Individuals with heritable cancer syndromes, such as CS/CSL, who do not carry mutations in the known predisposition genes, bring challenges to molecular diagnosis, predictive testing of family members, genetic counseling and preventive medical management. Identifying additional cancer susceptibility genes for CS/CSL would improve and facilitate the gene-specific personalized medical care. We have recently uncovered an alternative mechanism, germline hypermethylation of the tumor suppressor gene KLLN
(encoding KILLIN), accounting for one-third of PTEN
mutation-negative CS/CSL (21
). Germline KLLN
hypermethylation is associated with increased risks of breast and renal cancers over those with PTEN
mutations. Our current study not only validates the previous pilot observations that SDHx
variants occur in ~8% of PTEN
mutation-negative CS/CSL patients, but also validates the elevated breast and thyroid cancer frequencies over those with PTEN
, together with PTEN
, may form a panel of predisposition genes considered for genetic testing for CS/CSL, perhaps prioritized based on the individual patient's clinical phenotype at presentation and their family history. For example, if a CS/CSL individual has papillary thyroid carcinoma, then SDHx
testing should be considered first.
As with all inherited cancer syndromes to date, while we can counsel increased prevalence of specific cancers, we cannot predict which subset of those with PTEN mutations will develop each component cancer. Here we have found that 6% of PTEN mutation/variant-positive CS/CSL patients were also found to have germline SDHx variants, and the presence of SDHx variants appear to further modify PTEN mutation cancer risks over those of PTEN mutation in isolation. Because this is the first observation of SDHx variation modifying PTEN-related breast cancer risk, this will need to be independently validated before translation into the routine clinical armamentarium.
Among all 11 different SDHx
variants we detected, there are 4 novel variants not previously reported in either NCBI SNP database (http://www.ncbi.nlm.nih.gov/snp/
) or SDHx
mutation database (http://chromium.liacs.nl/lovd_sdh
Arg27Gly, Asn120Ser, Ala215Thr and SDHC
Ala66Val detected represents the first SDHC
variant in a CSL patient, a 54-year-old patient with invasive breast cancer, follicular thyroid cancer, uterine fibroids and skin hemangioma. Patients with SDHB
Arg27Gly, Asn120Ser or Ala215Thr variants all presented with invasive breast carcinoma and either malignant (papillary thyroid cancer) or benign thyroid lesions. The other patient with SDHD
His145Asn (rs121908984) variant first reported in our previous pilot study presented with both breast carcinoma and RCC. The fact that carriers of these variants all presented with malignant breast carcinoma suggest physiologic relevance. SDHB
Ala3Gly (rs11203289) and His57Arg (rs35962811) were reported in dbSNP but only in African American population, while our samples are derived from white individuals of European ancestry. The most frequent variants SDHB
Ser163Pro (rs33927012), SDHD
Gly12Ser (rs34677591) and SDHD
His50Arg (rs11214077) we seen in our CS/CSL individuals have also been reported in the database. Although these relatively common (1–5% frequency) variants were computationally predicted to be functionally benign (22
), our experimental data provide molecular evidence that they could have functional impact in cellular signaling regulation as well. The reason why bioinformatic analysis of prediction fails in SDHx
genes is because they are extremely well conserved throughout species (23
). With enormous numbers of variations uncovered by whole genome sequencing, it is essential to realize that functional analysis and clinical correlations must be performed to define the true pathogenic effect of DNA variations (24
), as we have done in the current study.
Changes in the mitochondrial metabolism have long been linked to cancer, known as the Warburg effect (25
). The mechanism(s) of disruption of mitochondrial function leading to neoplasia remain unclear. Succinate, the substrate of SDH, may function as a second messenger between the mitochondria (energy production body) and cytosol. Accumulation of succinate due to SDHx
mutations inhibit the prolyl-hydroxylase enzyme and contributes to stabilization of HIF1α in turn promoting transcription of genes containing hypoxic response elements believed to promote cancer (16
). Our data suggest that the hyperactivation of the HIF pathway is indeed involved in our CS/CSL development. It has been reported that the HIF signaling pathway can also be regulated by AKT and mTOR signaling downstream of PTEN (26
). It is notable that HIF1α expression in PTEN
mutation-positive samples versus SDHx
variant-only samples are different. We observed no obvious accumulation of HIF1α protein with PTEN
mutation, consistent with other reports that loss of function of PTEN most likely increases the HIF1α transactivation function by preventing HIF1α binding to HIF inhibitory factor (FIH), instead of stabilizing HIF1α protein expression (27
). This contrasts with our observation of SDHx
-related hyperactivation of HIF1α. This differential involvement of HIF signaling may partially explain the different levels of predisposition to breast, thyroid and/or renal carcinomas in PTEN
mutation positive or SDHx
variant positive, versus both.
As ‘energy factories’ through oxidative phosphorylation, mitochondria make endogenous ROS as byproducts of normal respiration. Under normal physiological conditions, complex II is not considered to be a site for ROS generation. However, the structure–function studies of bacterial Sdh and fumarate dehydrogenase indicated ROS production mainly resides at the FAD site of these enzymes (28
). The succinate-driven reverse-electron transfer through complex I was shown as another mechanism to yield the highest rates of H2
in isolated mitochondria (29
). Therefore, accumulated succinate from dysfunctional SDH complex not only could serve as a second messenger activating HIF signaling (as discussed above), but also could drive the intracellular ROS generation. The extra ROS stress has been reported with SDHx
), and also observed in our SDHx
variant carrier cells in general. Certain PTEN
mutation-positive cells do show elevated ROS (31
), which accounts for the additive increased ROS in samples with both PTEN
mutation and SDHx
variant. Excessive oxidation of DNA by ROS could be a major cause of DNA damage and genetic instability (32
), which leads to accumulation of mutations and deletions that eventually contribute to carcinogenesis.
, as a stress-induced tumor suppressor gene, plays important roles in programmed cell death (34
). The reduction in basal p53 levels in SDHx
variant carriers likely explains the corresponding escape of cell death, which otherwise should be regulated by the p53 pathway under normal cellular responses. A recent study also suggested that mitochondrial respiration deficiency may impair p53 expression and function (35
), which is consistent with our observation. We further investigated the underlying mechanism of this p53 impairment caused by SDHx
variation. The tightly regulated p53 protein levels are achieved via both ubiquitin-mediated degradation in 26S proteasomes based on interactions between p53 and Mdm2 (36
), and an ubiquitin-independent degradation in 20S proteasomes (18
). In the latter, p53 is degraded by 20S proteasomes through direct binding to NQO1. What is intriguing to us is that the function of NQO1 is tightly regulated by mitochondrial redox metabolites. As a key player here, NQO1 is a FAD-containing protein and its activity is highly dependent on intracellular NAD+
/NADH, also the product and substrate, respectively, of mitochondrial complex I (38
). Our data suggest that in our SDHx
variant cells, it is not the loss of absolute NQO1 expression but more specifically the loss of functional NQO1 binding to p53 that results in the reduction in p53 protein. The observation that increased FAD and lowered NAD+ concentrations in SDHx
variant cells validates the observation of inhibited NQO1 function. Presence of excessive FAD results in activated signaling down the AKT and MAPK pathways, mimicking PTEN dysfunction. Therefore, our findings reveal a novel mechanism that mitochondrial metabolites regulate cellular signaling, hence mechanistically linking mitochondrial dysfunction to tumorigenesis.
In conclusion, our genetic analyses revealed that germline SDHx variants are associated with elevated cancer risks in CS/CSL individuals, both alone and synergistically with germline PTEN mutations. Our functional data suggest that disruption of complex II could lead to mitochondrial metabolite imbalance, and in turn cause the stabilization of HIF1α, loss of baseline p53 levels and is at least partially responsible for ROS generation. The cross-talk between SDH and PTEN results in a multi-signaling cascade that contributes to tumorigenesis (Fig. ). As we are learning more about the heterogeneity of tumor formation, it is not surprising to observe multiple pathways crosstalk, contribute alone or simultaneously to the final outcome of differential organ-specific carcinogenesis. Together, our findings suggest the importance of considering SDHx as candidate predisposing genes and as candidate modifier genes for CS/CSL-related malignancy risks, and may also guide future tailored preventative or therapeutic approaches.
Proposed signaling model of CS/CSL tumorigenesis caused by SDHx variants.