Prognostic relevance of caspase 8 -652 6N InsDel and Asp302His polymorphisms for breast cancer

Programmed cell death, also referred to as apoptosis, physiologically occurs in multicellular organisms and its aberration has important implications in cancer biology. Among the death receptor signaling pathway, the initiator Caspase 8, a 55 kDa cysteine protease, plays an important role in intrinsic and extrinsic apoptosis induction. In terms of the intrinsic apoptosis pathway, caspase-8 activates the death inducing signaling complex (DISC), which in turn induces downstream effector caspase-3, finally resulting in apoptosis [1, 2]. Among the extrinsic pathway, caspase-8 cleaves the Bcl-2 related protein Bid, which in turn induces cytochrome c release from mitochondria and caspase-3 activation, likewise resulting in apoptosis [1, 2].

Nearly at the same time, two CASP8 polymorphisms, namely CASP8 -652 AGTAAG InsDel (-652 6N Del, rs3834129) and CASP8 Asp302His (rs1045485) were described in key publications [3, 4]. The non-coding CASP8 -652 6N InsDel polymorphism, a functional 6-bp deletion located in the promoter region of the CASP8 gene, has been associated with reduced CASP8 mRNA expression and concomitantly impaired caspase-8 activity and reduced “activation induced cell death” (AICD) in stimulated T-lymphocytes [3]. The second polymorphism, CASP8 Asp302His, is located in the coding region of caspase 8 and results in aspartic acid to histidine substitution (Fig. 1a). Although instructive data on the functionality of this polymorphism are missing, it was hypothesized that the Asp302His change could likewise impair caspase-8 function, possibly by negatively affecting its auto processing or its catalytic activity [5].

Given that the above mentioned caspase-8 variants were suggested to impair caspase-8 function and to interfere with cell death of T-lymphocytes, which has essential consequences on immune surveillance of malignancies, a variety of studies on different cancer entities investigated, whether these polymorphisms may influence cancer susceptibility. These studies, also including meta-analyses, were primarily performed on breast cancer, but also on other cancer entities, such as colorectal-, ovarian- or gastric cancer. The majority of investigators observed an association of the CASP8 -652 6N Del or the CASP8 Asp302His variant with reduced cancer susceptibility [3, 5‐12]. However these insights were not entirely conclusive, since some other studies failed to confirm a significant cancer protective effect of these caspase 8 variants [13‐17]. Furthermore, since they are in linkage disequilibrium, it remains unclear, whether both or only one of these polymorphisms has an impact on cancer risk.

Beside the extensively studied role of the above mentioned caspase 8 polymorphisms in cancer susceptibility, recent approaches also started to investigate, whether these polymorphisms also influence the outcome of cancer in patients with already existing disease. In this context, recent pilot investigations reported a negative prognostic impact for the CASP8 -652 Del allele or the CASP8 302His allele for patients with colon cancer or neuroblastoma, respectively [18, 19]. However, although breast cancer was primarily adressed by recent CASP8 -652 6N Del and CASP8 Asp302His susceptibility studies, surprisingly, prognostic relevance of these caspase 8 polymorphisms has not been investigated in breast cancer so far.

Hypothesizing that a functional polymorphism, which is involved in cancer susceptibility, is also likely to influence the outcome of a given cancer, we took advantage of a historic breast cancer cohort of clinically documented primary breast cancer patients and investigated prognostic significance of CASP8 -652 6N Ins/Del and CASP8 Asp302His in terms of an exploratory analysis.

In the present study, we investigated clinical relevance of two selected caspase 8 polymorphisms, namely CASP8 -652 6N InsDel and Asp302His, for patients with primary breast cancer. Intriguingly, in contrast to previous molecular epidemiological findings [4, 7, 26], describing an association of the CASP8 -652 6N deletion variant or the CASP8 Asp302His variant with decreased breast cancer susceptibility, we showed that these caspase 8 variants have a negative and allele-dose dependent prognostic impact on breast cancer overall survival. Moreover, we confirmed that clinical informativity of both polymorphisms is independent from each other and that these polymorphisms have, besides, an allele-dose dependent additive influence on OS.

Considering that activation induced cell-death of antitumor T-lymphocytes was shown to be involved into immune surveillance of cancer cells [3, 27], the functionally underlying death receptor-pathway emerged as an interesting target to seek novel candidate polymorphisms for cancer susceptibility. In this regard, CASP8 -652 6N InsDel has already been shown to have an influence on caspase 8 mRNA expression in stimulated T-lymphocytes, by disrupting a Specifity Protein 1 (Sp1) transcription factor binding site in the caspase 8 promoter region and, consequently, by functionally interfering with caspase 8 transcription [3]. Complementarily, we reported that breast cancer tissues of patients, bearing a homozygous -652 6N Del variant, displayed lowest relative CASP8 expression, which corroborates that this effect is similarly applicable for malignant breast cancer tissue. This finding was not necessarily anticipated. Although Sp1 sites are typically believed to represent constitutive promoter elements for basal transcription, recent studies showed that, especially in cancer, the Sp1 transcription factor can be strongly regulated by post-translational modifications that positively or negatively affect its activity on a wide array of genes [28, 29].

The CASP8 Asp302His variant, especially in form the His/His genotype, was a rare event in our study population, which is in accordance to previous independent observations [5]. Similarly, CASP8 302His variant was shown to confer reduced breast cancer susceptibility in an allele-dose dependent manner [4]. However, given that the functional effect of this polymorphism is largely unknown, the underlying effect on caspase 8 functionality and tumor progression is less clear. Nevertheless, aspartate 302 was shown to be conserved between mouse and human caspase 8 and is located on the protein surface. Therefore, it has already been hypothesized that the Asp302His change could likewise impair caspase 8 function, possibly by negatively affecting its auto processing capability or its catalytic activity [5]. However, albeit highly interesting, a detailed functional analysis of the polymorphisms, investigated herein, is beyond the objective of our present investigation.

As our key finding, we described both caspase 8 variants as a negative prognostic factor for breast cancer. At first glance, our finding may appear counterintuitive, since the CASP8 -652 InsDel or DelDel genotype has previously been associated with impaired immune surveillance of cancer cells and concomitantly decreased breast cancer susceptibility [3, 6, 7, 9]. However, our data are in accordance with a recent pilot investigation, reporting, albeit with borderline statistical significance, a negative prognostic impact of the CASP8 -652 6N Del allele for colorectal cancer patients [19]. Complementarily, in a very recent approach, CASP8 302His was associated with worse overall and event-free survival in patients with MYCN-amplified neuroblastoma tumors [18]. Apoptosis, with caspase 8 as one of its key regulators, is not only involved in AICD of antitumor T lymphocytes, but also constitutes an important defense mechanism against hyperproliferation and malignancy, which can be induced by e.g. DNA damage [30, 31]. Therefore, the acquired ability to resist apoptotic stimuli, caused by aberrations in key apoptotic pathways, is an essential characteristic for cells to become malignant and to develop a metastatic phenotype [31, 32]. Moreover, the death receptor pathway, with caspase 8 as key regulator, was shown to be de-regulated in malignant tumor cells, such as in breast cancer cells [33, 34], in dysplastic cells or in carcinomas in situ [35]. Therefore, in breast cancer, we hypothesize an ambiguous tumor biological relevance and a context dependent clinical informativity for CASP8 -652 InsDel: In healthy individuals, impaired caspase 8 activity and reduced apoptotic capacity seems to have primarily influence on immune escape (in terms of AICD) and obviously decreases breast cancer susceptibility. Contrarily, in patients with diagnosis of primary breast cancer, in which malignant cells have already accomplished immune escape, the so far protective effect of increased immune surveillance becomes obviously inferior. In this situation, increased resistance of tumor cells to apoptotic stimuli, conferred by the CASP8 InsDel or DelDel genotype, turns the balance and becomes a potentially pro-tumorigenic and negative prognostic factor, resulting in decreased OS. However, this concept is not necessarily transferable to other cancer entities, since the CASP8 InsDel and DelDel genotypes were contrarily described as favorable prognostic indicators for gastric cancer patients [36]. However, in the light of the complexity of death receptor signaling, these data are not surprising. It is known that the magnitude of pro-death events (such as caspase activation) and pro-survival events (such as Nuclear Factor (NF)-kB) may vary not only from one cell type to the next but also among individual cells of the same type due to intrinsic and extrinsic factors. Therefore, death receptor ligands may simultaneously activate opposing signals via the same receptors [37].

Moreover, albeit being in linkage disequilibrium, CASP8 -652 6N InsDel and CASP8 Asp302His showed an independent and additive prognostic impact on OS. Therefore, we may hypothesize that both polymorphisms may account for an additive or even synergistic effect on total caspase 8 activity in breast cancer cells.

To the best of our knowledge, this is the first report describing a prognostic impact of both CASP8 -652 6N InsDel and CASP8 Asp302His for breast cancer patients. However, considering the limited number of patients in our study, our statistically verified conclusions should be handled with care and our explorative approach needs to be clinically validated in larger and independent patient cohorts. Nevertheless, we performed multivariate analysis to reduce the risk of accidental findings, which revealed that our results remained significant after correction for the covariates. Moreover, two completely independent outcome studies in neuroblastoma and colon cancer showed a comparable effect of -652 6N InsDel and Asp302His, respectively [18, 19]. This provides further evidence for a real functional effect of these polymorphisms. Therefore, further larger (prospective) studies should be initiated to validate clinical utility of these two CASP8 polymorphisms for breast cancer and also to a broad scope of other malignancies.

AICD, activation induced cell death; CI, confidence intervall; DISC, death inducing signalling complex; HR, hazard ratio; NF, nuclear factor; OS, overall survival; PCR, polymerase chain reaction; Sp1, specifity protein 1