PIK3CA mutant tumors depend on oxoglutarate dehydrogenase.

Oncogenic PIK3CA mutations are found in a significant fraction of human cancers, but therapeutic inhibition of PI3K has only shown limited success in clinical trials. To understand how mutant PIK3CA contributes to cancer cell proliferation, we used genome scale loss-of-function screening in a large number of genomically annotated cancer cell lines. As expected, we found that PIK3CA mutant cancer cells require PIK3CA but also require the expression of the TCA cycle enzyme 2-oxoglutarate dehydrogenase (OGDH). To understand the relationship between oncogenic PIK3CA and OGDH function, we interrogated metabolic requirements and found an increased reliance on glucose metabolism to sustain PIK3CA mutant cell proliferation. Functional metabolic studies revealed that OGDH suppression increased levels of the metabolite 2-oxoglutarate (2OG). We found that this increase in 2OG levels, either by OGDH suppression or exogenous 2OG treatment, resulted in aspartate depletion that was specifically manifested as auxotrophy within PIK3CA mutant cells. Reduced levels of aspartate deregulated the malate-aspartate shuttle, which is important for cytoplasmic NAD+ regeneration that sustains rapid glucose breakdown through glycolysis. Consequently, because PIK3CA mutant cells exhibit a profound reliance on glucose metabolism, malate-aspartate shuttle deregulation leads to a specific proliferative block due to the inability to maintain NAD+/NADH homeostasis. Together these observations define a precise metabolic vulnerability imposed by a recurrently mutated oncogene.

Systematic interrogation of 3q26 identifies TLOC1 and SKIL as cancer drivers.

UNLABELLED: 3q26 is frequently amplified in several cancer types with a common amplified region containing 20 genes. To identify cancer driver genes in this region, we interrogated the function of each of these genes by loss- and gain-of-function genetic screens. Specifically, we found that TLOC1 (SEC62) was selectively required for the proliferation of cell lines with 3q26 amplification. Increased TLOC1 expression induced anchorage-independent growth, and a second 3q26 gene, SKIL (SNON), facilitated cell invasion in immortalized human mammary epithelial cells. Expression of both TLOC1 and SKIL induced subcutaneous tumor growth. Proteomic studies showed that TLOC1 binds to DDX3X, which is essential for TLOC1-induced transformation and affected protein translation. SKIL induced invasion through upregulation of SLUG (SNAI2) expression. Together, these studies identify TLOC1 and SKIL as driver genes at 3q26 and more broadly suggest that cooperating genes may be coamplified in other regions with somatic copy number gain. SIGNIFICANCE: These studies identify TLOC1 and SKIL as driver genes in 3q26. These observations provide evidence that regions of somatic copy number gain may harbor cooperating genes of different but complementary functions.

Genomic sequencing of colorectal adenocarcinomas identifies a recurrent VTI1A-TCF7L2 fusion.

Prior studies have identified recurrent oncogenic mutations in colorectal adenocarcinoma and have surveyed exons of protein-coding genes for mutations in 11 affected individuals. Here we report whole-genome sequencing from nine individuals with colorectal cancer, including primary colorectal tumors and matched adjacent non-tumor tissues, at an average of 30.7× and 31.9× coverage, respectively. We identify an average of 75 somatic rearrangements per tumor, including complex networks of translocations between pairs of chromosomes. Eleven rearrangements encode predicted in-frame fusion proteins, including a fusion of VTI1A and TCF7L2 found in 3 out of 97 colorectal cancers. Although TCF7L2 encodes TCF4, which cooperates with ß-catenin in colorectal carcinogenesis, the fusion lacks the TCF4 ß-catenin-binding domain. We found a colorectal carcinoma cell line harboring the fusion gene to be dependent on VTI1A-TCF7L2 for anchorage-independent growth using RNA interference-mediated knockdown. This study shows previously unidentified levels of genomic rearrangements in colorectal carcinoma that can lead to essential gene fusions and other oncogenic events.

Genomic instability and cancer: lessons learned from human papillomaviruses.

High-risk HPV E6 and E7 oncoproteins cooperate to subvert critical host cell cycle checkpoint control mechanisms in order to promote viral genome replication. This results not only in aberrant proliferation but also in host cellular changes that can promote genomic instability. The HPV-16 E7 oncoprotein was found to induce centrosome abnormalities thereby disrupting mitotic fidelity and increasing the risk for chromosome missegregation and aneuploidy. In addition, expression of the high-risk HPV E7 oncoprotein stimulates DNA replication stress as a potential source of DNA breakage and structural chromosomal instability. Proliferation of genomically unstable cells is sustained by several mechanisms including the accelerated degradation of claspin by HPV-16 E7 and the degradation of p53 by the high-risk HPV E6 oncoprotein. These results highlight the oncogenic potential of aberrant proliferation and opens new avenues for prevention of malignant progression, not only in HPV-associated cervical cancer but also in non-virally associated malignancies with disrupted cell cycle checkpoint control mechanisms.

Deficiencies in the Fanconi anemia DNA damage response pathway increase sensitivity to HPV-associated head and neck cancer.

Patients with the rare genetic disease, Fanconi anemia (FA), are highly susceptible to squamous cell carcinomas arising at multiple anatomic sites including the head and neck region. Human papillomaviruses (HPVs), particularly HPV16, are associated with ∼20% of head and neck squamous cell carcinomas (HNSCCs) in the general population. Some but not other investigators have reported that HNSCCs in FA patients are much more frequently positive for HPV. In addition, studies have demonstrated an interaction between the HPV16 E7 oncoprotein and the FA pathway, a DNA damage response pathway deficient in FA patients. On the basis of these studies, it was hypothesized that the FA pathway contributes to repair of DNA damage induced by HPV16 E7, providing one explanation for why FA patients are predisposed to HPV-associated HNSCCs. To determine the importance of the FA pathway in modulating the oncogenic abilities of E7, we crossed K14E7 transgenic (K14E7) and fancD2 knockout mice (FancD2(-/-)) to establish K14E7/FancD2(-/-) and K14E7/FancD2(+/+) mice and monitored their susceptibility to HNSCC when treated with a chemical carcinogen. K14E7/FancD2(-/-) mice had a significantly higher incidence of HNSCC compared with K14E7/FancD2(+/+) mice. This difference correlated with an increased proliferative index and the increase in expression of biomarkers that are used to assess levels of DNA damage. These animal studies support the hypotheses that FA patients have increased susceptibility to HPV-associated cancer and that the FA DNA damage response pathway normally attenuates the oncogenic potential of HPV16 E7.

Human papillomavirus 16 E7 oncoprotein attenuates DNA damage checkpoint control by increasing the proteolytic turnover of claspin.

The human papillomavirus (HPV) 16 E7 oncoprotein has been reported previously to stimulate DNA damage and to activate host cell DNA damage checkpoints. How HPV-16 E7 maintains proliferation despite activated DNA damage checkpoints is incompletely understood. Here, we provide evidence that cells expressing the HPV-16 E7 oncoprotein can enter mitosis in the presence of DNA damage. We show that this activity of HPV-16 E7 involves attenuation of DNA damage checkpoint control by accelerating the proteolytic turnover of claspin. Claspin mediates the activation of CHK1 by ATR in response to replication stress, and its degradation plays a critical role in DNA damage checkpoint recovery. Expression of a nondegradable mutant of claspin was shown to inhibit mitotic entry in HPV-16 E7-expressing cells. Multiple components of the SCF(beta-TrCP)-based claspin degradation machinery were found deregulated in the presence of HPV-16 E7, including cullin 1, beta-TrCP, Aurora A, and Polo-like kinase-1 (PLK1). In contrast, no difference in the expression level of the claspin deubiquitinating enzyme USP7 was detected. Levels of Aurora A and PLK1 as well as phosphorylated PLK1 at threonine 210, a prerequisite for DNA damage checkpoint recovery, remained detectable following replication stress in HPV-16 E7-expressing cells but not in control cells. In summary, our results suggest that the HPV-16 E7 oncoprotein alleviates DNA damage checkpoint responses and promotes mitotic entry by accelerating claspin degradation through a mechanism that involves deregulation of components of the SCF(beta-TrCP)-based claspin degradation machinery.

Centrosome overduplication, chromosomal instability, and human papillomavirus oncoproteins.

Centrosome aberrations are a frequent finding in human tumors. However, very little is known about the molecular mechanisms leading to disruption of centrosome duplication control and the functional consequences of aberrant centrosome numbers. The high-risk human papillomavirus Type 16 (HPV-16) E6 and E7 oncoproteins are overexpressed in HPV-associated malignancies of the anogenital tract and have been instrumental in delineating different pathways of centrosome amplification. Whereas the E6 oncoprotein was found to provoke centrosome accumulation, the HPV-16 E7 oncoprotein triggers a genuine disruption of the centrosome duplication cycle. Importantly, the E7 oncoprotein can rapidly cause centrosome overduplication through a pathway that involves the concurrent formation of multiple daughters at single maternal centrioles (centriole flowers). Several lines of evidence suggest that cyclin E/CDK2 complexes and Polo-like kinase 4 (PLK4) are crucial players in this process. These findings underscore that the HPV-16 E7 oncoprotein is a unique tool to dissect normal and abnormal centriole biogenesis and the underlying molecular circuitry.

HPV-16 E7 reveals a link between DNA replication stress, fanconi anemia D2 protein, and alternative lengthening of telomere-associated promyelocytic leukemia bodies.

Expression of the high-risk human papillomavirus (HPV-16) E7 oncoprotein extends the life span of primary human keratinocytes and partially restores telomere length in the absence of telomerase. The molecular basis of this activity is incompletely understood. Here, we show that HPV-16 E7 induces an increased formation of alternative lengthening of telomeres (ALT)-associated promyelocytic leukemia bodies (APBs) in early passage primary human keratinocytes as well as HPV-negative tumor cells. This activity was found to require sequences of HPV-16 E7 involved in degradation of the retinoblastoma tumor suppressor protein as well as regions in the COOH terminus. HPV-16 E7-induced APBs contained ssDNA and several proteins that are involved in the response to DNA replication stress, most notably the Fanconi anemia D2 protein (FANCD2) as well as BRCA2 and MUS81. In line with these results, we found that FANCD2-containing APBs form in an ATR-dependent manner in HPV-16 E7-expressing cells. To directly show a role of FANCD2 in ALT, we provide evidence that knockdown of FANCD2 rapidly causes telomere dysfunction in cells that rely on ALT to maintain telomeres. Taken together, our results suggest a novel link between replication stress and recombination-based telomere maintenance that may play a role in HPV-16 E7-mediated extension of host cell life span and immortalization.

The human papillomavirus type 16 E7 oncoprotein activates the Fanconi anemia (FA) pathway and causes accelerated chromosomal instability in FA cells.

Fanconi anemia (FA) patients have an increased risk for squamous cell carcinomas (SCCs) at sites of predilection for infection with high-risk human papillomavirus (HPV) types, including the oral cavity and the anogenital tract. We show here that activation of the FA pathway is a frequent event in cervical SCCs. We found that FA pathway activation is triggered mainly by the HPV type 16 (HPV-16) E7 oncoprotein and is associated with an enhanced formation of large FANCD2 foci and recruitment of FANCD2 as well as FANCD1/BRCA2 to chromatin. Episomal expression of HPV-16 oncoproteins was sufficient to activate the FA pathway. Importantly, the expression of HPV-16 E7 in FA-deficient cells led to accelerated chromosomal instability. Taken together, our findings establish the FA pathway as an early host cell response to high-risk HPV infection and may help to explain the greatly enhanced susceptibility of FA patients to squamous cell carcinogenesis at anatomic sites that are frequently infected by high-risk HPVs.

A role of the mitotic spindle checkpoint in the cellular response to DNA replication stress.

Replication stress is a frequent and early event during tumorigenesis. Whereas the cellular responses to a persistent block of replication fork progression have been extensively studied, relatively little is known about how cells respond to low-intensity replication stress. However, transient replication fork perturbations are likely to occur even more frequently in tumor cells than a permanent replication arrest. We report here that transient, low intensity replication stress leads to a rapid activation of the DNA replication checkpoint but to a significantly delayed apoptotic response in a small but significant number of cells. This late apoptotic response was independent of p53 and we found evidence for cell death during mitosis in a proportion of cells. To further explore the role of p53 in the response to replication stress, we analyzed mouse embryonic fibroblasts (MEFs) deficient of p53 in comparison to wild-type or p63- or p73-deficient MEFs. We detected a significant increase of apoptosis and morphological signs of failed mitosis such as multinucleation in p53-deficient MEFs following replication stress, but not in wild-type or p63- or p73-deficient cells. Multinucleated p53-deficient MEFs frequently retained cyclin B1 expression indicating a persistently activated mitotic spindle checkpoint. Collectively, our results suggest that the cellular response to replication stress involves the mitotic spindle checkpoint in a proportion of cells. These findings imply that the mitotic spindle checkpoint may act in concert with DNA damage and cell-cycle checkpoints as an early anti-tumor barrier and provide a possible explanation for its frequent relaxation in human cancer.