PD-1 Blockade in Tumors with Mismatch-Repair Deficiency.

BACKGROUND: Somatic mutations have the potential to encode "non-self" immunogenic antigens. We hypothesized that tumors with a large number of somatic mutations due to mismatch-repair defects may be susceptible to immune checkpoint blockade. METHODS: We conducted a phase 2 study to evaluate the clinical activity of pembrolizumab, an anti-programmed death 1 immune checkpoint inhibitor, in 41 patients with progressive metastatic carcinoma with or without mismatch-repair deficiency. Pembrolizumab was administered intravenously at a dose of 10 mg per kilogram of body weight every 14 days in patients with mismatch repair-deficient colorectal cancers, patients with mismatch repair-proficient colorectal cancers, and patients with mismatch repair-deficient cancers that were not colorectal. The coprimary end points were the immune-related objective response rate and the 20-week immune-related progression-free survival rate. RESULTS: The immune-related objective response rate and immune-related progression-free survival rate were 40% (4 of 10 patients) and 78% (7 of 9 patients), respectively, for mismatch repair-deficient colorectal cancers and 0% (0 of 18 patients) and 11% (2 of 18 patients) for mismatch repair-proficient colorectal cancers. The median progression-free survival and overall survival were not reached in the cohort with mismatch repair-deficient colorectal cancer but were 2.2 and 5.0 months, respectively, in the cohort with mismatch repair-proficient colorectal cancer (hazard ratio for disease progression or death, 0.10 [P<0.001], and hazard ratio for death, 0.22 [P=0.05]). Patients with mismatch repair-deficient noncolorectal cancer had responses similar to those of patients with mismatch repair-deficient colorectal cancer (immune-related objective response rate, 71% [5 of 7 patients]; immune-related progression-free survival rate, 67% [4 of 6 patients]). Whole-exome sequencing revealed a mean of 1782 somatic mutations per tumor in mismatch repair-deficient tumors, as compared with 73 in mismatch repair-proficient tumors (P=0.007), and high somatic mutation loads were associated with prolonged progression-free survival (P=0.02). CONCLUSIONS: This study showed that mismatch-repair status predicted clinical benefit of immune checkpoint blockade with pembrolizumab. (Funded by Johns Hopkins University and others; ClinicalTrials.gov number, NCT01876511.).

Pembrolizumab for patients with PD-L1-positive advanced gastric cancer (KEYNOTE-012): a multicentre, open-label, phase 1b trial.

BACKGROUND: Expression of PD-L1 has been shown to be upregulated in some patients with gastric cancer. As part of the phase 1b KEYNOTE-012 study, we aimed to assess the safety and activity of the anti-PD-1 antibody pembrolizumab in patients with PD-L1-positive recurrent or metastatic adenocarcinoma of the stomach or gastro-oesophageal junction. METHODS: This study was a multicentre, open-label, phase 1b trial done at 13 cancer research centres in the USA, Israel, Japan, South Korea, and Taiwan. We enrolled patients with PD-L1-positive recurrent or metastatic adenocarcinoma of the stomach or gastro-oesophageal junction. Patients received intravenous pembrolizumab at 10 mg/kg once every 2 weeks for 24 months or until progression or unacceptable toxic effects occurred. Response was assessed every 8 weeks in accordance with Response Evaluation Criteria in Solid Tumors version 1.1. The primary objectives were safety in patients who received at least one dose of pembrolizumab and the proportion of patients achieving overall responses in patients who received at least one pembrolizumab dose and who either had a post-baseline scan or who discontinued therapy because of clinical disease progression or a treatment-related adverse event before the first post-baseline scan. The study is registered with ClinicalTrials.gov, number NCT01848834, and is ongoing but no longer enrolling patients. FINDINGS: From Oct 23, 2013, to May 5, 2014, 39 patients were enrolled. 36 were evaluable for response by central assessment. Eight (22%, 95% CI 10-39) patients were judged to have had an overall response at central review; all responses were partial. All 39 patients were included in the safety analyses. Five (13%) patients had a total of six grade 3 or 4 treatment-related adverse events, consisting of two cases of grade 3 fatigue, one case each of grade 3 pemphigoid, grade 3 hypothyroidism, and grade 3 peripheral sensory neuropathy, and one case of grade 4 pneumonitis. No treatment-related deaths occurred. INTERPRETATION: In this population of patients with recurrent or metastatic PD-L1-positive gastric cancer, pembrolizumab had a manageable toxicity profile and promising antitumour activity, warranting further study in phase 2 and 3 trials. FUNDING: Merck & Co.

Ramucirumab monotherapy for previously treated advanced gastric or gastro-oesophageal junction adenocarcinoma (REGARD): an international, randomised, multicentre, placebo-controlled, phase 3 trial.

BACKGROUND: Vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2)-mediated signalling and angiogenesis can contribute to the pathogenesis and progression of gastric cancer. We aimed to assess whether ramucirumab, a monoclonal antibody VEGFR-2 antagonist, prolonged survival in patients with advanced gastric cancer. METHODS: We did an international, randomised, double-blind, placebo-controlled, phase 3 trial between Oct 6, 2009, and Jan 26, 2012, at 119 centres in 29 countries in North America, Central and South America, Europe, Asia, Australia, and Africa. Patients aged 24-87 years with advanced gastric or gastro-oesophageal junction adenocarcinoma and disease progression after first-line platinum-containing or fluoropyrimidine-containing chemotherapy were randomly assigned (2:1), via a central interactive voice-response system, to receive best supportive care plus either ramucirumab 8 mg/kg or placebo, intravenously once every 2 weeks. The study sponsor, participants, and investigators were masked to treatment assignment. The primary endpoint was overall survival. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00917384. FINDINGS: 355 patients were assigned to receive ramucirumab (n=238) or placebo (n=117). Median overall survival was 5·2 months (IQR 2·3-9·9) in patients in the ramucirumab group and 3·8 months (1·7-7·1) in those in the placebo group (hazard ratio [HR] 0·776, 95% CI 0·603-0·998; p=0·047). The survival benefit with ramucirumab remained unchanged after multivariable adjustment for other prognostic factors (multivariable HR 0·774, 0·605-0·991; p=0·042). Rates of hypertension were higher in the ramucirumab group than in the placebo group (38 [16%] vs nine [8%]), whereas rates of other adverse events were mostly similar between groups (223 [94%] vs 101 [88%]). Five (2%) deaths in the ramucirumab group and two (2%) in the placebo group were considered to be related to study drug. INTERPRETATION: Ramucirumab is the first biological treatment given as a single drug that has survival benefits in patients with advanced gastric or gastro-oesophageal junction adenocarcinoma progressing after first-line chemotherapy. Our findings validate VEGFR-2 signalling as an important therapeutic target in advanced gastric cancer. FUNDING: ImClone Systems.

Effect of biliary drainage on chemotherapy in patients with biliary tract cancer: an exploratory analysis of the BT22 study.

BACKGROUND/PURPOSE: Complications from biliary drainage in biliary tract cancer (BTC) may influence the relative dose intensity of chemotherapy or increase adverse events during chemotherapy. BT22 was a randomized phase II trial, the results of which were consistent with those of a phase III trial in non-Japanese that demonstrated the effectiveness of gemcitabine plus cisplatin combination therapy (GC) in BTC. The purpose of this exploratory analysis of the BT22 study was to identify the possible effects of biliary drainage on the efficacy and safety of GC or gemcitabine monotherapy (G). PATIENTS AND METHODS: The 83 BTC patients who received GC or G in BT22 were retrospectively analysed in two subgroups dependent upon whether biliary drainage was performed before study entry. Efficacy and safety of treatment (GC vs. G) were compared in these two groups. RESULTS: The GC arm had a higher 1-year survival rate and longer median survival time (MST) than the G arm independent of prior biliary drainage. Patients in the drainage subgroup developed cholangitis more frequently, however, the frequency of grade 3/4 adverse events did not differ between the treatment regimens with/without drainage. CONCLUSIONS: Biliary drainage before chemotherapy did not affect the therapeutic efficacy or tolerability of chemotherapy using G or GC.

Phase I study of LY2469298, an Fc-engineered humanized anti-CD20 antibody, in patients with relapsed or refractory follicular lymphoma.

Patients with follicular lymphoma (FL), where position 158 of FcγR-IIIa is heterozygous valine/phenylalanine or homozygous phenylalanine (F-carriers), have natural killer cells with lower binding affinity to IgG than valine homozygote patients. In addition, F-carriers show less efficacy with rituximab treatment than patients homozygous for valine. LY2469298 is a humanized IgG1 monoclonal antibody targeting CD20, with human germline framework regions, and specific amino acid substitutions engineered into the Fc region to increase effector function in antibody-dependent cell-mediated cytotoxicity. This dose-escalation, phase I study was conducted to assess the safety, pharmacokinetics and preliminary efficacy of LY2469298 in Japanese patients with previously treated, CD20-positive FL who had not relapsed or progressed within 120 days of prior rituximab. LY2469298 was administered by intravenous infusion at 100 or 375 mg/m(2) weekly for 4 weeks. Ten patients were enrolled (median age, 60 years); all had previously been treated with rituximab. Nine patients were F-carriers while one was homozygous for valine at position 158 of FcγRIIIa. No patients developed dose-limiting toxicities, and the most frequent adverse events were lymphopenia, pyrexia, leukopenia, chills and neutropenia. Five (50%) of the ten patients responded to LY2469298 treatment (three complete responses, one unconfirmed complete response and one partial response). Serum LY2469298 was eliminated in a biphasic manner and the pharmacokinetic profiles were not different from those in a preceding study in the United States. In conclusion, LY2469298 was well tolerated and clinical activity was observed in FL patients pretreated with rituximab, mostly consisting of F-carriers. Further investigation of FL is warranted.

Phase I dose escalation and pharmacokinetic study of oral enzastaurin (LY317615) in advanced solid tumors.

Enzastaurin is an oral serine/threonine kinase inhibitor that targets the protein kinase C (PKC) and phosphoinositide 3-kinase/AKT pathways to induce apoptosis and suppress proliferation of various cancer cell lines. This phase I study evaluated the tolerability and pharmacokinetics of enzastaurin in Japanese patients with advanced solid tumors and determined the recommended dose for phase II. Eligible patients had advanced solid tumors and an Eastern Cooperative Oncology Group performance status of 0-2. Patients received enzastaurin orally once daily until disease progression (PD) or unacceptable toxicity occurred. Enzastaurin was started at 250 mg/day followed by stepwise dose increases based on the incidence of dose-limiting toxicities (DLT). Twenty-three patients (seven patients: 250 mg; six patients: 375 mg; six patients: 500 mg; four patients: 750 mg) were enrolled and received enzastaurin. The major tumor types were non-small-cell lung cancer (n = 5) and breast cancer (n = 3). No DLT was reported at doses of 500 mg or less. Because two DLT (grade 2 QTc prolongation lasting for a week) were observed at 750 mg enzastaurin, this was determined as the maximum tolerated dose. Multiple daily doses at 500 mg achieved the target plasma concentration to inhibit PKC activity (1400 nmol/L). Enzastaurin was well tolerated up to 500 mg in Japanese patients with advanced solid tumors. The recommended dose for phase II was determined to be 500 mg daily for a 28-day cycle on the basis of safety and plasma exposures.

[Drug interactions in medical oncology].

Cytotoxic anti-neoplastic drugs are some of the strongest acting drugs. They have a complex pharmacological profile, narrow therapeutic window, steep dose-toxicity curve, and many pharmacokinetic and pharmacodynamic differences both within and between patients. This makes it difficult to avoid adverse effects. These drugs are approved for usage based on their clinical benefit to risk ratio. The recommended dose is usually close to the maximally-tolerated dose in order to achieve maximum therapeutic effect. Therefore, there is more concern about drug interactions affecting the pharmacokinetics of anti-neoplastic drugs than drugs in general. Any physician taking care of oncology patients must understand not only the pharmacokinetic profile(absorption, protein binding, metabolism and excretion)of the anti-neoplastic drugs their using, but also the many factors that affect the pharmacokinetic profile such as hepatic and renal function, and co-administered drugs. Expertise to achieve a good balance between safety and efficacy in medical treatment with proper knowledge in supportive care as well as an understanding of pharmacokinetics, pharmacodynamics and pharmacogenomics is essential for medical oncologists. In this review, we have summarized the drug-drug interactions important for the management of cancer patients. The types of interactions covered are pharmaceutical interactions and interactions at the level of absorption, protein binding, metabolism and excretion.

Safety and Antitumor Activity of the Anti-Programmed Death-1 Antibody Pembrolizumab in Patients With Advanced Esophageal Carcinoma.

Purpose The anti-programmed death-1 antibody pembrolizumab was evaluated in KEYNOTE-028, a multicohort, phase IB study of patients with programmed death ligand-1 (PD-L1)-positive advanced solid tumors. Results from the esophageal carcinoma cohort are reported herein. Patients and Methods Eligible patients with squamous cell carcinoma or adenocarcinoma of the esophagus or gastroesophageal junction in whom standard therapy failed and who had PD-L1-positive tumors received pembrolizumab 10 mg/kg every 2 weeks for up to 2 years or until confirmed disease progression or intolerable toxicity. Response was assessed every 8 weeks up to 6 months and every 12 weeks thereafter. Primary end points were safety and overall response rate, determined by investigator review per Response Evaluation Criteria in Solid Tumors (version 1.1). Results Among 83 patients with esophageal carcinoma and samples evaluable for PD-L1 expression, 37 (45%) had PD-L1-positive tumors, and 23 were enrolled. Median age was 65 years; 78% had squamous histology; and 87% received ≥ two prior therapies for advanced/metastatic disease. As of the data cutoff (February 20, 2017), median follow-up was 7 months (range, 1 to 33 months). Nine patients (39%) experienced treatment-related adverse events, most commonly decreased appetite, decreased lymphocyte count, generalized rash, and rash (two patients [9%] each). No grade 4 adverse events or deaths were attributed to pembrolizumab. Overall response rate was 30% (95% CI, 13% to 53%); median duration of response was 15 months (range, 6 to 26 months). A six-gene interferon-γ gene expression signature analysis suggested that delayed progression and increased response occur among pembrolizumab-treated patients with higher interferon-γ composite scores. Conclusion Pembrolizumab demonstrated manageable toxicity and durable antitumor activity in patients with heavily pretreated, PD-L1-positive advanced esophageal carcinoma.

Correlation of N-myc downstream-regulated gene 1 expression with clinical outcomes of colorectal cancer patients of different race/ethnicity.

AIM: To evaluate the role of N-myc downstream-regulated gene 1 (NDRG1) expression in prognosis and survival of colorectal cancer patients with different ethnic backgrounds. METHODS: Because NDRG1 is a downstream target of p53 and hypoxia inducible factor-1 alpha (HIF-1 alpha), we examined NDRG1 expression together with p53 and HIF-1 alpha by immunohistochemistry. A total of 157 colorectal cancer specimens including 80 from Japanese patients and 77 from US patients were examined. The correlation between protein expression with clinicopathological features and survival after surgery was analyzed. RESULTS: NDRG1 protein was significantly increased in colorectal tumor compared with normal epithelium in both Japanese and US patient groups. Expression of NDRG1 protein was significantly correlated with lymphatic invasion, venous invasion, depth of invasion, histopathological type, and Dukes' stage in Japanese colorectal cancer patients. NDRG1 expression was correlated to histopathological type, Dukes' stage and HIF-1 alpha expression in US-Caucasian patients but not in US-African American patients. Interestingly, Kaplan-Meier survival analysis demonstrated that NDRG1 expression correlated significantly with poorer survival in US-African American patients but not in other patient groups. However, in p53-positive US cases, NDRG1 positivity correlated significantly with better survival. In addition, NDRG1 expression also correlated significantly with improved survival in US patients with stages III and IV tumors without chemotherapy. In Japanese patients with stages II and III tumors, strong NDRG1 staining in p53-positive tumors correlated significantly with improved survival but negatively in patients without chemotherapy. CONCLUSION: NDRG1 expression was correlated with various clinicopathological features and clinical outcomes in colorectal cancer depending on the race/ethnicity of the patients. NDRG1 may serve as a biological basis for the disparity of clinical outcomes of colorectal cancer patients with different ethnic backgrounds.

Tumor suppressor p53 represses transcription of RECQ4 helicase.

RECQ4 is a member of the RecQ helicase family, which has been implicated in the regulation of DNA replication, recombination and repair. p53 modulates the functions of RecQ helicases including BLM and WRN. In this study, we demonstrate that p53 can regulate the transcription of RECQ4. Using nontransformed, immortalized normal human fibroblasts, we show that p53-dependent downregulation of RECQ4 expression occurred in G1-arrested cells, both in the absence or presence of exogenous DNA damage. Wild-type p53 (but not the tumor-derived mutant forms) repressed RECQ4 promoter activity. The camptothecin or etoposide-dependent p53-mediated repression was attenuated by trichostatin A (TSA), an inhibitor of histone deacetylases (HDACs). Repression of the RECQ4 promoter was accompanied with an increased accumulation of HDAC1, and the loss of SP1 and p53 binding to the promoter. The simultaneous formation of a camptothecin-dependent p53-SP1 complex indicated its occurrence outside of the RECQ4 promoter. These data suggest that p53-mediated repression of RECQ4 transcription during DNA damage results from the modulation of the promoter occupancy of transcription activators and repressors.

Leu-574 of human HIF-1alpha is a molecular determinant of prolyl hydroxylation.

Hypoxia-inducible factor (HIF)-1alpha, a master regulator of oxygen homeostasis, regulates genes crucial for cell growth and survival. In normoxia, HIF-1alpha is constantly degraded via the ubiquitin-proteasome pathway. The von Hippel-Lindau (VHL) E3 ubiquitin ligase binds HIF-1alpha through specific recognition of hydroxylated Pro-402 or Pro-564, both of which are modified by the oxygen-dependent HIF prolyl hydroxylases (PHDs/HPHs). Despite the identification of a conserved Leu-X-X-Leu-Ala-Pro motif, the molecular requirement of HIF-1alpha for PHDs/HPHs binding remains elusive. Recently, we demonstrated that Leu-574 of human HIF-1alpha--10 residues downstream of Pro-564--is essential for VHL recognition. We show here that the role of Leu-574 is to recruit PHD2/HPH2 for Pro-564 hydroxylation. An antibody specific for hydroxylated Pro-564 has been used to determine the hydroxylation status; mutation or deletion of Leu-574 results in a significant decrease in the ratio of the hydroxylated HIF-1alpha to the total amount. The nine-residue spacing between Pro-564 and Leu-574 is not obligatory for prolyl hydroxylation. Furthermore, mutation of Leu-574 disrupts the binding of PHD2/HPH2, a key prolyl hydroxylase for oxygen-dependent proteolysis of HIF-1alpha. Hence, our findings indicate that Leu-574 is essential for recruiting PHD2/HPH2, thereby providing a molecular basis for modulating HIF-1alpha activity.

Role of GLI1 and NDRG1 in Increased Resistance to Apoptosis Induction.

We examined the effects of GLI1 expression in PW mouse embryo fibroblasts and H441 lung carcinoma cells. Ectopic expression of GLI1 in PW cells induced anchorage-independent growth and increased resistance to staurosporine-induced apoptosis, and overexpression of GLI1 in H441 cells caused resistance to apoptosis induced by staurosporine and etoposide. GLI1 expression in both H441 and PW cells was associated with increased expression of NDRG1, a gene known to be downregulated by the MYC family of proteins, indicating that upregulation of NDRG1 by GLI1 is not cell-type specific. Consistent with suppression of NDRG1 by c-MYC and N-MYC, increased NDRG1 expression correlated with decreased expression of c-MYC and N-MYC in GLI1-expressing H441 and GLI1-expressing PW cells, respectively. Downregulation of GLI1 expression in A549 cells by siRNA transfection increased sensitivity to etoposide-induced apoptosis, and downregulation of NDRG1 expression in H441 cells by siRNA transfection increased sensitivity to etoposide-induced apoptosis. Of clinical significance, inhibition of GLI1 and NDRG1 expression may increase sensitivity of cancer cells to chemotherapeutic drugs. Strategies that aim to inhibit GLI1 function and NDRG1 expression may be useful for targeted therapy of cancers induced by the SHH-GLI signaling pathway.

Lessons from the comparison of two randomized clinical trials using gemcitabine and cisplatin for advanced biliary tract cancer.

There had been no standard chemotherapy established for advanced biliary tract cancer (BTC) until 2009, when the combination of cisplatin and gemcitabine (GC) was adopted as a first line standard chemotherapy option based on the results from two randomized studies: ABC-02, a UK investigator-initiated trial and the largest randomized phase III study in this tumor type with 410 patients; and BT22, a Japanese, industry-sponsored, randomized phase II study with 83 patients. In this review, investigators from both studies collaborated to compare protocols, patient characteristics, and outcomes of both studies including sub-analyses of study results. Although both studies showed GC combination therapy to be more effective than monotherapy, a detailed comparison revealed disparities between efficacy and safety end-points between the studies, which did not necessarily arise from different populations but from differences in protocol design. This review provides clinicians with insights for advanced BTC clinical study design and interpretation of historical studies.

A phase I study of enzastaurin combined with pemetrexed in advanced non-small cell lung cancer.

INTRODUCTION: Enzastaurin is an oral serine/threonine kinase inhibitor, which suppress signaling through protein kinase C-beta and the phosphatidylinositol 3-kinase/AKT pathway. Preclinical studies suggested synergic antitumor activity of enzastaurin and pemetrexed. We conducted this phase I study to evaluate the safety, pharmacokinetics, and clinical activity of this combination in patients with previously treated advanced non-small cell lung cancer. METHODS: An oral daily dose of 500 mg enzastaurin was administered once daily (QD) or twice daily (BID) in combination with 500 mg/m pemetrexed on day 1 in repeated 21-day cycles. Cycle 1 started with a 7-day enzastaurin lead-in treatment that preceded pemetrexed administration: a loading dose of 1125 mg enzastaurin on day 1 followed by a 500 mg total daily dose on days 2-7. RESULTS: Twelve patients were treated QD (n = 6) or BID (n = 6). One dose-limiting toxicity (grade 3 QTc prolongation) was reported in the QD cohort. Grade 3/4 hematological toxicities were slightly increased in the BID cohort compared with the QD cohort. After beginning the combination therapy, enzastaurin exposures decreased slightly but remained above the target plasma concentration of 1400 nmol/L. Compared with QD, there was a higher exposure with BID. The enzastaurin dosing regimen (QD or BID) had no effect on pemetrexed pharmacokinetics. Two patients had partial responses as defined by RECIST. Five patients received more than 10 cycles of treatment without disease progression. CONCLUSIONS: Both schedules of enzastaurin in combination with pemetrexed were well tolerated and clinically active in patients with advanced non-small cell lung cancer.

A landmark point analysis with cytotoxic agents for advanced NSCLC.

INTRODUCTION: As a result of recent publications, we hypothesized that period of 8 weeks after initiation of treatment is a useful landmark point for cytotoxic agents for advanced non-small cell lung cancer (NSCLC). To test this hypothesis, we conducted landmark analyses with clinical trials employing cytotoxic agents. Our goal was to assess the proper design of clinical trials with cytotoxic agents for NSCLC for maximizing patients' benefit. METHODS: We conducted landmark analyses of a phase II study of pemetrexed in locally advanced or metastatic NSCLC and a phase III study of Four-Arm Cooperative Study for advanced NSCLC. A total of 806 patients who received chemotherapy (pemetrexed, cisplatin and irinotecan, paclitaxel and carboplatin, cisplatin and gemcitabine, cisplatin and vinorelbine) were included in this assessment. RESULTS: Tumor-shrinkage rate at 8 weeks was significantly associated with longer survival in the study with pemetrexed (p = 0.043), whereas tumor-shrinkage rate at 4 weeks did not correlated with survival (p = 0.139). Similarly, using the Four-Arm Cooperative Study data, the optimal landmark point was 8 weeks (p = 0.002), not 4 weeks (p = 0.190). CONCLUSION: The landmark point for NSCLC was 8 weeks with all cytotoxic agents in our analysis when the therapy was given as a frontline or subsequent therapy. Our result suggests the concept of a disease-specific landmark point, which may lead to a change of phase II/III clinical study design to evaluate cytotoxic agents and clinical investigators, and their sponsors may consider an early look to assess the efficacy of cytotoxic agents for NSCLC.

Hypoxic suppression of the cell cycle gene CDC25A in tumor cells.

Hypoxia, a key microenvironmental factor for tumor development, not only stimulates angiogenesis and glycolysis for tumor expansion, but also induces cell cycle arrest and genetic instability for tumor progression. Several independent studies have shown hypoxic blockade of cell cycle progression at the G1/S transition, arising from the inactivation of S-phase-promoting cyclin E-CDK2 kinase complex. Despite these findings, the biochemical pathways leading to the cell cycle arrest remain poorly defined. We recently showed that hypoxic activates the expression of CDNK1A encoding the CDK2 inhibitor p21Cip1, through a novel HIF-1alpha-Myc pathway that involves Myc displacement from the CDNK1A promoter by the hypoxia-inducible transcription factor HIF-1alpha. In pursuit of further understanding of the hypoxic effects on cell cycle in tumor cells, here we report that hypoxia inhibits the expression of CDC25A, another cell cycle gene encoding a tyrosine phosphatase that maintains CDK2 activity. In accordance with the HIF-1alpha-Myc pathway, hypoxia requires HIF-1alpha for CDC25A repression, resulting in a selective displacement of an activating Myc from the CDC25A promoter without affecting a canonical Myc binding in the intron. Intriguingly, HIF-1alpha alone fails to recapitulate the hypoxic effect, indicating that HIF-1alpha is necessary but insufficient for the hypoxic repression. Taken together, our studies indicate that hypoxia inhibits cell cycle progression by controlling the expression of various cell cycle genes.

Genetic instability: the dark side of the hypoxic response.

Under low oxygen tension, the activated transcription factor HIF-1alpha upregulates an array of hypoxia-inducible genes via heterodimerization with ARNT and binding to the hypoxia-responsive element in the promoter. Alternatively, HIF-1alpha regulates hypoxia-responsive genes by functionally antagonizing the oncoprotein Myc via protein-protein interactions. This so-called HIF-1alpha-Myc mechanism apparently not only accounts for the gene upregulation, but also for the gene downregulation during hypoxia, depending upon the activating and repressive nature of Myc in gene expression. Indeed, our recent study demonstrated that both mismatch repair genes, MSH2 and MSH6, are inhibited by this mechanism in a p53-dependent manner. In particular, the constitutively bound transcription factor Sp1 serves as a molecular switch by recruiting HIF-1alpha in hypoxia to displace the transcription activator Myc from the promoter. Therefore, our findings shed light on the mechanisms underlying hypoxia-induced genetic instability, an "adverse"effect of the hypoxic response, and yet a germane process to tumor survival and progression.

HIF-1alpha induces genetic instability by transcriptionally downregulating MutSalpha expression.

Hypoxia promotes genetic instability by undefined mechanisms. The transcription factor HIF-1alpha is crucial for the cellular response to hypoxia and is frequently overexpressed in human cancers, resulting in the activation of genes essential for cell survival. Here, we demonstrate that HIF-1alpha is responsible for genetic instability at the nucleotide level by inhibiting MSH2 and MSH6, thereby decreasing levels of the MSH2-MSH6 complex, MutSalpha, which recognizes base mismatches. HIF-1alpha displaces the transcriptional activator Myc from Sp1 binding to repress MutSalpha expression in a p53-dependent manner; Sp1 serves as a molecular switch by recruiting HIF-1alpha to the gene promoter under hypoxia. Furthermore, in human sporadic colon cancers, HIF-1alpha overexpression is statistically associated with the loss of MSH2 expression, especially when p53 is immunochemically undetectable. These findings indicate that the regulation of DNA repair is an integral part of the hypoxic response, providing molecular insights into the mechanisms underlying hypoxia-induced genetic instability.

Dynamic balancing of the dual nature of HIF-1alpha for cell survival.

In hypoxic cells, HIF-1alpha escapes from oxygen-dependent proteolysis and binds to the hypoxia-responsive element (HRE) for transcriptional activation of target genes involved in angiogenesis and glycolysis. We recently demonstrated that the G(1) checkpoint gene p21(cip1)is activated by HIF-1alpha with a novel mechanism that involves the HIF-1alpha PAS domains to displace Myc binding from p21(cip1) promoter. This HIF-1alpha-Myc pathway may account for up- and down-regulation of other hypoxia-responsive genes that lack the HRE. Moreover, the role of HIF-1alpha in cell cycle control indicates a dual, yet seemingly conflicting, nature of HIF-1alpha: promoting cell growth and arrest in concomitance. We speculate that a dynamic balance between the two processes is achieved by a "stop-and-go" strategy to maintain cell growth and survival. Tumor cells may adopt such scheme to evade the killing by chemotherapeutic agents.

Microsatellite analysis of fecal DNA for colorectal cancer detection.

BACKGROUND AND OBJECTIVES: The advent of noninvasive methods of testing for colorectal cancer that have a high level of specificity and sensitivity is eagerly awaited. METHODS: Thirty patients with sporadic colorectal cancer and 11 patients with hereditary nonpolyposis colon cancer (HNPCC) enrolled in this study. We analyzed the loss of heterozygosity (LOH) in matched genomic DNA extracted from blood and surgical specimens (tumor and tumor-free colonic mucosa), and the corresponding oral rinse and stool specimens using seven microsatellite loci (APC, p53, DCC, hMLH1, D9S162, D9S171, and IFNA). To reduce the normal colonocyte DNA contamination of the stool samples, we compared three different methods for fecal genomic DNA extraction. As normal controls, we analyzed the LOH using the oral rinse and stool samples from 15 individuals without cancer. RESULTS: The LOH determined from the oral rinse and the stool samples matched those determined from the blood and the neoplastic tissue. All patients with HNPCC had microsatellite alterations at hMLH-1 in tumor DNA and corresponding fecal DNA. There were significant differences in the frequency of p53-LOH and D9S171-LOH between the group with sporadic disease and those with HNSCC (P = 0.0031 and 0.0294, respectively). Two cases with D9S162-LOH were detected in individuals without cancer. For the patients with sporadic disease, using p53 and adenomatous polyposis coli (APC), the sensitivity of the fecal DNA analysis was 96.7% (95% CI, 83-100) with a specificity of 100%. CONCLUSION: We demonstrate that LOH analysis using oral rinse and stool samples may be a suitable screening tool for colorectal cancer.