependent on Oct-4 as was mature Noxa mRNA (Supplementary Figure 3).

This result indicates that Oct-4 regulates Noxa gene transcription. Puma, on the other hand,

was only slightly reduced upon Oct-4 depletion (Figure 3A, left panels). Together, these data

suggest an Oct-4-dependent transcriptional mechanism resulting in high constitutive Noxa

protein levels.

The effect of Oct-4 on constitutive expression of Noxa in Oct-4-positive and Oct-4-depleted

EC cells was even more pronounced at the protein level (Figure 3A, right panel).

Furthermore, the absolute levels of Noxa protein in Oct-4-positive cells were 9.2fold

(NTERA-2D1 vs. PBMC) and 11.2fold (2102EP vs. PBMC) higher as compared to those of

stimulated hematopoietic cells (Figure 3A, right panel) which have been shown to exhibit

constitutive Noxa protein (20), whereas the difference in transcript levels was only 2.3fold

(NTERA-2D1 vs. PBMC) and 1.3fold (2102EP vs. PBMC) (Figure 3A, left panel). It is

therefore likely that Oct-4 also modulates Noxa protein translation and/or stability by a yet

unknown mechanism. In contrast to Noxa, Puma levels were found to be higher only in one of

the two Oct-4-positive cell lines when compared to differentiated cells (Figure 3A, right

panel).

To further investigate the correlation of Oct-4 and Cisplatin sensitivity with Noxa and Puma,

we analyzed constitutive protein levels of both BH3-only proteins (Figure 3B, upper left

panel) together with Cisplatin IC50 values (Figure 3B, lower left panel) in an extended TGCT

cell line panel consisting of 5 Oct-4-negative and 5 Oct-4-positive cell lines. We confirmed a

correlation of Cisplatin sensitivity with Oct-4 (Spearman r=-0.842, p=0.004; Figure 3B, lower

right panels). Importantly, Noxa was significantly correlated with Oct-4 expression

(Spearman r=0.891, p=0.001; Figure 3B, upper right panels) as well as with Cisplatin

sensitivity (Spearman r=-0.806, p=0.007; Figure 3B, lower right panels). On the other hand,




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while Puma protein levels were also positively correlated with Oct-4 status, this correlation

was weaker than for Noxa (Spearman r=0.685, p=0.035; Figure 3B, upper right panel), and no

significant correlation was seen with Cisplatin sensitivity (Spearman r=-0.527, p>0.05; Figure

3B, lower right panel). Notably, similar results were observed with Nutlin-3 (Supplementary

Figure 4). These data further support the conjecture that Oct-4 lowers the apoptotic threshold

in TGCT primarily by maintaining high constitutive Noxa protein levels.

In addition, we sought to extend our in vitro findings to cancer patient samples. Indeed, we

could confirm a close correlation between Oct-4 and Noxa protein levels in primary tissue

samples derived from 5 ECs and 8 seminomas (Spearman r=0.832; p<0.0001; Figure 3C).

Accumulation of both Noxa and p53 is required to induce cell death in Oct-4-depleted

TGCT cells

We have previously shown that accumulation of p53 is a central determinant in TGCT

hypersensitivity (10). In the present study, we established that Oct-4-positive TGCT cells are

characterized by high constitutive Noxa protein levels, which seems to be a requirement for

their p53-mediated hypersensitivity. Therefore, in Oct-4-depleted cells, pharmacological

induction of both Noxa and p53 protein should result in cell death. In order to test this

hypothesis, NTERA-2D1 and 2102EP cells, respectively, were depleted of Oct-4 by RNAi-

mediated gene silencing (Figure 4, upper panels). In contrast to Cisplatin or Nutlin-3 (Figure

1A), treatment with the proteasome inhibitor MG132 resulted in efficient induction of

apoptosis in both Oct-4-depleted cell lines (Figure 4, lower panels). Importantly, MG132

treatment induced simultaneous accumulation of Noxa and p53 protein (Figure 4, middle

panels). Prevention of accumulation of either protein by RNAi-mediated knockdown led to

increased survival upon MG132 treatment in NTERA-2D1 and 2102EP cells (Figure 4, lower

panels). These data further demonstrate that both Noxa and p53 protein are required to induce

apoptosis in TGCT cells.




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Discussion:

Cisplatin-based chemotherapy has been a successful cure for TGCT for decades. A variety of

molecular determinants of these neoplasms were proposed to account for their exquisite

sensitivity to Cisplatin such as a reduced DNA repair capacity, a deregulated G1/S transition

or marginal p21 expression (2; 3; 21). Much of the research done in this field has focused on

p53 which is almost exclusively expressed in its wild type conformation in TGCT (22). We

recently demonstrated a central role for p53 in TGCT hypersensitivity to Cisplatin and

suggested an intrinsic sensitivity of these tumors to activation of p53 which leads to robust

induction of apoptosis in this specific cellular context (10). In the present study we found that

this sensitivity is dependent on the presence of Oct-4, although p53 activation is not directly

influenced by this mediator of pluripotency. Rather, by maintaining high constitutive Noxa

protein levels, Oct-4 provides a cellular context that primes TGCT cells to p53-dependent

apoptosis.

TGCT derive from intrinsically apoptosis-prone tissue. Therefore, their unique responsiveness

to chemotherapy might be due to inherent biological characteristics (23). An intrinsic

molecular determinant of the pluripotent compartment of TGCT is the expression of

regulators of pluripotency such as Oct-4. Recent work related loss of Oct-4 to an increase in

Cisplatin resistance (6). In the present study we show that Oct-4 depletion not only protected

cells from Cisplatin-induced cell death but also led to a reduction of the apoptotic response to

other genotoxic and non-genotoxic p53 inducers (Figure 1A and Supplementary Figure 2).

These data imply that Oct-4 mediates the general intrinsic hypersensitivity to p53 activators in

pluripotent EC cells. Several studies have attributed the increased susceptibility to apoptosis

to overexpression of p53 (24; 25). However, we and others could show that high p53 protein

levels are not a characteristic of TGCT (10; 26). Here we demonstrate that Oct-4 had no

impact on constitutive p53 levels or on its accumulation upon Cisplatin treatment (Figure 1C).

Furthermore, Oct-4 depletion did not influence the Cisplatin-induced transactivation of 46




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representative bona fide p53 target genes (Figure 1B). These data indicate that the

transcriptional p53 response is not substantially altered in Oct-4-positive hypersensitive

TGCT cells. In addition to its capability to launch the apoptotic program through

transactivation of proapoptotic target genes, p53 can directly induce apoptosis in the

cytoplasm by interacting with Bcl-2 family proteins. Subcellular fractionation revealed that

besides an accumulation of p53 in the nucleus, Cisplatin treatment resulted in a similar

accumulation in the cytoplasm (Figure 1C), suggesting that p53 could function independent of

its transcriptional activity to launch the apoptotic program in TGCT; this was also proposed as

a proapoptotic mechanism in ESC (27). However, Oct-4 depletion did not significantly

influence subcellular distribution of p53 (Figure 1C). Together, these data demonstrate that

neither transcriptional nor cytoplasmic functions of p53 are dependent on the presence of Oct-

4 in TGCT cells. Since Oct-4 is central for Cisplatin sensitivity, it must provide a cellular

context allowing an effective proapoptotic p53 response.

Recently, Letai and co-workers established a model to explain and predict chemotherapeutic

response by mitochondrial priming (19). According to this hypothesis, occupation of anti-

apoptotic Bcl-2 family proteins by their proapoptotic counterparts determines the proximity of

a cell to the threshold of apoptosis (28). Our data demonstrate that the presence of Oct-4 in

pluripotent EC cells dictates a Bcl-2 family profile dominated by high constitutive Noxa

protein levels which augments the cell’s commitment to apoptosis. We found that Noxa

protein levels are tightly correlated with both Oct-4 status and Cisplatin sensitivity in a panel

of cell lines and also in samples from primary TGCT.

The relative levels of other Bcl-2 proteins have also previously been proposed to determine

hypersensitivity in TGCT. Several studies suggested that the enhanced sensitivity to

genotoxic agents is dependent on a high Bax/Bcl-2 ratio (29) and low levels of Bcl-2 and Bcl-

xL (30; 31). A chemoprotective role of Bcl-xL in these tumors has also been proposed (32).

However, we found no correlation between the protein levels of Bcl-2, Bax and Bak and Oct-




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4 status (Figure 2A). Indeed, recent data demonstrated that Bcl-2, Bcl-xL, Bax or Bak levels

were similar among TGCT cell lines with differential Cisplatin sensitivity (15; 33). Together,

these data indicate that Noxa is the only Bcl-2 family protein that is tightly correlated to both

Oct-4 status and Cisplatin sensitivity, suggesting that constitutive Noxa protein is a key

determinant of the exclusive sensitivity of Oct-4 positive EC cells to Cisplatin. Moreover, we

show that the general sensitivity of TGCT to genotoxic and non-genotoxic agents is also

dependent on Noxa (Supplementary Figure 2). The prominent role of Noxa for TGCT

hypersensitivity is further supported by the finding that Noxa protein was found to be

correlated with good clinical prognosis in patients with EC (34).

Noxa was shown to preferentially bind to Mcl-1 and A1 (35). The apoptotic potential of this

BH3-only protein when overexpressed was therefore suggested to be quite variable due to its

inability to antagonize Bcl-2 and Bcl-xL (36). However, as mentioned above, TGCT exhibit

low expression of Bcl-2 and Bcl-xL (30; 31), potentially providing an ideal premise for an

increased apoptotic potential of Noxa overexpression. Moreover, our results demonstrate a

considerable shift of the Noxa/Mcl-1 ratio in favor of Mcl-1 upon Oct-4 depletion (Figure

2A), enforcing a higher apoptotic threshold. A similar dependence on the Noxa/Mcl-1 balance

was shown for HeLa and pancreatic cancer cells treated with camptothecin (37; 38). It is of

note that Noxa was recently shown to be capable of binding Bcl-xL upon DNA damage and

thus facilitate apoptosis induction (39), a conceivable mechanism that broadens Noxa’s

proapoptotic capacity. In addition, it seems most likely that while Noxa inhibits the anti-

apoptotic functions of Mcl-1, a robust induction of other BH3-only proteins by p53 sequesters

Bcl-xL and activates Bax and Bak. This hypothesis is supported by the fact that Puma was

found to be as important as was Noxa for the hypersensitivity to Cisplatin (Figure 2B).

However, constitutive Puma protein levels could not to be linked to Cisplatin sensitivity

(Figure 3B). These results indicate that Puma exerts its prominent proapoptotic function in

TGCT cells only upon induction by p53 as previously described for other cell types (40).




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Due to the established function of Oct-4 as a transcription factor it is tempting to speculate

that transcription of the Noxa gene is directly promoted by Oct-4. Interestingly, in a recent

study, an Oct-4 binding site was mapped within 8 kb of the Noxa promoter (41). Moreover,

Noxa mRNA was shown to be upregulated in ESCs (42) and its expression was found to be

correlated with Oct-4 in this cell type (43; 44). The finding that Noxa pre-mRNA was

downregulated upon Oct-4 depletion to a similar extent as the corresponding mature mRNA

(Supplementary Figure 3) suggests that Oct-4 may indeed directly regulate the transcription of

the Noxa gene. However, since Oct-4 is involved in the transactivation of many genes,

including other transcription factors (44), it remains possible that this regulation might as well

include indirect effects.

Noxa was commonly seen as a transcriptionally activated stress-responsive gene (36) rather

than a protein present at high levels in unstressed cells. However, upregulated Noxa protein

levels were reported to restrain lymphocyte expansion and can be triggered by glucose

deprivation (20). Together with the high constitutive protein levels reported in the present

work (Figure 3A), these data suggest an additional regulation at the protein level, e.g. through

the ubiquitin-proteasome system (45; 46). Since Noxa protein regulation by Oct-4 was

demonstrated to be more pronounced when compared to regulation at the mRNA level (Figure

3A), altered Noxa protein stability cannot be excluded as a possible additional explanation for

high constitutive Noxa protein levels.

We established that loss of high constitutive Noxa protein levels in Oct-4-depleted cells

causes resistance to Cisplatin and Nutlin-3. However, these cells were still sensitive to

proteasomal inhibition by MG132 (Figure 4). Importantly, MG132 resulted in stabilization of

Noxa protein reaching levels comparable to that observed in Oct-4-positive cells.

Concomitantly, p53 was stabilized upon proteasomal inhibition. RNAi-mediated knockdown

of Noxa or p53, respectively, rescued Oct-4-depleted cells from cell death upon MG132

treatment (Figure 4). These data further indicate that hypersensitivity of TGCT is dependent




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on both high levels of Noxa and induction of p53.

We propose that, under basal non-stressed conditions, EC cells may buffer the high apoptotic

pressure driven by high constitutive Noxa levels by a parallel increase in its anti-apoptotic

binding partner Mcl-1. Upon cellular stress, p53-mediated induction of additional BH3-only

proteins such as PUMA (10) and concomitant Mcl-1 degradation (47) may then shift the

balance and lead to a rapid and robust induction of apoptosis.

In conclusion, our present study demonstrates that Oct-4-dependent high constitutive Noxa

levels determine Cisplatin hypersensitivity in TGCT. Despite the predominant role of p53 in

TGCT hypersensitivity, Oct-4 does not directly influence p53 activation but rather provides

an extremely low apoptotic threshold by maintaining high constitutive Noxa protein levels.

Therefore, in response to cellular stress, accumulation of p53 triggers a rapid and massive

induction of apoptosis.




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Acknowledgements:

We are grateful to Prof. Hans-Joachim Schmoll (Martin-Luther-University of Halle-

Wittenberg, Germany) for the initiation of the collaboration between Stuttgart and Halle as

well as Tabea Lieberich (Stuttgart) and Franziska Reipsch (Halle) for technical assistance.

Grant Support:

This work was financially supported by the Robert Bosch Foundation (project

11.5.8000.0094.0 and O2A) and the Federal State of Saxony Anhalt (FKZ 3646A/0907).

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Figure legends:

Figure 1: Oct-4 depletion does not compromise the p53 activity in response to Cisplatin.

Cells were transfected with indicated siRNA and incubated for 48 h prior to Cisplatin or

Nutlin-3 treatment. Controls reflect cells treated with a non-targeting siRNA. (A) p53

hypersensitivity is dependent on Oct-4 in NTERA-2D1 cells. Left panel: verification of Oct-4

knockdown by Western Blot. Right panels: quantification of survival upon Cisplatin by flow

cytometry. Cells were stained with Annexin-V/FITC and PI upon treatment with Cisplatin

(16 h) or Nutlin-3 (24 h). Graph reflects means ±SD of survival upon Cisplatin or Nutlin-3

from 3 independent experiments. (B) Oct-4 depletion does not influence the transcriptional

response of p53 to Cisplatin. Cells were incubated with or without Cisplatin for 6 h. To

evaluate p53 transactivation activity upon Cisplatin treatment in dependency of Oct-4,

transcripts of 46 bona fide p53 target genes were quantified by qPCR. For this, RNA was

isolated, transcribed to cDNA and applied to qPCR analysis using the primers listed in

Supplementary Table 1. Graph shows means of fold changes upon Cisplatin of detected

transcripts from 3 independent experiments, dots depict fold change values of each transcript

in cells pre-treated with control (X axis) or Oct-4 siRNA (Y axis). (C) Oct-4 depletion does

not affect p53 accumulation and localization. Cells were treated with Cisplatin for indicated

times and subjected to subcellular fractionation. p53 protein was analyzed by Western Blot

along with tubulin, representing the cytoplasmic compartment and TBP representing the

nuclear compartment. Graphs reflect densitometric quantification of p53 protein levels

normalized to tubulin or TBP, respectively.

Figure 2: Composition of Bcl-2 family proteins is dependent on Oct-4 and determines

Cisplatin hypersensitivity. Cells were transfected with indicated siRNA and incubated for

48 h. Controls reflect cells treated with a non-targeting siRNA. (A) Noxa, Puma and Bim

expression is dependent on Oct-4. Transfected cells were lysed and subjected to Western Blot




23

analysis to determine protein levels of Bcl-2 family members. (B) Silencing of Noxa and

Puma but not Bim leads to a marked reduction in Cisplatin sensitivity comparable to that

observed upon Oct-4 knockdown. Left panel: quantification of survival upon Cisplatin by

flow cytometry. Transfected cells were treated with Cisplatin for 16 h (NTERA-2D1) or 12 h

(2102EP) and stained with Annexin-V/FITC and PI. Graph reflects means ±SD of survival

upon Cisplatin from 3 independent experiments. Right panel: verification of indicated

knockdowns by Western Blot

Figure 3: Noxa protein levels are tightly correlated with Oct-4 status and Cisplatin

sensitivity in TGCT cell lines and patient samples. (A) Oct-4-positive EC cells express

high constitutive Noxa mRNA and protein levels. NTERA-2D1 and 2102EP cells were

transfected with indicated siRNA and incubated for 48 h prior to analysis. Controls reflect cell

treated with a non-targeting siRNA. The other cells were left untreated and used as a control.

Left panels: RNA was isolated, transcribed to cDNA and applied to qPCR analysis. Graph

reflects means ±SD of Noxa and Puma transcript from 3 independent experiments. Right

panel: cells were harvested and lysed to analyze Oct-4, Noxa and Puma protein levels by

Western Blot. (B) Constitutive Noxa protein levels are highly correlated with Oct-4 and

Cisplatin hypersensitivity in TGCT cell lines. Upper left panel: cells were harvested and lysed

to analyze Oct-4, Noxa and Puma protein levels by Western Blot. Lower left panel: IC50 of

Cisplatin were determined by SRB microculture colorimetric assay. Graph reflects means

±SD from 3 independent experiments. Upper right panels: dot blots reflect correlation of Oct-

4 with Noxa or Puma protein as determined by densitometric analysis. Lower right panels: dot

blots reflect correlation of Oct-4, Noxa and Puma protein levels with IC50 of Cisplatin. (C)

Noxa and Oct-4 protein levels are highly correlated in cancer patient samples. Whole protein

lysates were obtained from frozen patient samples of 5 EC, 8 seminoma and a lung tumor and

applied to Western Blot analysis to determine Oct-4 and Noxa protein levels. Lysates from




24

NTERA-2D1 and 1777NRpmet cell lines served as positive or negative control, respectively.

Graph reflects correlation of Oct-4 and Noxa protein levels.

Figure 4: Accumulation of both Noxa and p53 is required to induce cell death in Oct-4-

depleted TGCT cells

Cells were transfected with indicated siRNAs and incubated for 48 h before MG132

treatment. Controls reflect cells treated with a non-targeting siRNA. Efficient gene silencing

was verified by Western Blot analysis (upper and middle panels). Transfected cells were

treated with MG132 for 20 h (NTERA-2D1) or 16 h (2102EP), protein lysates were prepared

and subjected to Western Blot analysis to determine protein levels of p53 and Noxa (middle

panels) and stained with Annexin-V/FITC and PI for cell death analysis by flow cytometry

(lower panels). Graph reflects means ±SD of survival upon MG132 from 3 independent

experiments.




Published OnlineFirst January 9, 2013.Cancer Res Matthias Gutekunst, Thomas Mueller, Andrea Weilbacher, et al. determined by high constitutive Noxa levels mediated by Oct4Cisplatin hypersensitivity of testicular germ cell tumors is

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