The role of genetic diversity in cancer

The role of genetic heterogeneity within neoplasms is increasingly recognized as important for understanding the dynamics of cancer progression, cancer stem cells, and therapeutic resistance, and there is interest in intratumoral heterogeneity measurements as potential biomarkers for risk stratification. In this issue of the JCI, Park et al. characterize this genetic diversity in carcinoma in situ and in invasive regions from 3 types of human breast cancers and lay the groundwork for translation of these measures to the clinic. Although there are clear suggestions that diversity may be important in the establishment and progression of cancers (1), it is unclear how diversity should be defined. Should we use genetic or phenotypic markers? How can we account for epigenetic diversity? Should we look at large samples or small ones, and how many biopsies should we sample in order to accurately estimate the underlying diversity of the neoplasm? The problems of defining diversity and characterizing the number of cancer clones are not new and have parallels to problems encountered in other fields. Ecologists have created various measures to estimate the number and abundance of species (2); even in literature, calculating the number of words Shakespeare knew from his existing corpus (3) is analogous to determining the number of cancer clones in a tumor from a small number of samples. Heterogeneity in neoplasms has long been recognized (4–7), but has only recently been systematically investigated, and much can be gained by applying these methodologies to cancer and precancerous lesions (8–10). As Park et al. clearly demonstrate in their study in this issue of the JCI (11), this is not merely an academic exercise, as the level of heterogeneity in a neoplasm may have important clinical implications as a biomarker of risk stratification.     

Diabetes and risk of prostate cancer: a study using the National Health Insurance

OBJECTIVE

The link between diabetes and prostate cancer is rarely studied in Asians.

RESEARCH DESIGN AND METHODS

The trend of age-standardized prostate cancer incidence in 1995–2006 in the Taiwanese general population was calculated. A random sample of 1,000,000 subjects covered by the National Health Insurance in 2005 was recruited. A total of 494,630 men for all ages and 204,741 men ≥40 years old and without prostate cancer at the beginning of 2003 were followed to the end of 2005. Cumulative incidence and risk ratio between diabetic and nondiabetic men were calculated. Logistic regression estimated the adjusted odds ratios for risk factors.

RESULTS

The trend of prostate cancer incidence increased significantly (P < 0.0001). The cumulative incidence markedly increased with age in either the diabetic or nondiabetic men. The respective risk ratio (95% CI) for all ages and age 40–64, 65–74, and ≥75 years was 5.83 (5.10–6.66), 2.09 (1.60–2.74), 1.35 (1.07–1.71), and 1.39 (1.12–1.71). In logistic regression for all ages or for age ≥40 years, age, diabetes, nephropathy, ischemic heart disease, dyslipidemia, living region, and occupation were significantly associated with increased risk, but medications including insulin and oral antidiabetic agents were not.

CONCLUSIONS

Prostate cancer incidence is increasing in Taiwan. A positive link between diabetes and prostate cancer is observed, which is more remarkable in the youngest age of 40–64 years. The association between prostate cancer and comorbidities commonly seen in diabetic patients suggests a more complicated scenario in the link between prostate cancer and diabetes at different disease stages.The association between diabetes and prostate cancer has been inconsistently reported, even though two meta-analyses suggested that diabetic patients have a lower risk of prostate cancer of 9% (1) and 16% (2), respectively.While the two meta-analyses were examined, many studies were case-control and only three focused on the follow-up of cohorts of diabetic patients (3–5). Among the three cohorts, the cases of prostate cancer were 9 (3), 498 (4), and 2,455 (5), respectively; and only the last (5) showed a significant 9% risk reduction in diabetic patients. Except for the first study being conducted in residents with diabetes in Rochester, Minnesota (3), the diabetic patients in the other two were from hospitalized patients in Denmark (4) and Sweden (5), respectively. The meta-analyses have limitations including a mixture of case-control and cohort designs, a mixture of incident and dead cases, a small number of prostate cancer in most studies, and different sources of subjects with potential selection bias. Although the contamination of type 1 diabetes is possibly minimal because >90% of overall patients have type 2 diabetes, residual confounding could not be excluded if the two types of diabetes are not differentiated.Although some recent studies still suggested a lower risk of prostate cancer in diabetic patients including Caucasians (6,7), Iranians (8), Israelis (9), African Americans, Native Hawaiians, and Japanese Americans (6), the lower risk in African Americans and Native Hawaiians (6) was not significant. Two Japanese studies did not find any significant association (10,11). The Ohsaki Cohort Study suggested that diabetes was not predictive for total prostate cancer, but diabetic patients did show a higher risk of advanced cancer (11).Because diabetic patients are prone to develop cancer involving pancreas, liver, breast, colorectum, bladder, and endometrium (12–15) and the protective effect of diabetes on prostate cancer requires confirmation, this study evaluated the possible link between diabetes and prostate cancer, and the potential risk factors, by using the reimbursement database of the National Health Insurance (NHI) in Taiwan.     

Lower risk of cancer in patients on metformin in comparison with those on sulfonylurea derivatives: results from a large population-based follow-up study

OBJECTIVE

Numerous studies have suggested a decreased risk of cancer in patients with diabetes on metformin. Because different comparison groups were used, the effect magnitude is difficult to estimate. Therefore, the objective of this study was to further analyze whether, and to what extent, use of metformin is associated with a decreased risk of cancer in a cohort of incident users of metformin compared with users of sulfonylurea derivatives.

RESEARCH DESIGN AND METHODS

Data for this study were obtained from dispensing records from community pharmacies individually linked to hospital discharge records from 2.5 million individuals in the Netherlands. The association between the risk of cancer in those using metformin compared with those using sulfonylurea derivatives was analyzed using Cox proportional hazard models with cumulative duration of drug use as a time-varying determinant.

RESULTS

Use of metformin was associated with a lower risk of cancer in general (hazard ratio 0.90 [95% CI 0.88–0.91]) compared with use of sulfonylurea derivatives. When specific cancers were used as end points, similar estimates were found. Dosage-response relations were identified for users of metformin but not for users of sulfonylurea derivatives.

CONCLUSIONS

In our study, cumulative exposure to metformin was associated with a lower risk of specific cancers and cancer in general, compared with cumulative exposure to sulfonylurea derivatives. However, whether this should indeed be seen as a decreased risk of cancer for the use of metformin or as an increased risk of cancer for the use sulfonylurea derivatives remains to be elucidated.As the drug of first choice in type 2 diabetes, metformin is the most widely prescribed oral glucose-lowering drug (OGLD) (1,2). However, the decision to prescribe metformin also depends on patient characteristics: metformin use is contraindicated in those with renal failure, cardiac, or hepatic failure (2).A statistically nonsignificant relationship between use of metformin and the risk of colon cancer was described in 2004 (3). However, 1 year later, metformin was found to be associated with a decreased risk of cancer in general in a case-control study in a diabetic population (4). Numerous studies followed; among which studies confirming the association between use of metformin and a decreased risk of cancer in general (5–8) or in specific cancers (5,6,9–14). However, for breast cancer (5,6) and prostate cancer (5,14), the decreased risk was not consistently demonstrated; for other cancers, no association with use of metformin was found (6,12). Hence, there is heterogeneity among published studies on cancer in patients with diabetes on metformin (15), partly because different comparison groups were used, such as nonmetformin users, users of other OGLDs, or users of insulin. Higher endogenous insulin levels have been linked to an increased risk of certain cancers (16). Moreover, specifically for insulin glargine, the debate whether this specific insulin increases the risk of cancer is ongoing (17–21).Owing to factors such as different drugs used to attain metabolic control, the duration of diabetes, and the presence of other diseases, the assessment of cancer risk in diabetic patients remains difficult. Therefore, the objective of this study was to analyze whether, and to what extent, use of metformin is associated with a decreased risk of cancer in a cohort of incident users of metformin compared with use of sulfonylurea derivatives.     

Homocysteine-mediated thrombosis and angiostasis in vascular pathobiology

The mechanisms by which homocysteine contributes to atherothrombosis are complex and their in vivo relevance uncertain. In this issue of the JCI, Jacovina and colleagues report a unique in vivo mechanism by which homocysteine may contribute to vascular disease (see the related article beginning on page 3384). This group had previously reported that homocysteine impairs endothelial cell surface plasminogen activation by posttranslationally modifying annexin A2, the coreceptor for plasminogen and tissue plasminogen activator. They now show that an annexin A2–deficient mouse rendered hyperhomocysteinemic by dietary means has impaired fibrinolysis, perivascular fibrin persistence, and attenuated angiogenesis (angiostasis). Potential mechanisms by which homocysteine-dependent changes in endothelial phenotype link thrombosis to angiostasis are reviewed and their relationship to homocysteine-dependent vascular disease considered. Over the past 30 years, evidence from many in vitro and in vivo studies supports an association between mild to moderate hyperhomocysteinemia and vascular dysfunction and disease (1). A key mechanism that predisposes to vascular disease in hyperhomocysteinemia is endothelial dysfunction (2). Homocysteine induces endothelial dysfunction in part by promoting oxidant stress, as illustrated in cellular studies (3, 4) and in genetic animal models (2, 5, 6). The mechanisms for homocysteine-induced oxidant stress are complex and include inhibiting the translation of glutathione peroxidase-1 (7), a major antioxidant enzyme in vascular cells that regulates mitochondrial reactive oxygen species flux (8) and whose overexpression rescues the normal vascular phenotype of hyperhomocysteinemic mice (9); upregulation of NADPH oxidase expression (10); enhanced expression of inducible nitric oxide synthase (10) and uncoupling of nitric oxide synthases (11); and decreased glutathione levels owing to decreased cysteine synthesis (12), thereby shifting the redox balance of endothelial cells toward oxidant stress. Among the specific phenotypic properties of the endothelial cell that go awry in hyperhomocysteinemia are its antithrombotic properties. Hyperhomocysteinemia produces a prothrombotic state (13), some mechanisms for which include enhanced platelet activation, enhanced coagulation (likely as a consequence of increased tissue factor expression; ref. 14), and attenuated fibrinolysis. Two recognized mechanisms for reduced fibrinolysis include posttranslational modification of fibrinogen by homocysteinylation, rendering the fibrin derived from fibrinogen relatively resistant to plasmin (15), and increased activity of thrombin-activatable fibrinolysis inhibitor (TAFI) (16). In this issue of the JCI, Jacovina and colleagues (17) report the existence of another antifibrinolytic mechanism caused by homocysteine. In the group’s earlier work (18, 19), they demonstrated that the calcium-regulated, phospholipid-binding protein annexin A2 is an endothelial coreceptor for TPA and plasminogen that facilitates plasminogen activation by enhancing the catalytic efficiency of TPA. They next demonstrated that homocysteine forms a mixed disulfide bond with the Cys9 residue of annexin A2, which decreases the binding affinity of the receptor for TPA. As a result of this posttranslational modification, endothelial cell surface plasminogen activation is markedly attenuated (18). These observations were next confirmed in vivo using Anxa2^–/– mice, which showed increased perivascular fibrin deposition and impaired fibrin clearance. Fibrin formation and plasmin-dependent remodeling are not only important in the hemostatic response to vascular injury, but also for luminal recanalization and angiogenesis during the healing phase. Consistent with this role for fibrin and plasmin, the Anxa2^–/– mice showed significantly impaired angiogenesis in a range of vascular injury models (neoangiogenesis) (19). In their latest study, reported in this issue of the JCI (17), the investigators found that moderate hyperhomocysteinemia induced by feeding mice methionine also leads to impaired fibrinolysis with perivascular fibrin persistence as well as attenuated angiogenesis (angiostasis). In this animal model, they observed significantly reduced TPA–annexin A2 binding and plasminogen activation consistent with the importance of endothelial cell surface plasmin activity for normal fibrin clearance and angiogenesis. The pathophenotype was readily reversed by infusion of wild-type recombinant annexin A2 into the hyperhomocysteinemic mice (17).     

Effect of elevated basal insulin on cancer incidence and mortality in cancer incident patients: the Israel GOH 29-year follow-up study

OBJECTIVE

Diabetes is associated with many forms of cancer. Recent evidence has suggested that some treatments for diabetes are associated with an increased cancer risk. Less is known about the association between endogenous insulin in the prediabetes state and cancer risk.

RESEARCH DESIGN AND METHODS

We investigated cumulative cancer incidence and cancer incidence density over 29 years, according to basal insulin, in a cohort of 1,695 nondiabetic men and women of four ethnic origins, aged 51.8 ± 8.0 years at baseline. Total mortality among the 317 subjects (18.7%) who developed cancer at least 2 years after baseline was assessed.

RESULTS

In a Cox proportional hazards model, the all-site hazard ratio of cancer incidence comparing the highest insulin quartile with the other three quartiles was 1.09 (95% CI 0.85–1.40), adjusted for age, sex, and ethnicity. BMI, smoking, and fasting blood glucose were not statistically significant in this model. Basal insulin level was not significantly associated with cancer of specific sites (breast, prostate, colon/rectum, or bladder). Fasting insulin in the upper quartile conferred a 37% increased risk for total mortality among cancer patients, adjusting for age, sex, and ethnic origin (95% CI 0.94–2.00, P = 0.097) compared with that of the lower quartiles. Male sex, older age, and North African origins were associated with a greater risk of mortality during follow-up time.

CONCLUSIONS

This long-term cohort study may suggest a role for basal elevated insulin levels, mainly as a negative predictor in cancer prognosis.The American Diabetes Association and the American Cancer Society recently issued a consensus report showing cancer incidence to be associated with diabetes (1). Type 2 diabetes has been associated with increased incidence, in the range of 1.2–2.5 of cancers of the pancreas (2), breast (3), colon (4), and bladder (5). In addition, a recent meta-analysis of 23 studies found diabetes to be associated with an increased mortality hazard ratio (HR) of 1.41 (95% CI 1.28–1.55) among individuals with cancer (6). Furthermore, some treatments for diabetes have been implicated in increasing the risk of malignancy (7). The development of some types of insulin has been discontinued secondary to increased mitogenic side effects (8). The affinity of binding to the IGF-1 receptor (IGF-1R) has been implicated.We and others have shown basal hyperinsulinemia to predict type 2 diabetes (9,10). Further, elevated levels of circulating insulin and C-peptide have been associated with an increased risk of colorectal and pancreatic cancers (11). Though the risk of breast cancer was less certain in the latter study, two recent analyses of the Women''s Health Initiative found a positive association between insulin levels and breast cancer (12,13).Studies in animals (14–17) and in vitro (18,19) suggest a role for insulin in tumor progression. Glucose tolerance status from 2-h glucose tolerance tests has been shown to associate with the risk of cancer mortality (4). However, the effect of elevated basal insulin on cancer prognosis has not been investigated in vivo.The current study examined the effect, up to 29 years later (mean follow-up time 21.7 ± 6.5 years), of elevated levels of basal insulin on the cumulative incidence of cancer and on cancer survival in a cohort of the Jewish population, representing the four main ethnic origins of immigration to Israel.     

A New Mechanism of NK Cell Cytotoxicity Activation: The CD40–CD40 Ligand Interaction

NK recognition is regulated by a delicate balance between positive signals initiating their effector functions, and inhibitory signals preventing them from proceeding to cytolysis. Knowledge of the molecules responsible for positive signaling in NK cells is currently limited. We demonstrate that IL-2–activated human NK cells can express CD40 ligand (CD40L) and that recognition of CD40 on target cells can provide an activation pathway for such human NK cells. CD40-transfected P815 cells were killed by NK cell lines expressing CD40L, clones and PBLderived NK cells cultured for 18 h in the presence of IL-2, but not by CD40L-negative fresh NK cells. Cross-linking of CD40L on IL-2–activated NK cells induced redirected cytolysis of CD40-negative but Fc receptor-expressing P815 cells. The sensitivity of human TAP-deficient T2 cells could be blocked by anti-CD40 antibodies as well as by reconstitution of TAP/MHC class I expression, indicating that the CD40-dependent pathway for NK activation can be downregulated, at least in part, by MHC class I molecules on the target cells. NK cell recognition of CD40 may be important in immunoregulation as well as in immune responses against B cell malignancies.NK cells represent a distinct lineage of lymphocytes that are able to kill a variety of tumor (1), virus-infected (2), bone marrow transplanted (3), and allogeneic target cells (4). NK cells do not express T cell receptors or immunoglobulins and are apparently normal in mice with defects in the recombinase machinery (5, 6).Our knowledge about NK cell specificity has increased considerably in the last years. NK cells can probably interact with target cells by a variety of different cell surface molecules, some involved in cell adhesion, some activating the NK cytolytic program (7, 8), and other ones able to inhibit this activation by negative signaling (as reviewed in reference 9).A common feature of several inhibitory NK receptors is the capability to bind MHC class I molecules (10, 11), as predicted by the effector inhibition model within the missing self hypothesis of recognition by NK cells (12–14). Interestingly, the MHC class I receptors identified so far belong to different gene families in mouse and man; these are the p58/p70/NKAT or killer cell inhibitory receptors (KIR)¹ of the immunoglobulin superfamily in man and the Ly49 receptors of the C-type lectin family in the mouse. There is also evidence that MHC class I molecules can be recognized as triggering signals in NK cells of humans, rats as well as mice (13). The inhibitory receptors allow NK cells to kill tumor or normal cell targets with deficient MHC class I expression (12, 14). This does not exclude that other activating pathways can override inhibition by MHC class I molecules (15) and, even in their absence, there must be some activating target molecules that initiate the cytolytic program. Several surface molecules are able to mediate positive signals in NK cells. Some of these structures, like NKRP1 (16), CD69 (17), and NKG2 (18) map to the NK complex region (NKC) of chromosome 6 in mice and of chromosome 12 in humans (13). CD2 (19) and CD16 (20) molecules can also play a role in the activation pathway.NK cells resemble T cells in many respects, both may arise from an immediate common progenitor (21, 22), and share the expression of several surface molecules (23). NK cells produce cytokines resembling those secreted by some helper T cell subsets (24) and contain CD3 components in the cytoplasm (21). The expression of some surface structures, involved in TCR-dependent T cell costimulation, like CD28 in human (25), has been described on NK cells, but the functional relevance of these molecules for NK activation processes has not been fully established.Another T cell molecule of interest is CD40L, which interacts with CD40, a 50-kD membrane glycoprotein expressed on B cells (26), dendritic cells (27), and monocytes (28). CD40 is a member of the tumor necrosis factor/nerve growth factor receptor family (29) which includes CD27 (30), CD30 (31), and FAS antigen (32). Murine and human forms of CD40L had been cloned and found to be membrane glycoproteins with a molecular mass of ∼39 kD induced on T cells after activation (33). Also mast cells (34), eosinophils (35), and B cells (36) can be induced to express a functional CD40L. The CD40L–CD40 interaction has been demonstrated to be necessary for T cell–dependent B cell activation (33, 37). Mutations in the CD40L molecule cause a hyper-IgM immunodeficiency condition in man (38, 39, 40). On the other hand, CD40–CD40L interactions also orchestrate the response of regulatory T cells during both their development (41, 42) and their encounter with antigen (43, 44).NK cells have also been suggested to play a role in B cell differentiation and immunoglobulin production (45). Therefore, it was of interest to investigate whether NK cells could use a CD40-dependent pathway in their interactions with other cells. Therefore, we have investigated the ability of target cells expressing CD40 to induce activation of NK cytotoxicity.     

TRAF-interacting Protein (TRIP): A Novel Component of the Tumor Necrosis Factor Receptor (TNFR)- and CD30-TRAF Signaling Complexes That Inhibits TRAF2-mediated NF-κB Activation

Through their interaction with the TNF receptor–associated factor (TRAF) family, members of the tumor necrosis factor receptor (TNFR) superfamily elicit a wide range of biological effects including differentiation, proliferation, activation, or cell death. We have identified and characterized a novel component of the receptor–TRAF signaling complex, designated TRIP (TRAF-interacting protein), which contains a RING finger motif and an extended coiled-coil domain. TRIP associates with the TNFR2 or CD30 signaling complex through its interaction with TRAF proteins. When associated, TRIP inhibits the TRAF2-mediated NF-κB activation that is required for cell activation and also for protection against apoptosis. Thus, TRIP acts as a receptor–proximal regulator that may influence signals responsible for cell activation/proliferation and cell death induced by members of the TNFR superfamily.Members of the TNF receptor (TNFR)¹ superfamily play important roles in the induction of diverse signals leading to cell growth, activation, and apoptosis (1). Whether the signals induced by a given receptor leads to cell activation or death is, however, highly cell-type specific and tightly regulated during differentiation of cells. For example, the TNFRs can exert costimulatory signals for proliferation of naive lymphocytes but also induce death signals required for deletion of activated T lymphocytes (1). The cytoplasmic domains of these receptors lack intrinsic catalytic activity and also exhibit no significant homology to each other or to other known proteins. Exceptions to this include Fas(CD95) and TNFR1 that share a significant homology within an 80–amino acid region of their cytoplasmic tails (called the “death domain”; 2, 3). Therefore, it is suggested that the TNFR family members can initiate different signal transduction pathways by recruiting different types of intracellular signal transducers to the receptor complex (1).Indeed, several types of intracellular signal transducers have been identified that initiate distinct signal transduction pathways when recruited to the members of TNFR superfamily (4–19). Recent biochemical and molecular studies showed that a class of signal-transducing molecules are recruited to Fas(CD95) or TNFR1 via interaction of the death domains (2, 3, 6, 12, 17, 20). For example, Fas(CD95) and TNFR1 recruit FADD(MORT1)/RIP or TRADD/FADD (MORT1)/RIP through the interactions of their respective death domains (2, 3, 6, 12, 17, 20, 21). Clustering of these signal transducers leads to the recruitment of FLICE/ MACH, and subsequently, to cell death (13, 14).The TNFR family members can also recruit a second class of signal transducers called TRAFs (TNFR-associated factor), some of which are responsible for the activation of NF-κB or JNK (9, 20, 22). TRAF proteins were identified by their biochemical ability to interact with TNFR2, CD40, CD30, or LT-βR (4, 5, 10, 11, 15, 23–27). These receptors interact directly with TRAFs via a short stretch of amino acids within their cytoplasmic tails, but do not interact with the death domain containing proteins (4, 5, 15, 24–27). To date, five members of the TRAF family have been identified as signaling components of the TNFR family members. All TRAF members contain a conserved TRAF domain, ∼230 amino acids in length, that is used for either homo- or heterooligomerization among the TRAF family, for interactions with the cytoplasmic regions of the TNFR superfamily, or for interactions with downstream signal transducers (4, 5, 8, 10, 11, 19, 23–25, 28). In addition to the TRAF domain, most of the TRAF family members contain an NH₂-terminal RING finger and several zinc finger structures, which appear to be important for their effector functions (4, 5, 10, 11, 23–25).Several effector functions of TRAFs were revealed by recent experiments based on a transfection system. TRAF2, first identified by its interaction with TNFR2 (4), was subsequently shown to mediate NF-κB activation induced by two TNF receptors, CD40 and CD30 (9, 28–30). TRAF5 was also implicated in NF-κB activation mediated by LTβR (10), whereas TRAF3 (also known as CRAF1, CD40bp, or LAP1; references 5, 11, 24, and 25) was shown to be involved in the regulation of CD40-mediated CD23 upregulation in B cells (5). The role of other TRAF members in the TNFR family–mediated signal transduction is not clear. They may possess some effector functions as yet to be revealed, or work as adapter proteins to recruit different downstream signal transducers to the receptor complex. For example, TRAF1 is required for the recruitment of members of the cellular inhibitor of apoptosis protein (c-IAP) family to the TNFR2-signaling complex (7). In addition to the signal transduction by the TNFR family members, TRAFs may regulate other receptor-mediated signaling pathways. For example, TRAF6 is a component of IL-1 receptor (IL1R)–signaling complex, in which it mediates the activation of NF-κB by IL-1R (31). Since TRAFs form homo- or heterooligomers, it is suggested that the repertoire of TRAF members in a given cell type may differentially affect the intracellular signals triggered by these receptors. This may be accomplished by the selective interaction of TRAFs with a specific set of downstream signal transducers. Although many aspects of TRAF-mediated effector functions leading to cellular activation have been defined, it needs to be determined whether TRAF proteins will also mediate the apoptotic signals induced by the “death-domain-less” members of the TNFR superfamily (1, 27, 32–36).Here we report the isolation and characterization of a novel component of the TNFR superfamily/TRAFs signaling complex, named TRIP (TRAF-interacting protein). TRIP associates with the receptor/TRAF signaling complex, and inhibits the TRAF2-mediated NF-κB activation. Biochemical studies indicate that TRIP associates with the TNFR2 or CD30 receptor complex via its interaction with TRAF proteins, suggesting a model which can explain why the ligation of these receptors can promote different cell fates: proliferation or death.     

Diabetes and lung cancer among postmenopausal women

OBJECTIVE

Epidemiological evidence of diabetes as a lung cancer risk factor is limited and conflicting. Therefore, we assessed associations among diabetes, diabetes therapy, and lung cancer risk in postmenopausal women participating in the Women’s Health Initiative (WHI) study.

RESEARCH DESIGN AND METHODS

Postmenopausal women (n = 145,765), ages 50–79 years, including 8,154 women with diabetes at study entry were followed for a mean of 11 years with 2,257 lung cancers diagnosed. Information on diabetes therapy was collected via two methods (self-reported information on treatment history collected on a questionnaire at baseline and a face-to-face review of current medication containers that participants brought to the baseline visit). Lung cancers were confirmed by central medical record and pathology report review. Cox proportional hazards regression models adjusted for lung cancer risk factors were used to estimate hazard ratios (HRs) (95% CI) for diagnosis of diabetes and treatment of disease as risk factors for lung cancer.

RESULTS

Compared with women without diabetes, women with self-reported treated diabetes had a significantly higher risk of lung cancer (HR 1.27 [95% CI 1.02–1.59]), with risks increasing for women with diabetes requiring insulin treatment (1.71 [1.15–2.53]). However, we did not observe a significant association between lung cancer risk and diabetes not treated with medication or with duration of diabetes.

CONCLUSIONS

Postmenopausal women with treated diabetes, especially those using insulin, have a significantly higher risk of lung cancer. The influence of diabetes severity and specific classes of therapy for diabetes on lung cancer risk require future study.The prevalence of diabetes has been rapidly growing worldwide and has become a major public health concern. Epidemiological studies have shown that diabetes is associated with increased risk of several types of cancer, notably liver, pancreatic, endometrial, and colorectal cancers (1).Lung cancer is the leading cause of cancer-related death globally and in the U.S. (2). Preclinical studies support a role for diabetes and/or hyperglycemia in lung cancer development and growth (3–8). However, epidemiological evidence on the association of diabetes with lung cancer is limited and conflicting (1). Whereas some studies have reported significant (9) or nonsignificant higher risks of lung cancer associated with diabetes (10–13), especially among women (12,13), others have reported an inverse association between diabetes and lung cancer (14–17). These inconsistent results could stem from a number of factors including small sample size, different study design, or potential misclassification of exposures or confounding.The primary barrier to a more clear understanding of the association has been the lack of prospective studies of sufficient size and duration. The Women’s Health Initiative (WHI) is well positioned to overcome this barrier: it is a large prospective cohort study of postmenopausal women in which detailed information on diabetes and potential risk factors was collected at baseline, with 2,257 lung cancer cases adjudicated by centrally trained physicians through September 2010. In this study, we assessed associations among diabetes, diabetes therapy, and lung cancer risk in women participating in the WHI.     

Anti-Pneumocystis Activities of Aromatic Diamidoxime Prodrugs

Aromatic dicationic compounds, such as pentamidine, have potent antimicrobial activities. Clinical use of these compounds has been restricted, however, by their toxicity and limited oral activity. A novel approach, using amidoxime derivatives as prodrugs, has recently been proposed to overcome these limitations. Although results were presented for amidoxime derivatives of only one diamidine, pentamidine, the authors in the original proposal claimed that amidoxime derivatives would work as effective prodrugs for all pharmacologically active diamidines. Nine novel amidoxime derivatives were synthesized and tested in the present study for activity against Pneumocystis carinii in corticosteroid-suppressed rats. Only three of the nine compounds had significant oral anti-Pneumocystis activity. The bisbenzamidoxime derivatives of three direct pentamidine analogs had excellent oral and intravenous activities and reduced acute host toxicity. These compounds are not likely candidates for future drug development, however, because they have chronic toxic effects and the active amidine compounds have multiple sites susceptible to oxidative metabolism, which complicates their pharmacology and toxicology. Novel diamidoximes from three other structural classes, containing different groups linking the cationic moieties, lacked significant oral or intravenous anti-Pneumocystis activity, even though the corresponding diamidines were very active intravenously. Both active and inactive amidoximes were readily metabolized to the corresponding amidines by cell-free liver homogenates. Thus, the amidoxime prodrug approach may provide a strategy to exploit the potent antimicrobial and other pharmacological activities of selected, but certainly not all, aromatic diamidines.

Aromatic dicationic compounds, including bisbenzamidines and dicationically substituted bisbenzimidazoles and carbazoles, have excellent experimental anti-Pneumocystis activities (14, 40, 46, 48, 50, 51) and are also active against other microbial pathogens, including protozoan parasites (2–5, 10, 38, 41, 43, 44), fungi (45), and some viruses (25–27, 49, 53). Aromatic dications also possess other pharmacological properties, including antiinflammatory and anticoagulant activities (29–37). Two problems hindering development of these compounds as new drugs, however, are limited oral bioavailability and toxicity (24, 38, 48, 51).Recent studies of pentamidine metabolism (7–9, 21–23) have led to a novel approach to overcome the limited oral bioavailability and acute toxicity. Aromatic diamidoximes are hypothesized to be orally bioavailable prodrugs that are readily reduced by drug-metabolizing enzymes to the active aromatic amidines (19, 21, 22), resulting in excellent antimicrobial activity with reduced acute host toxicity.Amidoximes were first shown by Lamb and White to be active against experimental African trypanosomiasis (42) and then later were shown to be active against other microorganisms (1, 17, 18, 28). Although activities were often reported for both amidoximes and corresponding amidines, no mention was made in these early publications that metabolic activation was required for in vivo activity of the amidoximes. Moreover, no systematic studies were performed to determine which analogs were orally active and if the amidoxime derivatives had increased oral activity compared to the amidines. Thus, the concept of amidoximes as prodrugs of amidines was not raised in earlier studies.The hypothesis that amidoximes might be useful prodrugs resulted from research examining the metabolism of pentamidine (6–9, 21, 22). Two primary oxidative metabolites identified were the mono- and diamidoximes, formed by N-hydroxylation of pentamidine. Although the diamidoxime derivative of pentamidine has little or no activity against three protozoan parasites in vitro, both the mono- and diamidoximes were active against African trypanosomes and Leishmania spp. when given to experimental animals subcutaneously (19, 21–23, 39). The diamidoxime given orally to rats was absorbed from the gut and converted to pentamidine, a reaction subsequently shown to be catalyzed by an oxygen-independent hepatic reductase activity (21, 22). These observations led to the proposal that amidoxime derivatives, in general, are effective, orally absorbed prodrugs for all pharmacologically active amidine-containing compounds (19). However, the only amidoximes tested were derivatives of pentamidine. We recently demonstrated that two novel amidoximes of 2,5-bis[4-amidinophenyl]furan were highly active orally and intravenously (13). Moreover, Weller and coworkers demonstrated that amidoximes of potent monoamidine fibrinogen receptor antagonists greatly enhanced their oral bioavailability (54).With this promising background, we began to synthesize potential amidoxime prodrugs of our most active, least toxic diamidines. Results presented here, however, demonstrate that amidoximes are not effective prodrugs for all aromatic dicationic compounds. The nature of the linker between the two amidoxime moieties plays a key role in determining if a particular diamidoxime has oral anti-Pneumocystis activity. Diamidoxime derivatives of the very promising bisbenzimidazole and carbazole classes of dications, and bisbenzamidoximes that contain additional nitrogen atoms in the aliphatic linkers, had little or no anti-Pneumocystis activity, even though the parent diamidines had excellent intravenous activity. Variability in activity does not appear to be caused by differences in enzymatic reductase activity, since both active and inactive diamidoximes were metabolized by cell-free liver homogenates.     

Reduced risk of colorectal cancer with metformin therapy in patients with type 2 diabetes: a meta-analysis

OBJECTIVE

Both in vitro and in vivo studies indicate that metformin inhibits cancer cell growth and reduces cancer risk. Recent epidemiological studies suggest that metformin therapy may reduce the risks of cancer and overall cancer mortality among patients with type 2 diabetes. However, data on its effect on colorectal cancer are limited and inconsistent. We therefore pooled data currently available to examine the association between metformin therapy and colorectal cancer among patients with type 2 diabetes.

RESEARCH DESIGN AND METHODS

The PubMed and SciVerse Scopus databases were searched to identify studies that examined the effect of metformin therapy on colorectal cancer among patients with type 2 diabetes. Summary effect estimates were derived using a random-effects meta-analysis model.

RESULTS

The analysis included five studies comprising 108,161 patients with type 2 diabetes. Metformin treatment was associated with a significantly lower risk of colorectal neoplasm (relative risk [RR] 0.63 [95% CI 0.50–0.79]; P < 0.001). After exclusion of one study that investigated colorectal adenoma, the remaining four studies comprised 107,961 diabetic patients and 589 incident colorectal cancer cases during follow-up. Metformin treatment was associated with a significantly lower risk of colorectal cancer (0.63 [0.47–0.84]; P = 0.002). There was no evidence for the presence of significant heterogeneity between the five studies (Q = 4.86, P = 0.30; I² = 18%).

CONCLUSIONS

From observational studies, metformin therapy appears to be associated with a significantly lower risk of colorectal cancer in patients with type 2 diabetes. Further investigation is warranted.Colorectal cancer is one of the most frequent malignant tumors and a leading cause of cancer-related death worldwide (1). The incidence of colorectal cancer continues to increase in economically transitioning countries such as Asia, Eastern Europe, and selected countries in South America (2,3), whereas a declining trend has been noted in several developed countries in recent years (1).Type 2 diabetes is also a common disease, and it is well established that type 2 diabetes is associated with a higher risk of colorectal cancer (4–8). Metformin is a relative of isoamylene guanidine and has been recommended as the initial glucose-lowering therapy for diabetes. Emerging evidence from both in vitro and in vivo studies indicates that metformin may inhibit cancer cell growth and reduce cancer risks. Previous research suggests that metformin may be involved in the tumor suppressor pathway by indirectly activating AMP-activated protein kinase (9)—a key sensor of cellular ATP and AMP balance—and plays a role on activating tumor suppressor genes, e.g., LKB1. Subsequent in vitro studies have shown that metformin inhibits cancer cell proliferation (10,11) and selectively kills cancer stem cells (12). Animal experiments concur with these findings. Rodent models have shown that metformin suppresses colonic epithelial proliferation and colorectal aberrant crypt foci formation (13,14). Similarly, animal models of colon cancer have shown that metformin inhibits colon carcinoma growth (11,15). Given these encouraging findings, interest has arisen that metformin could potentially serve as a new antineoplasm drug to prevent colorectal cancer.Results from preliminary studies conducted in humans are encouraging. In a short-term randomized clinical trial among nondiabetic patients with rectal aberrant crypt foci, a significant decrease in the mean number of aberrant crypt foci was observed after metformin treatment for 1 month as compared with no significant changes in the control group (16). Findings from several epidemiological studies also support an antineoplastic role of metformin on cancer risks (17,18). If metformin therapy ultimately proves effective on reducing the risk of colorectal cancer, it would likely be recommended for the overwhelming majority of diabetes patients for both blood glucose control and cancer prevention. Nonetheless, despite accumulating evidence from population studies that indicate a lower risk of cancer at large with metformin therapy (17,19,20), data on its effect on colorectal cancer are limited and inconsistent. Accordingly, we performed a meta-analysis to pool studies currently available to examine the effect of metformin treatment on colorectal cancer risk among patients with type 2 diabetes.     

Long-Term Cardiovascular Risk in Type 2 Diabetic Compared With Nondiabetic First Acute Myocardial Infarction Patients: A population-based cohort study in southern Europe

OBJECTIVE

The aim of this study was to determine whether long-term cardiovascular risk differs in type 2 diabetic patients compared with first acute myocardial infarction patients in a Mediterranean region, considering therapy, diabetes duration, and glycemic control.

RESEARCH DESIGN AND METHODS

A prospective population-based cohort study with 10-year follow-up was performed in 4,410 patients aged 30–74 years: 2,260 with type 2 diabetes without coronary heart disease recruited in 53 primary health care centers and 2,150 with first acute myocardial infarction without diabetes recruited in 10 hospitals. We compared coronary heart disease incidence and cardiovascular mortality rates in myocardial infarction patients and diabetic patients, including subgroups by diabetes treatment, duration, and A1C.

RESULTS

The adjusted hazard ratios (HRs) for 10-year coronary heart disease incidence and for cardiovascular mortality were significantly lower in men and women with diabetes than in myocardial infarction patients: HR 0.54 (95% CI 0.45–0.66) and 0.28 (0.21–0.37) and 0.26 (0.19–0.36) and 0.16 (0.10–0.26), respectively. All diabetic patient subgroups had significantly fewer events than myocardial infarction patients: the HR of cardiovascular mortality ranged from 0.15 (0.09–0.26) to 0.36 (0.24–0.54) and that of coronary heart disease incidence ranged from 0.34 (0.26–0.46) to 0.56 (0.43–0.72).

CONCLUSIONS

Lower long-term cardiovascular risk was found in type 2 diabetic and all subgroups analyzed compared with myocardial infarction patients. These results do not support equivalence in coronary disease risk for diabetic and myocardial infarction patients.The prevalence of diabetes is reaching epidemic proportions in developed countries (1). For example, the U.S. has 18 million diabetic patients, Spain has >2 million diabetic patients, and management of the disease costs >$132 and >$3.3 billion per year, respectively (2).Some studies (3–5), several of them with great influence on important guidelines for cardiovascular prevention (3), suggest that the cardiovascular risk of diabetic patients is similar to that of coronary heart disease secondary prevention patients. Other reports, however, do not confirm these observations (6–10).Part of the discrepancy may stem from differences in the duration of diabetes, type of treatment, and baseline glucose control of diabetic patients included in the studies (3–5). These limit comparability, given the fact that time of evolution and treatment required to attain appropriate glycemic control are key determinants of prognosis (10–16).Among population-based cohort studies that compared the prognosis of diabetic patients with that of myocardial infarction patients without diabetes (3–10), only two analyzed the role of diabetes duration (11,12). Even these studies did not include unstable angina among the end points and risk was not stratified by type of treatment. To our knowledge, the effect of type 2 diabetes on coronary heart disease incidence has barely been studied in southern Europe, a region known for low cardiovascular mortality (17). The aim of this study was to determine whether long-term cardiovascular risk differed between type 2 diabetic patients and first acute myocardial infarction patients and to assess the influence of diabetes duration, type of treatment, and glycemic control at baseline.     

FGFR3-targeted mAb therapy for bladder cancer and multiple myeloma

Gain-of-function mutations in FGF receptor 3 (FGFR3) have been implicated in severe skeletal dysplasias and in a variety of cancers. In their study in this issue of the JCI, Qing et al. used specific shRNA probes to demonstrate that FGFR3 functions as an important driver of bladder carcinoma cell proliferation (see the related article beginning on page 1216). A unique anti-FGFR3 mAb was shown to exhibit antitumor activity in human bladder carcinoma cells in vitro and in mouse bladder cancer or multiple myeloma xenograft tumor models bearing either wild-type or mutant FGFR3. These results suggest that clinical development of anti-FGFR3 mAbs should be considered for targeted therapy of cancer and other diseases. FGFs are members of a large family of growth factors that play an important role in the control of diverse cellular processes (e.g., cell proliferation, differentiation, survival, and migration) during embryonic development and in the maintenance of cellular homeostasis in virtually all organs and tissues (1, 2). FGFs act in concert with heparan sulfate proteoglycans (HSPGs) to activate a family of four receptor tyrosine kinases (RTKs) designated FGF receptors 1–4 (FGFR1–4). Each FGFR consists of an extracellular region composed of three Ig-like domains designated D1, D2, and D3; a short stretch of amino acids in the D1–D2 linker region, designated the acid box; and a single transmembrane domain followed by an intracellular tyrosine kinase domain with additional regulatory sequences (1). Diversity in ligand-binding specificity and tissue expression patterns of FGFR1–3 are further enhanced by alternative RNA splicing in the C-terminal half of D3 to generate either the IIIb or the IIIc splice isoform. It was previously shown that the IIIb isoform is expressed in epithelial cells and the IIIc isoform is expressed in mesenchymal cells (1). Interestingly, FGFs that activate the epithelial IIIb FGFR isoforms are expressed in mesenchymal cells, whereas FGFs that activate the mesenchymal IIIc FGFR isoforms are produced in epithelial cells (1). A variety of human skeletal dysplasias associated with severe impairment in cranial, digital, and skeletal development have been shown to be driven by gain-of-function mutations in FGFR1–3 (1). Gain-of-function mutations in FGFRs have also been identified in a variety of human cancers, including myeloproliferative syndromes, lymphomas, glioblastoma, as well as prostate, bladder, and mammary carcinomas (1). Importantly, the t(4;14)(p16.3;q32) chromosomal translocation that results in FGFR3 overexpression in 20% of multiple myeloma patients is correlated with poor clinical response and patient survival (3). FGFR3 overexpression has also been identified in bladder carcinoma (4). Moreover, somatic gain-of-function mutations in FGFR3 have been identified in 65% of papillary and in 20% of muscle-invasive bladder carcinomas (4). These studies suggest that FGFR3 could be considered a candidate for targeted therapies for the treatment of cancers and skeletal dysplasias driven by gain-of-function FGFR3 mutations. Several small molecule inhibitors of the tyrosine kinase activity of FGFR3 and other members of the FGFRs have been described, some of which are currently in clinical development (5, 6). However, potent small molecule inhibitors that specifically interfere with the tyrosine kinase activity of FGFR3 are difficult to develop because of the close structural similarity of the FGFR3 tyrosine kinase domain to that of other members of the FGFR family and other RTKs. An alternative approach for developing a selective inhibitor of FGFR3 that does not interfere with the activity of other FGFRs and RTKs is to raise inhibitory mAbs that selectively bind to the extracellular domain of FGFR3.     

Neutrophils give us a shock

Systemic anaphylaxis is generally recognized as a severe allergic reaction caused by IgE-mediated activation of mast cells, leading to massive release of vasoactive mediators that induce acute hypotension and shock. However, experimental evidence in mice suggests that this view is too simple. Using a variety of techniques to manipulate immune cell makeup, Jönsson et al. come to the conclusion in this issue of the JCI that recognition of IgG1 and IgG2 antibodies by FcγRIII and FcγRIV receptors on neutrophils is a major pathway for induction of anaphylaxis. These exciting results suggest that we have to reevaluate our models for anaphylaxis in humans, which will have a direct impact on our therapeutic approaches for prevention of this potential deadly hypersensitivity reaction. I turned to Wikipedia when I was searching for a way to explain the basics of anaphylaxis and came across the following statement: “True anaphylaxis is caused by degranulation of mast cells or basophils mediated by immunoglobulin E (IgE).” This is the classic teaching, present in all the immunology textbooks, but the paper in this issue of the JCI by Jönsson et al. (1) informs us that this view is, at best, incomplete. Instead, we learn that anaphylaxis can be mediated by neutrophils recognizing IgG/antigen complexes. In addition to turning around our understanding of anaphylaxis, this paper adds to the growing list of neutrophil functions besides just bacterial killing and protease production (2). Lately we have learned that neutrophils are major sources of cytokines and chemokines (3, 4). They play a direct role in influencing the recruitment and activation of monocytes/macrophages, T cells, and NK cells during inflammation (5–7). Neutrophils have been implicated as the primary initiators of immune complex–mediated diseases (8, 9). And now the shocking news (pun intended!) that they are major players in initiating anaphylaxis.     

Location,location, location: important for jet-lagged circadian loops

It is now believed that frequent jet lag or shifts of daily rhythms caused by rotating shift work can lead to deleterious health outcomes. Indeed, many serious health problems, including breast cancer, stroke, and cardiovascular disease, have been linked to an occupational history of shift work. This has heightened interest in better understanding the biological responses to jet lag and shift work, with the hope that this will pave the way to developing compounds that can help people avoid their negative health consequences. In this context, a report in this issue of the JCI takes us to a new level of understanding of the molecular control of the resetting of the multitude of internal biological clocks disrupted in a mouse model of jet lag. Jet lag can seem like a minor discomfort, but studies are now suggesting that frequent jet lag or shifts of daily rhythms as a result of rotating shift work can lead to many deleterious health outcomes. Breast cancer, reproductive disorders, cardiovascular disease, stroke, and metabolic syndrome are all linked to an occupational history of shift work (1–3). Experiments modeling chronic jet lag have shown that it can speed up tumor growth (4) and bring on early mortality in aged mice (5), as well as lower the survival rate for hamsters with impaired heart function (6). The International Agency for Research on Cancer, a unit of the World Health Organization, has declared that night shift work is also a probable carcinogen (7). These serious health concerns heighten our interest in better understanding the biological responses to jet lag and shift work.