High Expression of Fas-Associated Factor 1 Indicates a Poor Prognosis in Non-Small-Cell Lung Cancer.

Fas-associated factor 1 (FAF1) is a death-promoting protein identified as an interaction partner of the death receptor Fas. The downregulation and mutation of FAF1 have been reported in a variety of human tumors, but there have been few studies on lung cancer. Here, we investigated the prognostic significance of FAF1 expression in non-small-cell lung cancer (NSCLC), and whether aberrant FAF1 expression may be involved in the pathogenesis and prognosis of NSCLC. FAF1 expression was examined in NSCLC specimens as well as human lung cancer cell lines. In addition, changes in cell viability and apoptosis upon regulating FAF1 expression were investigated in lung cancer cell lines. As a result, high FAF1 expression was significantly associated with a poor prognosis in NSCLC. In lung cancer cell lines, FAF1 downregulation hindered cell viability and tended to promote early apoptosis. In conclusion, this is the first study of the clinical significance of FAF1 in NSCLC, showing that FAF1 overexpression is associated with a poor prognosis in NSCLC and that FAF1 acts as a dangerous factor rather than an apoptosis promoter in NSCLC.

Non-CpG sites preference in G:C > A:T transition of TP53 in gastric cancer of Eastern Europe (Poland, Romania and Hungary) compared to East Asian countries (China and Japan).

AIM: Mutation spectrum of TP53 in gastric cancer (GC) has been investigated world-widely, but a comparison of mutation spectrum among GCs from various regions in the world are still sparsely documented. In order to identify the difference of TP53 mutation spectrum in GCs in Eastern Europe and in East Asia, we sequenced TP53 in GCs from Eastern Europe, Lujiang (China), and Yokohama, Kanagawa (Japan) and identified the feature of TP53 mutations of GC in these regions. SUBJECTS AND METHOD: In total, 689 tissue samples of GC were analyzed: 288 samples from East European populations (25 from Hungary, 71 from Poland and 192 from Romania), 268 from Yokohama, Kanagawa, Japan and 133 from Lujiang, Anhui province, China. DNA was extracted from FFPE tissue of Chinese, East European cases; and from frozen tissue of Japanese GCs. PCR products were direct-sequenced by Sanger method, and in ambiguous cases, PCR product was cloned and up to 8 clones were sequenced. We used No. NC_000017.11(hg38) as the reference sequence of TP53. Mutation patterns were categorized into nine groups: six base substitutions, insertion, deletion and deletion-insertion. Within G:C > A:T mutations the mutations in CpG and non-CpG sites were divided. The Cancer Genome Atlas data (TCGA, ver.R20, July, 2019) having somatic mutation list of GCs from Whites, Asians, and other ethnicities were used as a reference for our data. RESULTS: The most frequent base substitutions were G:C > A:T transition in all the areas investigated. The G:C > A:T transition in non-CpG sites were prominent in East European GCs, compared with Asian ones. Mutation pattern from TCGA data revealed the same trend between GCs from White (TCGA category) vs Asian countries. Chinese and Japanese GCs showed higher ratio of G:C > A:T transition in CpG sites and A:T > G:C mutation was more prevalent in Asian countries. CONCLUSION: The divergence in mutation spectrum of GC in different areas in the world may reflect various pathogeneses and etiologies of GC, region to region. Diversified mutation spectrum in GC in Eastern Europe may suggest GC in Europe has different carcinogenic pathway of those from Asia.

Structural basis of transcription regulation by CNC family transcription factor, Nrf2.

Several basic leucine zipper (bZIP) transcription factors have accessory motifs in their DNA-binding domains, such as the CNC motif of CNC family or the EHR motif of small Maf (sMaf) proteins. CNC family proteins heterodimerize with sMaf proteins to recognize CNC-sMaf binding DNA elements (CsMBEs) in competition with sMaf homodimers, but the functional role of the CNC motif remains elusive. In this study, we report the crystal structures of Nrf2/NFE2L2, a CNC family protein regulating anti-stress transcriptional responses, in a complex with MafG and CsMBE. The CNC motif restricts the conformations of crucial Arg residues in the basic region, which form extensive contact with the DNA backbone phosphates. Accordingly, the Nrf2-MafG heterodimer has approximately a 200-fold stronger affinity for CsMBE than canonical bZIP proteins, such as AP-1 proteins. The high DNA affinity of the CNC-sMaf heterodimer may allow it to compete with the sMaf homodimer on target genes without being perturbed by other low-affinity bZIP proteins with similar sequence specificity.

m6A demethylase ALKBH5 promotes tumor cell proliferation by destabilizing IGF2BPs target genes and worsens the prognosis of patients with non-small-cell lung cancer.

The modification of N6-methyladenosine (m6A) in RNA and its eraser ALKBH5, an m6A demethylase, play an important role across various steps of human carcinogenesis. However, the involvement of ALKBH5 in non-small-cell lung cancer (NSCLC) development remains to be completely elucidated. The current study revealed that the expression of ALKBH5 was increased in NSCLC and increased expression of ALKBH5 worsened the prognosis of patients with NSCLC. In vitro study revealed that ALKBH5 knockdown suppressed cell proliferation ability of PC9 and A549 cells and promoted G1 arrest and increased the number of apoptotic cells. Furthermore, ALKBH5 overexpression increased the cell proliferation ability of the immortalized cell lines. Microarray analysis and western blotting revealed that the expression of CDKN1A (p21) or TIMP3 was increased by ALKBH5 knockdown. These alterations were offset by a double knockdown of both ALKBH5 and one of the IGF2BPs. The decline of mRNAs was, at least partly, owing to the destabilization of these mRNAs by one of the IGF2BPs. In conclusions, the ALKBH5-IGF2BPs axis promotes cell proliferation and tumorigenicity, which in turn causes the unfavorable prognosis of NSCLC.

Confined Hot Electron Relaxation at the Molecular Heterointerface of the Size-Selected Plasmonic Noble Metal Nanocluster and Layered C60.

The plasmonic response of metallic nanostructures plays a key role in amplifying photocatalytic and photoelectric conversion. Since the plasmonic behavior of noble metal nanoparticles is known to generate energetic charge carriers such as hot electrons, it is expected that the hot electrons can enhance conversion efficiency if they are transferred into a neighboring molecule or semiconductor. However, the method of transferring the energized charge carriers from the plasmonically generated hot electrons to the neighboring species remains controversial. Herein, we fabricated a molecularly well-defined heterointerface between the size-selected plasmonic noble-metal nanoclusters (NCs) of Agn (n = 3-55)/Aun (n = 21) and the organic C60 film to investigate hot electron generation and relaxation dynamics using time-resolved two-photon photoemission (2PPE) spectroscopy. By tuning the NC size and the polarization of the femtosecond excitation photons, the plasmonic behavior is characterized by 2PPE intensity enhancement by 10-100 times magnitude, which emerge at n ≥ 9 for Agn NCs. The 2PPE spectra exhibit contributions from low-energy electrons forming coherent plasmonic currents and hot electrons with an excitation energy up to photon energy owing to two-photon excitation of an occupied state of the Agn NC below the Fermi level. The time-resolved pump-probe measurements demonstrate that plasmon dephasing generates hot electrons which undergo electron-electron scattering. However, no photoemission occurs via the charge transfer state forming Agn+C60- located in the vicinity of the Fermi level. Thus, this study reveals the mechanism of ultrafast confined hot electron relaxation within plasmonic Agn NCs at the molecular heterointerface.

Microsatellite frameshift variants in SGO1 of gastric cancer are not always associated with MSI status.

AIMS: Although frameshift variants in the microsatellite area of shugoshin 1 (SGO1) have been reported in the context of microsatellite instability-high (MSI-H)/deficient mismatch repair gastrointestinal cancer, most have been evaluated only in early stage I-III patients, and only two of its five microsatellite regions have been evaluated. Therefore, we investigated the frequency and MSI status of microsatellite frameshift variants in gastric cancer cases, including stage IV. METHODS: In a total of 55 cases, 30 gastric cancer resection and 25 non-resection cases, DNA was extracted from both tumour and normal parts and PCR was performed. The variant was confirmed by TA cloning, and MSI was evaluated using GeneMapper software. RESULTS: A frameshift variant of c.973delA was observed in 16 of the 45 evaluable cases. Its frequency was 35.6%. Of the 25 cases that could be assessed for MSI status, two cases of MSI-H were associated with the c.973delA SGO1 variant. However, c.973delA SGO1 variant was also observed in four cases of microsatellite stable. CONCLUSION: Our study shows that SGO1 frameshift variants are not always associated with MSI status.

Direct observation of photocarrier electron dynamics in C60 films on graphite by time-resolved two-photon photoemission.

Time-resolved two-photon photoemission (TR-2PPE) spectroscopy is employed to probe the electronic states of a C60 fullerene film formed on highly oriented pyrolytic graphite (HOPG), acting as a model two-dimensional (2D) material for multi-layered graphene. Owing to the in-plane sp2-hybridized nature of the HOPG, the TR-2PPE spectra reveal the energetics and dynamics of photocarriers in the C60 film: after hot excitons are nascently formed in C60 via intramolecular excitation by a pump photon, they dissociate into photocarriers of free electrons and the corresponding holes, and the electrons are subsequently detected by a probe photon as photoelectrons. The decay rate of photocarriers from the C60 film into the HOPG is evaluated to be 1.31 × 1012 s-1, suggesting a weak van der Waals interaction at the interface, where the photocarriers tentatively occupy the lowest unoccupied molecular orbital (LUMO) of C60. The photocarrier electron dynamics following the hot exciton dissociation in the organic thin films has not been realized for any metallic substrates exhibiting strong interactions with the overlayer. Furthermore, the thickness dependence of the electron lifetime in the LUMO reveals that the electron hopping rate in C60 layers is 3.3 ± 1.2 × 1013 s-1.

Synovial sarcoma cell lines showed reduced DNA repair activity and sensitivity to a PARP inhibitor.

Synovial sarcoma is a soft-tissue sarcoma and a rare type of cancer. Unfortunately, effective chemotherapies for synovial sarcomas have not been established. In this report, we show that synovial sarcoma cell lines have reduced repair activity for DNA damage induced by ionizing radiation (IR) and a topoisomerase II inhibitor (etoposide). We also observed reduced recruitment of RAD51 homologue (S. cerevisiae; RAD51) at sites of double-strand breaks (DSBs) in synovial sarcoma cell lines that had been exposed to IR. These findings showed that synovial sarcoma cell lines are defective in homologous recombination (HR) repair. Furthermore, we found that a poly-(ADP-ribose) polymerase (PARP) inhibitor (AZD2281; olaparib) effectively reduced the growth of synovial sarcoma cell lines in the presence of an alkylating agent (temozolomide). Our findings offer evidence that treatment combining a PARP inhibitor and an alkylating agent could have therapeutic benefits in the treatment of synovial sarcoma.

Ruptured Pseudoaneurysm after Gamma Knife Surgery for Vestibular Schwannoma.

Ruptured aneurysms of anterior inferior cerebellar artery (AICA) after radiotherapy for vestibular schwannoma (VS) are rare, and no definite treatment has been established for distal AICA pseudoaneurysms. We describe a 61-year-old man who underwent Gamma Knife surgery (GKS) for left VS. Follow-up magnetic resonance imaging (MRI) revealed partial regression of the tumor. Twelve years after GKS, he suffered from subarachnoid hemorrhage. Initial angiogram showed no vascular lesions; second left vertebral angiogram, 10 days after admission, demonstrated a pseudoaneurysm in the lateral pontine segment of the left AICA. The proximal portion of the AICA was occluded by a coil. Postoperative MRI revealed an infarction on the left side of the pons and brachium pontis. Although the patient suffered from mild postoperative cerebellar ataxia and facial and abducens nerve palsy, he was discharged 1 month postoperatively requiring no assistance with activities of daily living. Twelve months later, he recovered satisfactorily with a modified Rankin Scale grade of 1, and no recanalization of the aneurysm was found on MR angiography. Endovascular parent artery occlusion for ruptured aneurysms at distal AICA carries the risk of brain stem infarction, but should be considered when no other option is available such as after radiotherapy for VS.

Chemical characterization of an alkali-like superatom consisting of a Ta-encapsulating Si16 cage.

Chemical characterization was performed for an alkali-like superatom consisting of a Ta-encapsulating Si16 cage, Ta@Si16, deposited on a graphite substrate using X-ray photoelectron spectroscopy (XPS) to element-specifically clarify the local electronic structure of the cage atoms. The XPS spectra derived from Ta 4f and Si 2p core levels have been well modeled with a single chemical component, revealing the formation of a symmetric Si cage around the Ta atom in the deposited nanoclusters. On chemical treatments by heating or oxygen exposure, it is found that the deposited Ta@Si16 is thermally stable up to 700 K and is also exceptionally less reactive toward oxygen compared to other Ta-Si nanoclusters, although some heat degradation and oxidation accompany the treatments. These results show the promising possibility of applying Ta@Si16 as a building block to fabricate cluster-assembled materials consisting of naked nanoclusters.

A rare polymorphic variant of NBS1 reduces DNA repair activity and elevates chromosomal instability.

Failure to expeditiously repair DNA at sites of double-strand breaks (DSB) ultimately is an important etiologic factor in cancer development. NBS1 plays an important role in the cellular response to DSB damage. A rare polymorphic variant of NBS1 that resulted in an isoleucine to valine substitution at amino acid position 171 (I171V) was first identified in childhood acute lymphoblastic leukemia. This polymorphic variant is located in the N-terminal region that interacts with other DNA repair factors. In earlier work, we had identified a remarkable number of structural chromosomal aberrations in a patient with pediatric aplastic anemia with a homozygous polymorphic variant of NBS1-I171V; however, it was unclear whether this variant affected DSB repair activity or chromosomal instability. In this report, we demonstrate that NBS1-I171V reduces DSB repair activity through a loss of association with the DNA repair factor MDC1. Furthermore, we found that heterozygosity in this polymorphic variant was associated with breast cancer risk. Finally, we showed that this variant exerted a dominant-negative effect on wild-type NBS1, attenuating DSB repair efficiency and elevating chromosomal instability. Our findings offer evidence that the failure of DNA repair leading to chromosomal instability has a causal impact on the risk of breast cancer development.

Macrophage migration inhibitory factor and stearoyl-CoA desaturase 1: potential prognostic markers for soft tissue sarcomas based on bioinformatics analyses.

The diagnosis and treatment of soft tissue sarcomas (STSs) has been particularly difficult, because STSs are a group of highly heterogeneous tumors in terms of histopathology, histological grade, and primary site. Recent advances in genome technologies have provided an excellent opportunity to determine the complete biological characteristics of neoplastic tissues, resulting in improved diagnosis, treatment selection, and investigation of therapeutic targets. We had previously developed a novel bioinformatics method for marker gene selection and applied this method to gene expression data from STS patients. This previous analysis revealed that the extracted gene combination of macrophage migration inhibitory factor (MIF) and stearoyl-CoA desaturase 1 (SCD1) is an effective diagnostic marker to discriminate between subtypes of STSs with highly different outcomes. In the present study, we hypothesize that the combination of MIF and SCD1 is also a prognostic marker for the overall outcome of STSs. To prove this hypothesis, we first analyzed microarray data from 88 STS patients and their outcomes. Our results show that the survival rates for MIF- and SCD1-positive groups were lower than those for negative groups, and the p values of the log-rank test are 0.0146 and 0.00606, respectively. In addition, survival rates are more significantly different (p = 0.000116) between groups that are double-positive and double-negative for MIF and SCD1. Furthermore, in vitro cell growth inhibition experiments by MIF and SCD1 inhibitors support the hypothesis. These results suggest that the gene set is useful as a prognostic marker associated with tumor progression.

Identification of PITX1 as a TERT suppressor gene located on human chromosome 5.

Telomerase, a ribonucleoprotein enzyme that maintains telomere length, is crucial for cellular immortalization and cancer progression. Telomerase activity is attributed primarily to the expression of telomerase reverse transcriptase (TERT). Using microcell-mediated chromosome transfer (MMCT) into the mouse melanoma cell line B16F10, we previously found that human chromosome 5 carries a gene, or genes, that can negatively regulate TERT expression (H. Kugoh, K. Shigenami, K. Funaki, J. Barrett, and M. Oshimura, Genes Chromosome Cancer 36:37-47, 2003). To identify the gene responsible for the regulation of TERT transcription, we performed cDNA microarray analysis using parental B16F10 cells, telomerase-negative B16F10 microcell hybrids with a human chromosome 5 (B16F10MH5), and its revertant clones (MH5R) with reactivated telomerase. Here, we report the identification of PITX1, whose expression leads to the downregulation of mouse tert (mtert) transcription, as a TERT suppressor gene. Additionally, both human TERT (hTERT) and mouse TERT (mtert) promoter activity can be suppressed by PITX1. We show that three and one binding site within the hTERT and mtert promoters, respectively, that express a unique conserved region are responsible for the transcriptional activation of TERT. Furthermore, we showed that PITX1 binds to the TERT promoter both in vitro and in vivo. Thus, PITX1 suppresses TERT transcription through direct binding to the TERT promoter, which ultimately regulates telomerase activity.

Up-regulation of PSF1 promotes the growth of breast cancer cells.

PSF1 is a subunit of the GINS complex that functions along with the MCM2-7 complex and Cdc45 in eukaryotic DNA replication. Although mammalian PSF1 is predominantly expressed in highly proliferating cells and organs, little is known about the roles of PSF1 in mature cells or cancer cells. We found that PSF1 was expressed at relatively high levels in breast tumor cells, but at low levels in normal breast cells. Knockdown of PSF1 expression using small interfering RNA (siRNA) slowed the growth of breast cancer cell lines by delaying DNA replication but did not affect proliferation of normal human mammary epithelial cells. Reduced PSF1 expression also inhibited anchorage-independent growth in breast cancer cell lines. These results suggest that PSF1 over-expression is specifically involved in breast cancer cell growth. Therefore, PSF1 inhibition might provide new therapeutic approaches for breast cancer.

CADM1 interacts with Tiam1 and promotes invasive phenotype of human T-cell leukemia virus type I-transformed cells and adult T-cell leukemia cells.

CADM1 encodes a multifunctional immunoglobulin-like cell adhesion molecule whose cytoplasmic domain contains a type II PSD95/Dlg/ZO-1 (PDZ)-binding motif (BM) for associating with other intracellular proteins. Although CADM1 lacks expression in T lymphocytes of healthy individuals, it is overexpressed in adult T-cell leukemia-lymphoma (ATL) cells. It has been suggested that the expression of CADM1 protein promotes infiltration of leukemic cells into various organs and tissues, which is one of the frequent clinical manifestations of ATL. Amino acid sequence alignment revealed that Tiam1 (T-lymphoma invasion and metastasis 1), a Rac-specific guanine nucleotide exchange factor, has a type II PDZ domain similar to those of membrane-associated guanylate kinase homologs (MAGUKs) that are known to bind to the PDZ-BM of CADM1. In this study, we demonstrated that the cytoplasmic domain of CADM1 directly interacted with the PDZ domain of Tiam1 and induced formation of lamellipodia through Rac activation in HTLV-I-transformed cell lines as well as ATL cell lines. Our results indicate that Tiam1 integrates signals from CADM1 to regulate the actin cytoskeleton through Rac activation, which may lead to tissue infiltration of leukemic cells in ATL patients.

PH domain-only protein PHLDA3 is a p53-regulated repressor of Akt.

p53 And Akt are critical players regulating tumorigenesis with opposite effects: whereas p53 transactivates target genes to exert its function as a tumor suppressor, Akt phosphorylates its substrates and transduces downstream survival signals. In addition, p53 and Akt negatively regulate each other to balance survival and death signals within a cell. We now identify PHLDA3 as a p53 target gene that encodes a PH domain-only protein. We find that PHLDA3 competes with the PH domain of Akt for binding of membrane lipids, thereby inhibiting Akt translocation to the cellular membrane and activation. Ablation of endogenous PHLDA3 results in enhanced Akt activity and decrease of p53-dependent apoptosis. We also demonstrate the suppression of anchorage-independent cell growth by PHLDA3. Loss of the PHLDA3 genomic locus was frequently observed in primary lung cancers, suggesting a role of PHLDA3 in tumor suppression. Our results reveal a new mode of coordination between the p53 and Akt pathways.

Adenylate cyclase-associated protein 1 overexpressed in pancreatic cancers is involved in cancer cell motility.

Pancreatic cancer has the worst prognosis among cancers due to the difficulty of early diagnosis and its aggressive behavior. To characterize the aggressiveness of pancreatic cancers on gene expression, pancreatic cancer xenografts transplanted into severe combined immunodeficient mice served as a panel for gene-expression profiling. As a result of profiling, the adenylate cyclase-associated protein 1 (CAP1) gene was shown to be overexpressed in all of the xenografts. The expression of CAP1 protein in all 73 cases of pancreatic cancer was recognized by immunohistochemical analyses. The ratio of CAP1-positive tumor cells in clinical specimens was correlated with the presence of lymph node metastasis and neural invasion, and also with the poor prognosis of patients. Immunocytochemical analyses in pancreatic cancer cells demonstrated that CAP1 colocalized to the leading edge of lamellipodia with actin. Knockdown of CAP1 by RNA interference resulted in the reduction of lamellipodium formation, motility, and invasion of pancreatic cancer cells. This is the first report demonstrating the overexpression of CAP1 in pancreatic cancers and suggesting the involvement of CAP1 in the aggressive behavior of pancreatic cancer cells.

Expression of Aurora B and alternative variant forms in hepatocellular carcinoma and adjacent tissue.

Surgical resection is the effective treatment modality for hepatocellular carcinoma (HCC); however, rapid recurrence of the tumors are frequently observed even after apparently curative resection. The recurrence and prognostic assessment of patients with HCC after resection is an important clinical issue. We recently reported that aberrant expression of Aurora B is observed in primary HCC, and that it can be a predictive factor for HCC recurrence exceeding Milan criteria after curative hepatectomy. In this study we investigated the expression of the newly observed Aurora B splicing variant forms in HCC, and their roles in hepatocarcinogenisis. The expression of Aurora B and splicing variant forms were screened in 125 HCC patients (94 chronic hepatitis with cirrhosis background liver specimens), 18 metastatic liver cancer patients and 16 normal liver specimens by cDNA microarray, reverse transcription -- polymerase chain reaction (RT-PCR) and Real time quantitative Reverse Transcription PCR (qRT-PCR). The results showed that expression of Aurora B splicing variant 2 (AURKB-Sv2) variant form was absent in normal liver and was higher in metastatic liver cancer than HCC. This aberrant expression was associated with the advanced stages of HCC (P < 0.01), correlated with a poor outcome (P = 0.008) and short disease-free period (P = 0.018). Furthermore, AURKB-Sv2 variant form is associated with a higher level of serum alpha-fetoprotein, protein induced by vitamin K absence or antagonist-II (PIVKAII), tumor capsular invasion, multiple tumor formation and at an age younger than those with other variant forms (P < 0.05). The results thus suggest that AURKB-Sv2 variant form is more significantly associated with the advanced stages of HCC than others and is a marker of poor prognosis. Founded in the tumor capsular invasion and multiple tumor regions, suggests that this might play a role in enhancing multiple malignant tumor formation and recurrence of HCC in hepatocarcinogenesis. This is the first study to report clinicopathological significance of aberrant expression of AURKB-Sv2 variant form in hepatocellular carcinoma.

Bach1 inhibits oxidative stress-induced cellular senescence by impeding p53 function on chromatin.

Cellular senescence is one of the key strategies to suppress expansion of cells with mutations. Senescence is induced in response to genotoxic and oxidative stress. Here we show that the transcription factor Bach1 (BTB and CNC homology 1, basic leucine zipper transcription factor 1), which inhibits oxidative stress-inducible genes, is a crucial negative regulator of oxidative stress-induced cellular senescence. Bach1-deficient murine embryonic fibroblasts showed a propensity to undergo more rapid and profound p53-dependent premature senescence than control wild-type cells in response to oxidative stress. Bach1 formed a complex that contained p53, histone deacetylase 1 and nuclear co-repressor N-coR. Bach1 was recruited to a subset of p53 target genes and contributed to impeding p53 action by promoting histone deacetylation. Because Bach1 is regulated by oxidative stress and heme, our data show that Bach1 connects oxygen metabolism and cellular senescence as a negative regulator of p53.

Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy.

The nuclear factor E2-related factor 2 (Nrf2) is a master transcriptional activator of genes encoding numerous cytoprotective enzymes that are induced in response to environmental and endogenously derived oxidative/electrophilic agents. Under normal, nonstressed circumstances, low cellular concentrations of Nrf2 are maintained by proteasomal degradation through a Keap1-Cul3-Roc1-dependent mechanism. A model for Nrf2 activation has been proposed in which two amino-terminal motifs, DLG and ETGE, promote efficient ubiquitination and rapid turnover; known as the two-site substrate recognition/hinge and latch model. Here, we show that in human cancer, somatic mutations occur in the coding region of NRF2, especially among patients with a history of smoking or suffering from squamous cell carcinoma; in the latter case, this leads to poor prognosis. These mutations specifically alter amino acids in the DLG or ETGE motifs, resulting in aberrant cellular accumulation of Nrf2. Mutant Nrf2 cells display constitutive induction of cytoprotective enzymes and drug efflux pumps, which are insensitive to Keap1-mediated regulation. Suppression of Nrf2 protein levels by siRNA knockdown sensitized cancer cells to oxidative stress and chemotherapeutic reagents. Our results strongly support the contention that constitutive Nrf2 activation affords cancer cells with undue protection from their inherently stressed microenvironment and anti-cancer treatments. Hence, inactivation of the Nrf2 pathway may represent a therapeutic strategy to reinforce current treatments for malignancy. Congruously, the present study also provides in vivo validation of the two-site substrate recognition model for Nrf2 activation by the Keap1-Cul3-based E3 ligase.