Loss of glucose 6-phosphate dehydrogenase function increases oxidative stress and glutaminolysis in metastasizing melanoma cells

The pentose phosphate pathway is a major source of NADPH for oxidative stress resistance in cancer cells but there is limited insight into its role in metastasis, when some cancer cells experience high levels of oxidative stress. To address this, we mutated the substrate binding site of glucose 6-phosphate dehydrogenase (G6PD), which catalyzes the first step of the pentose phosphate pathway, in patient-derived melanomas. G6PD mutant melanomas had significantly decreased G6PD enzymatic activity and depletion of intermediates in the oxidative pentose phosphate pathway. Reduced G6PD function had little effect on the formation of primary subcutaneous tumors, but when these tumors spontaneously metastasized, the frequency of circulating melanoma cells in the blood and metastatic disease burden were significantly reduced. G6PD mutant melanomas exhibited increased levels of reactive oxygen species, decreased NADPH levels, and depleted glutathione as compared to control melanomas. G6PD mutant melanomas compensated for this increase in oxidative stress by increasing malic enzyme activity and glutamine consumption. This generated a new metabolic vulnerability as G6PD mutant melanomas were more dependent upon glutaminase than control melanomas, both for oxidative stress management and anaplerosis. The oxidative pentose phosphate pathway, malic enzyme, and glutaminolysis thus confer layered protection against oxidative stress during metastasis.

The pentose phosphate pathway is an important source of NADPH for oxidative stress resistance (1–5). The oxidative branch of the pentose phosphate pathway contains two enzymes that generate NADPH from NADP⁺, glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (PGD) (SI Appendix, Fig. S1). NADPH is an important source of reducing equivalents for oxidative stress resistance because it is used by cells to convert oxidized glutathione (GSSG) to glutathione (GSH), an abundant redox buffer. Complete deficiency for G6PD is embryonic-lethal in mice (2, 6, 7) but hypomorphic G6PD mutations are common in certain human populations, perhaps because they protect against malaria (8, 9). These partial loss-of-function G6PD mutations are well tolerated in adults, though they sensitize red blood cells to hemolysis from oxidative stress under certain circumstances (10).Several studies have reported a lower incidence and mortality for certain cancers in people with hypomorphic mutations in G6PD (11–14), suggesting that cancer cells depend upon G6PD to manage oxidative stress. Cells experience high levels of oxidative stress during certain phases of cancer development and progression, including during metastasis (15–17). Antioxidant mechanisms thus promote the survival of cells during oncogenic transformation (18, 19) as well as during metastasis (15, 16). For example, relative to primary cutaneous tumors, metastasizing melanoma cells exhibit increased dependence upon the folate pathway (15), monocarboxylate transporter-1 (MCT1) (20), and glutathione peroxidase-4 (GPX4) (21), each of which directly or indirectly attenuate oxidative stress. By better understanding the mechanisms that confer oxidative stress resistance in cancer cells, it may be possible to develop pro-oxidant therapies that inhibit cancer progression by exacerbating the oxidative stress experienced by cancer cells.G6PD (22) or PGD deficiency (23–25) reduce the growth of some cancers, including melanoma, but G6PD deficiency has little effect on primary tumor formation by K-Ras–driven epithelial cancers (26). This is at least partly because loss of G6PD in these cancers leads to compensatory increases in the function of other NADPH-generating enzymes, including malic enzyme and isocitrate dehydrogenase (1, 27). Nonetheless, pentose phosphate pathway function may increase during metastasis (20, 28–30) and higher G6PD expression is associated with worse outcomes in several cancers (31–33), raising the question of whether metastasizing cells are particularly dependent upon G6PD. G6PD is not essential for metastasis in a breast cancer cell line but it reduces their capacity to form metastatic tumors (26).Melanoma cells show little evidence of oxidative stress in established primary tumors but exhibit increased levels of reactive oxygen species (ROS) and dependence upon antioxidant mechanisms during metastasis (15, 20, 21). To test if these cells are more dependent upon the pentose phosphate pathway during metastasis, we generated three G6PD mutant melanomas, including two patient-derived xenografts and one human melanoma cell line. Reduced G6PD function had little effect on the formation or growth of primary subcutaneous tumors but significantly increased ROS levels and reduced spontaneous metastasis. G6PD mutant melanomas compensated by increasing malic enzyme activity and glutamine consumption, both to increase oxidative stress resistance and to replenish tricarboxylic acid (TCA) cycle intermediates through anaplerosis. Melanoma cells thus have redundant layers of protection against oxidative stress during metastasis, including the abilities to alter fuel consumption and antioxidant pathway utilization.     

The truth about snitches: an archival analysis of informant testimony

Informants are witnesses who often testify in exchange for an incentive (i.e. jailhouse informant, cooperating witness). Despite the widespread use of informants, little is known about the circumstances surrounding their use at trial. This study content-analyzed trials from 22 DNA exoneration cases involving 53 informants. Because these defendants were exonerated, the prosecution informant testimony is demonstrably false. Informant characteristics including motivation for testifying, criminal history, relationship with the defendant and testimony were coded. Most informants were prosecution jailhouse informants; however, there were also defence jailhouse informants and prosecution cooperating witnesses. Regardless of informant type, most denied receiving an incentive, had criminal histories, were friends/acquaintances of the defendant and had testimonial inconsistencies. In closing statements, attorneys relied on informant testimony by either emphasizing or questioning its reliability. The impact of informant testimony on jurors’ decisions is discussed in terms of truth-default theory (TDT), the fundamental attribution error and prosecutorial vouching.Key words: jailhouse informant, cooperating witness, truth-default theory, secondary confessions

In the Nassau County Courthouse on 12 November 1986, Samuel Newsome took the witness stand and promised to tell the truth, the whole truth and nothing but the truth. He testified that he had heard the defendant, Dennis Halstead, confess to the rape and murder of a young girl while the two were playing spades in jail together. Newsome had a long criminal history, and the defence attorney suggested at trial that the criminal charges against him were being dropped in exchange for his testimony. The prosecution had only circumstantial evidence and therefore their star witness was Newsome. He proved to be an effective witness, providing a detailed account of how the victim was raped and killed, including where she had been picked up, the vehicle that was used and even that the defendant had smiled while committing the act. Despite his adamant claims of innocence, Halstead was convicted of rape and murder and sentenced to 33⅓ years to life. Nineteen years later, DNA evidence conclusively proved that Halstead could not have been the perpetrator; he was exonerated on 29 December 2005. Research has shown that false testimony from informants like Samuel Newsome is one of the leading causes of the growing number of wrongful convictions (Gross et al., 2005; Warden, 2005).Since DNA testing became available in the late 1980s, there have been over 350 DNA exonerations of wrongfully convicted individuals. The Innocence Project lists informant testimony among the major contributing factors to wrongful convictions, along with eyewitness misidentification, faulty forensic science, false confessions, government misconduct and inadequate defence (http://www.innocenceproject.org). In capital cases, false testimony from informants is the leading cause of wrongful convictions, with informant testimony present in 49.5% of cases since the mid-1970s (Warden, 2005). According to the Innocence Project, informant testimony contributed to more than 15% of the wrongful convictions that were later overturned through DNA testing (http://www.innocenceproject.org). Courts have repeatedly indicated that offering incentives to informants provides enormous motivation to give false testimony and evidence (Giglio v. United States, 1972; United States v. Singleton, 1998).Generally, accurate informant testimony is an effective means of securing convictions against guilty defendants. Ideally, false informant testimony should be detected as false by the judges and juries charged with hearing criminal cases. However, as the many wrongful convictions clearly demonstrate, false informant testimony can be compelling evidence against innocent defendants. In order to better understand the influence of informant testimony, DNA exoneration cases involving 53 informants were content analyzed. These cases provide a critical context in which to evaluate and understand informant testimony because any testimony implicating the defendant is demonstrably false, as proven by the subsequent DNA exoneration.Informant witnessesInformants, in the most general sense, are individuals who provide information about criminal activity. Sometimes informants are recruited by the government to infiltrate criminal circles and collect incriminating evidence, and sometimes informants initiate contact with the government because they possess incriminating information and have the hope of gaining some incentive in exchange for reporting it. This paper investigates the latter type; that is, informants who collect or encounter incriminating evidence by themselves rather than being planted by the government. The evidence given by these informants has often been referred to as ‘bartered testimony’, because the informant has negotiated a deal in return for their cooperation (Roth, 2016).Within this definition of informant there are three subtypes: jailhouse informants, cooperating witnesses and accomplice witnesses (Roth, 2016). Jailhouse informants are individuals who gain evidence about a fellow inmate’s case while they are both in custody and who often come forward in return for some promised incentive. Jailhouse informants’ testimony typically includes a secondary confession, meaning that they claim to have heard the defendant confess to committing the crime (Neuschatz et al., 2008). Cooperating witnesses are citizens with incriminating evidence about a defendant’s case but who require an incentive to testify. Cooperating witnesses typically learn about a case through some connection to the defendant (e.g. they are a friend) or through their own experiences (e.g. as an eyewitness). For example, an eyewitness may be reluctant to testify for fear of retaliation, thus prosecutors may provide some type of incentive (e.g. relocation funds) to secure their testimony. Finally, accomplice witnesses are individuals who allegedly committed the crime with the defendant but testify against the defendant for some leniency in their own case. The present research is focused on jailhouse informants and cooperating witnesses (but not accomplice witnesses), and therefore the term ‘informant’ is used to refer to these two types collectively.Despite the prevalence of informants, especially in capital cases, their contribution to wrongful convictions has not been sufficiently studied (Roth, 2016). By contrast, eyewitness identification – another leading cause of wrongful conviction – has received a great deal of attention from both the scientific and legal communities. A search of PsycINFO revealed 786 peer-reviewed studies pertaining to eyewitness identification; however, a search for jailhouse informants and cooperating witnesses produced only 9 peer-reviewed studies, 6 of which address the impact of informant testimony on juror decision-making. What little research has been done demonstrated that jurors’ verdicts are uninfluenced by informants’ incentives for testifying (i.e. no incentive vs. a five-year reduction in sentence) or by their role in the case (i.e. jailhouse informant, accomplice witness or civilian; Neuschatz et al., 2012). These studies have also demonstrated that jurors hold a number of erroneous beliefs about informant testimony (Key et al., 2018), are unable to detect that informants may be lying and rely heavily on informants’ testimony when making verdict decisions (Wetmore et al., 2014). Therefore, it is necessary to understand the information that informants provide and the reasons why jurors may be unable to detect their deception. The current analysis addresses these two questions by documenting what informants testified about and how attorneys used the testimony in their arguments to jurors.Deception detectionThere are several findings from the deception detection and attribution literatures that elucidate why jurors tend to believe that informants are telling the truth: they rely on inappropriate cues, exist in a truth-default state, identify when testimony matches the fact pattern of the case and succumb to the fundamental attribution error (Gilbert & Malone, 1995). First, in order for jurors to assess informant testimony accurately they must be able to identify cues that indicate truth telling and deception. Unfortunately, people are generally poor at detecting deception, as accuracy rates are usually at chance levels in studies which have examined this ability (see Vrij et al., 2010). One reason for this is that people often rely on cues which they believe to be indicative of deception, but in fact are not. For instance, it has been found that behaviors which are often believed to be associated with lying (e.g. averting one’s eyes, shifting in one’s seat or fidgeting) are in fact not at all related to deception (DePaulo et al., 2003; Vrij et al., 2000).Second, not only do people rely on the wrong cues but they are also naturally inclined to believe that other people are telling the truth – that is, they have a truth bias. According to Levine (2014), people naturally exist in a truth-default state, meaning that they initially evaluate all incoming messages as truthful unless there is a reason to suspect deception. Furthermore, people do not actively look for deception; rather, they have to be compelled to do so. The inclination to believe that people are being truthful and honest may be strengthened in the courtroom, where witnesses must take an oath to tell the truth and where jurors rely on prosecutorial vouching – that is, the belief that prosecutors rigorously vet their witnesses and would not let a dishonest witness testify (Covey, 2014; Roth, 2016).Third, it may be especially difficult to detect deception when it is embedded in a narrative that is factually accurate. Vrij et al. (2010) have suggested that rather than telling lies which are completely untrue, people often change a few details of an otherwise true account. This strategy tends to be true of criminals (see Hartwig et al., 2007). For example, it has been found that when criminals assume a false identity they only change a small portion of their actual identity (Wang et al., 2004). Informants may adopt a similar strategy, weaving a few lies into an otherwise factually accurate depiction of the case (e.g. facts gathered from media coverage, non-public facts given by the police), making them more believable and making it more difficult for jurors to detect their deception. Along with the presence of accurate facts and details in their accounts, informants are also more likely to be believed if they are thought to be testifying against their self-interest (e.g. they may be harmed in jail or a family member may be put in danger as a result of testifying). According to Kassin (2015), statements made against self-interest are reflexively believed.Lastly, determining whether the evidence points to deception or truth may be very difficult; not only can deception be embedded in accurate facts but individuals often make the fundamental attribution error, or have a correspondence bias, when evaluating others – even informants (Neuschatz et al., 2008; Ross, 1977). The fundamental attribution error refers to the tendency for individuals to overestimate dispositional factors and underestimate situational factors when explaining other people’s behavior (Ross, 1977). Therefore, when evaluating an informant’s motives for testifying, jurors will identify dispositional reasons (e.g. trying to do the right thing) rather than relying on situational factors (e.g. incentives to cooperate).Given the issues laid out above, we coded all the DNA exoneration cases involving informants in order to provide some insight into why their testimony was believed by the jurors. Unfortunately, it is impossible to know from this set of cases whether or not the jurors detected the appropriate cues or how they evaluated the evidence. However, it is possible to address various aspects of the cases that relate specifically to the narratives provided by the informants and attorneys, whether statements against self-interest were made or not and whether dispositional or situational explanations for testifying were present. In order to evaluate the narrative components of the case, we coded the informants’ testimony for the number of case details and their accuracy, the presence of inconsistencies, how they purportedly heard the confession (if at all), whether or not they received an incentive and whether or not they testified against their self-interest. In addition, we also coded the closing arguments made by the prosecuting and defence attorneys to further evaluate the case facts presented during the trial, the presence of inconsistencies in the informants’ testimony and whether or not any ulterior motives or alternative explanations for the informants’ testimony were explored.It is clear that in some, if not all, of the cases examined herein, the juries failed to detect the deceptive nature of the informants’ testimony. Nonetheless, it is still an important first step in better understanding the impact of informant testimony to analyze what the informants actually said at trial and how they were portrayed by both the prosecution and the defence. This is important because the way in which attorneys portray informants likely influences jurors’ perceptions of their truthfulness, which may in turn affect verdict decisions. Because there is scant research on the content of informant testimony, this study is by necessity exploratory. Even so, some predictions could be derived from the deception detection and attribution literatures. First, because it has been shown that the majority of informants are repeat players (Roth, 2016), we hypothesized that the informants in the present study would also be found to have prior convictions and previous involvement in the criminal justice system (Hypothesis 1). Second, we expected to find that the informants made statements against their self-interest in order to increase their credibility (Hypothesis 2). Kassin (2015) argues that false confessions are persuasive because people reflexively believe that statements made against self-interest are true; therefore, to the extent that jurors perceive informants to be making such statements, they may be more likely to accept their testimony as truthful. Third, we predicted that the informants would be found to have given dispositional reasons for their testimony (e.g. they felt bad for the family) rather than situational explanations (e.g. receiving an incentive; Hypothesis 3). Finally, we expected to find that the prosecutors and defence attorneys specifically addressed the informants’ motivations in their cross-examinations and closing arguments (Hypothesis 4).     

The modified radical vulvectomy with groin dissection: an eight-year experience

Fifty-one patients were admitted to a single practice at St. Joseph's Hospital between April 1, 1978, and April 1, 1986 with a diagnosis of squamous cell carcinoma of the vulva greater than 1 mm in depth. Five advanced lesions were treated with combinations of radiation and surgery. Four patients had recurrent squamous cell carcinoma. Of 42 patients treated surgically with intention of cure, 14 were treated with complete radical vulvectomy and bilateral inguinofemoral lymphadenectomies, and 28 patients were treated with complete radical vulvectomy and bilateral inguinofemoral lymphadenectomies, and 28 patients were treated in 26 instances with bilateral inguinofemoral lymphadenectomies in one of five different excision patterns individualized to the site of primary tumor. None of the 28 patients have had a recurrence. Five had positive nodes. Eight have died of unrelated causes. Lesions in 25 cases were stage I or II and in three cases they were stage III. Modified radical vulvectomy and bilateral groin dissection is a safe approach for most patients with stage I or II and occasionally even stage III lesions.     

Sexuality Among Middle-Aged and Older Adults With Diagnosed and Undiagnosed Diabetes: A national, population-based study

OBJECTIVE

To describe sexual activity, behavior, and problems among middle-age and older adults by diabetes status.

RESEARCH DESIGN AND METHODS

This was a substudy of 1,993 community-residing adults, aged 57–85 years, from a cross-sectional, nationally representative sample (N = 3,005). In-home interviews, observed medications, and A1C were used to stratify by diagnosed diabetes, undiagnosed diabetes, or no diabetes. Logistic regression was used to model associations between diabetes conditions and sexual characteristics, separately by gender.

RESULTS

The survey response rate was 75.5%. More than 60% of partnered individuals with diagnosed diabetes were sexually active. Women with diagnosed diabetes were less likely than men with diagnosed diabetes (adjusted odds ratio 0.28 [95% CI 0.16–0.49]) and other women (0.63 [0.45–0.87]) to be sexually active. Partnered sexual behaviors did not differ by gender or diabetes status. The prevalence of orgasm problems was similarly elevated among men with diagnosed and undiagnosed diabetes compared with that for other men, but erectile difficulties were elevated only among men with diagnosed diabetes (2.51 [1.53 to 4.14]). Women with undiagnosed diabetes were less likely to have discussed sex with a physician (11%) than women with diagnosed diabetes (19%) and men with undiagnosed (28%) or diagnosed (47%) diabetes.

CONCLUSIONS

Many middle-age and older adults with diabetes are sexually active and engage in sexual behaviors similarly to individuals without diabetes. Women with diabetes were more likely than men to cease all sexual activity. Older women with diabetes are as likely to have sexual problems but are significantly less likely than men to discuss them.Advances in treatment for diabetes have prolonged and improved quality of life for many of the ∼12 million affected individuals aged ≥60 years in the U.S. Clinical guidelines for diabetes care include assessment and treatment of erectile problems in men (1). Sexual problems may be a warning sign of diabetes or a consequence that can lead to depression, lack of adherence to treatment, and strained intimate relationships. In contrast, older women''s sexual issues have been largely overlooked in screening for and treating diabetes (1,2). Failure to recognize and address sexual issues among middle-aged and older adults with diabetes may impair quality of life and adaptation to the disease.Some adults with diabetes maintain sexual relationships throughout their lives (3). Prior studies have focused on the pathophysiological effects of diabetes on male sexual function, primarily erection and sexual desire. The effects of diabetes on women''s sexual functioning are poorly understood and probably multifactorial (2). Sexual problems in adults with diabetes have been associated with age, disease duration, and comorbidity (1). The effects of chronic hyperglycemia, degree of diabetes control, or use of glucose-lowering drugs are less clear (4), in part because individuals with undiagnosed or preclinical diabetes are typically aggregated with control subjects in other studies (1). Psychosocial correlates of sexual problems in individuals with diagnosed diabetes have been found in younger adults. Studies including older adults find associations with depression (1), vulnerability, lifestyle restrictions due to disease management (5), and marital conflict (6).Prior data on sexuality in individuals with diabetes were derived primarily from studies that are small, have not included very old individuals or aggregated individuals ≥65 years, lacked a comparison group, and relied on convenience or other nongeneralizable samples (1,2). Comprehensive, population-based data are needed to further physicians'' understanding of the sexual norms and problems of older adults with diagnosed and undiagnosed diabetes. Virtually nothing is known about sexual function among individuals with undiagnosed diabetes; this information could be relevant for diagnosis, motivation to engage in treatment, and prevention of sexual and nonsexual diabetes-related complications. The National Social Life, Health and Aging Project (NSHAP) provides disease-specific data on the sexual activity, behaviors, and problems of middle-aged and older adults affected by diabetes.     

Effects of Tick-Control Interventions on Tick Abundance,Human Encounters with Ticks,and Incidence of Tickborne Diseases in Residential Neighborhoods,New York,USA

Tickborne diseases (TBDs) such as Lyme disease result in ≈500,000 diagnoses annually in the United States. Various methods can reduce the abundance of ticks at small spatial scales, but whether these methods lower incidence of TBDs is poorly understood. We conducted a randomized, replicated, fully crossed, placebo-controlled, masked experiment to test whether 2 environmentally safe interventions, the Tick Control System (TCS) and Met52 fungal spray, used separately or together, affected risk for and incidence of TBDs in humans and pets in 24 residential neighborhoods. All participating properties in a neighborhood received the same treatment. TCS was associated with fewer questing ticks and fewer ticks feeding on rodents. The interventions did not result in a significant difference in incidence of human TBDs but did significantly reduce incidence in pets. Our study is consistent with previous evidence suggesting that reducing tick abundance in residential areas might not reduce incidence of TBDs in humans.     

Single-Center Comparison of Overall Survival and Toxicities in Patients with Infiltrative Hepatocellular Carcinoma Treated with Yttrium-90 Radioembolization or Drug-Eluting Embolic Transarterial Chemoembolization

Purpose

To compare overall survival and toxicities after yttrium-90 (⁹⁰Y) radioembolization and chemoembolization with drug-eluting embolics (DEE) in patients with infiltrative hepatocellular carcinoma (HCC).