Effect of Obesity-Linked FTO rs9939609 Variant on Physical Activity and Dietary Patterns in Physically Active Men and Women.

Single nucleotide polymorphisms (SNPs) in the fat mass and obesity-associated (FTO) locus are associated with obesity, but lifestyle factors may modulate the obesity risk related to FTO. This study examined the physical activity and dietary patterns of 528 physically active white men and women (mean (SD): 34.9 (9.5) years, 26.6 (4.3) kg·m-2) carrying different risk variants of FTO SNP rs9939609. Sex, age, and anthropometric measurements (stature, body mass, and waist circumference) were self-reported using an online questionnaire, and body mass index and waist-to-height ratio were calculated. Physical activity and eating behaviour were assessed using the International Physical Activity Questionnaire (IPAQ) and Three-Factor Eating Questionnaire (TFEQ), respectively. Body mass, body mass index (BMI), waist circumference, and waist-to-height ratio were not significantly different between individuals expressing different FTO rs9939609 risk variants (all P ≥ 0.66). The cohort was physically active (4516 (3043) total MET min·week-1), although homozygous risk allele carriers (AA) displayed higher TFEQ cognitive restraint compared with nonrisk allele carriers (TT) (ES = 0.33 and P=0.03). In conclusion, obesity-related parameters were not different in physically active individuals expressing different risk variants of FTO rs9939609, although homozygous risk allele carriers exhibited higher cognitive restraint.

Immunotherapy Not Working? Check Your Microbiota.

Gut microbes have ascended to prominence as key modulators of host immunity, raising the possibility that they could influence the outcome of cancer immunotherapy. Two recent studies address this question by identifying specific gut-resident bacteria as drivers of checkpoint blockade immunotherapy in pre-clinical tumor models.

Emerging cytokine networks in colorectal cancer.

Cytokine networks are crucial aspects of tumour immunology, particularly for colorectal cancer (CRC), in which inflammation and antitumour immunity are key determinants of disease progression. In this Review, we highlight new insights into the functions of well-known cytokines in CRC, describe recently discovered roles for a growing number of novel players, and emphasize the complexity and therapeutic implications of the cytokine milieu. We also discuss how cancer mutations and epigenetic adaptations influence the oncogenic potential of cytokines, a relatively unexplored area that could yield crucial insights into tumour immunology and facilitate the effective application of cytokine-modulatory therapies for CRC.

Intratumoural inflammation and endocrine resistance in breast cancer.

It is becoming clear that inflammation-associated mechanisms can affect progression of breast cancer and modulate responses to treatment. Estrogen receptor alpha (ERα (ESR1)) is the principal biomarker and therapeutic target for endocrine therapies in breast cancer. Over 70% of patients are ESR1-positive at diagnosis and are candidates for endocrine therapy. However, ESR1-positive tumours can become resistant to endocrine therapy. Multiple mechanisms of endocrine resistance have been proposed, including suppression of ESR1. This review discusses the relationship between intratumoural inflammation and endocrine resistance with a particular focus on inflammation-mediated suppression of ESR1.

Model-directed engineering of "difficult-to-express" monoclonal antibody production by Chinese hamster ovary cells.

Despite improvements in volumetric titer for monoclonal antibody (MAb) production processes using Chinese hamster ovary (CHO) cells, some "difficult-to-express" (DTE) MAbs inexplicably reach much lower process titers. These DTE MAbs require intensive cell line and process development activity, rendering them more costly or even unsuitable to manufacture. To rapidly and rationally identify an optimal strategy to improve production of DTE MAbs, we have developed an engineering design platform combining high-yielding transient production, empirical modeling of MAb synthesis incorporating an unfolded protein response (UPR) regulatory loop with directed expression and cell engineering approaches. Utilizing a panel of eight IgG1 λ MAbs varying >4-fold in volumetric titer, we showed that MAb-specific limitations on folding and assembly rate functioned to induce a proportionate UPR in host CHO cells with a corresponding reduction in cell growth rate. Derived from comparative empirical modeling of cellular constraints on the production of each MAb we employed two strategies to increase production of DTE MAbs designed to avoid UPR induction through an improvement in the rate/cellular capacity for MAb folding and assembly reactions. Firstly, we altered the transfected LC:HC gene ratio and secondly, we co-expressed a variety of molecular chaperones, foldases or UPR transactivators (BiP, CypB, PDI, and active forms of ATF6 and XBP1) with recombinant MAbs. DTE MAb production was significantly improved by both strategies, although the mode of action was dependent upon the approach employed. Increased LC:HC ratio or CypB co-expression improved cell growth with no effect on qP. In contrast, BiP, ATF6c and XBP1s co-expression increased qP and reduced cell growth. This study demonstrates that expression-engineering strategies to improve production of DTE proteins in mammalian cells should be product specific, and based on rapid predictive tools to assess the relative impact of different engineering interventions.

Markers of T cell infiltration and function associate with favorable outcome in vascularized high-grade serous ovarian carcinoma.

BACKGROUND: When T cells infiltrate the tumor environment they encounter a myriad of metabolic stressors including hypoxia. Overcoming the limitations imposed by an inadequate tumor vasculature that contributes to these stressors may be a crucial step to immune cells mounting an effective anti-tumor response. We sought to determine whether the functional capacity of tumor infiltrating lymphocytes (TIL) could be influenced by the tumor vasculature and correlated this with survival in patients with ovarian cancer. METHODOLOGY AND PRINCIPAL FINDINGS: In 196 high-grade serous ovarian tumors, we confirmed that the tumor vascularity as measured by the marker CD31 was associated with improved patient disease-specific survival. We also found that tumors positive for markers of TIL (CD8, CD4 and forkhead box P3 (FoxP3)) and T cell function (granzyme B and T-cell restricted intracellular antigen-1 (TIA-1)) correlated significantly with elevated vascularity. In vitro, hypoxic CD8 T cells showed reduced cytolytic activity, secreted less effector cytokines and upregulated autophagy. Survival analysis revealed that patients had a significant improvement in disease-specific survival when FoxP3 expressing cells were present in CD31-high tumors compared to patients with FoxP3 expressing cells in CD31-low tumors [HR: 2.314 (95% CI 1.049-5.106); p = 0.0377]. Patients with high vascular endothelial growth factor (VEGF) expressing tumors containing granzyme B positive cells had improved survival compared to patients with granzyme B positive cells in VEGF-low tumors [HR: 2.522 (95% CI 1.097-5.799); p = 0.0294]. SIGNIFICANCE: Overall, this data provides a rationale for developing strategies aimed at improving the adaptability and function of TIL to hypoxic tumor conditions.

The autophagy protein LC3A correlates with hypoxia and is a prognostic marker of patient survival in clear cell ovarian cancer.

Clear cell ovarian cancer histotypes exhibit metabolic features associated with resistance to hypoxia and glucose deprivation-induced cell death. This metabolic characteristic suggests that clear cell ovarian cancers activate survival mechanisms not typical of other epithelial ovarian cancers. Here we demonstrate that microtubule-associated protein 1 light chain 3A (LC3A), a marker of autophagy, is related to hypoxia and poor prognosis in clear cell ovarian cancer. In 485 ovarian tumours, we found that LC3A was significantly associated with poor progression-free (p = 0.0232), disease-specific (p = 0.0011) and overall patient survival (p = 0.0013) in clear cell ovarian cancer patients, but not in other subtypes examined. LC3A was an independent prognostic marker of reduced disease-specific [hazard ratio (HR): 2.55 (95% CI 1.21-5.37); p = 0.014] and overall survival [HR: 1.95 (95% CI 1.00-3.77); p = 0.049] in patients with clear cell ovarian carcinoma. We also found a strong link between autophagy and hypoxia as LC3A staining revealed a significant positive association with the hypoxia-related proteins carbonic anhydrase-IX and HIF-1α. The functional link between hypoxia and autophagy was demonstrated using clear cell and high-grade serous cell lines that were subjected to hypoxia or hypoxia + glucose deprivation. Clear cell carcinoma lines displayed greater autophagy induction and were subsequently more sensitive to inhibition of autophagy under hypoxia compared to the high-grade serous lines. Together, our findings indicate that hypoxia-induced autophagy may be crucial to the clinical pathology of clear cell ovarian cancer and is a potential explanation for histological subtype differences in patient disease progression and outcomes.

Oncostatin M suppresses oestrogen receptor-α expression and is associated with poor outcome in human breast cancer.

The most important clinical biomarker for breast cancer management is oestrogen receptor alpha (ERα). Tumours that express ER are candidates for endocrine therapy and are biologically less aggressive, while ER-negative tumours are largely treated with conventional chemotherapy and have a poor prognosis. Despite its significance, the mechanisms regulating ER expression are poorly understood. We hypothesised that the inflammatory cytokine oncostatin M (OSM) can downregulate ER expression in breast cancer. Recombinant OSM potently suppressed ER protein and mRNA expression in vitro in a dose- and time-dependent manner in two human ER+ breast cancer cell lines, MCF7 and T47D. This was dependent on the expression of OSM receptor beta (OSMRß) and could be blocked by inhibition of the MEKK1/2 mitogen-activated protein kinases. ER loss was also necessary for maximal OSM-induced signal transduction and migratory activity. In vivo, high expression of OSM and OSMR mRNA (determined by RT-PCR) was associated with reduced ER (P<0.01) and progesterone receptor (P<0.05) protein levels in a cohort of 70 invasive breast cancers. High OSM and OSMR mRNA expression was also associated with low expression of ESR1 (ER, P<0.0001) and ER-regulated genes in a previously published breast cancer gene expression dataset (n=321 cases). In the latter cohort, high OSMR expression was associated with shorter recurrence-free and overall survival in univariate (P<0.0001) and multivariate (P=0.022) analyses. OSM signalling may be a novel factor causing suppression of ER and disease progression in breast cancer.

Tumor-infiltrating lymphocytes predict response to anthracycline-based chemotherapy in estrogen receptor-negative breast cancer.

INTRODUCTION: Infiltration of breast tumors by tumor-infiltrating lymphocytes (TIL) has been associated with sensitivity to anthracycline-based chemotherapy. However, it is unclear whether this is true within the estrogen receptor-alpha (ER)-negative subset of breast tumors that frequently manifest high TIL levels. METHODS: The association of TIL with short-term and long-term clinical response to anthracycline-based therapy was assessed in two independent ER-negative breast cancer cohorts in which patients were categorized as TIL-high or TIL-low. We defined an eight-gene lymphocyte mRNA expression signature (including CD19, CD3D, CD48, GZMB, LCK, MS4A1, PRF1, and SELL) and used unsupervised hierarchical clustering to examine the association between TIL and short-term response to neoadjuvant chemotherapy in a previously published cohort of ER-negative tumors (n = 113). We also examined the association between TIL and long-term chemotherapeutic efficacy in a second cohort of ER-negative tumors (n = 255) with longer than 6 years of median follow-up by using tissue microarrays and immunohistochemistry (IHC) for detection of CD3, CD8, CD4, CD20, and TIA-1. RESULTS: In patients with ER-negative tumors treated with neoadjuvant anthracycline-based chemotherapy, pathologic complete responses (pCRs) were achieved by 23 (74%) of 31 TIL-high patients and 25 (31%) of 80 TIL-low patients (odds ratio (OR), 6.33; 95% confidence interval (CI), 2.49 to 16.08; P < 0.0001). Multivariate logistic regression with standard clinicopathologic features demonstrated that only tumor size (P = 0.037) and TIL status (P = 0.001) were independent predictors of anthracycline response. In the second cohort, adjuvant anthracycline-based therapy was associated with increased disease-free survival (DFS) only in patients with high levels of intraepithelial CD3+ TIL (P = 0.0023). In contrast, outcomes after CMF treatment (cyclophosphamide, methotrexate, and fluorouracil) showed no association with CD3 status. In both cohorts, cytotoxic T-cells were the primary TIL subtype associated with anthracycline sensitivity. Finally, TIL significantly predicted anthracycline sensitivity for both the Her2-positive and triple-negative tumor phenotypes. CONCLUSIONS: ER-negative breast cancers with high levels of TIL have heightened sensitivity to anthracycline-based chemotherapy, as assessed by the immediate response to neoadjuvant therapy and long-term outcome following adjuvant therapy. Investigations of TIL-based predictive tests to identify patients likely to benefit from anthracycline-based treatments are warranted.

Intratumoral Immune Responses Can Distinguish New Primary and True Recurrence Types of Ipsilateral Breast Tumor Recurrences (IBTR).

Ipsilateral breast tumor recurrence (IBTR) is an increasingly common clinical challenge. IBTRs include True Recurrences (TR; persistent disease) and New Primaries (NP; de novo tumors), but discrimination between these is difficult. We assessed tumor infiltrating leukocytes (TIL) as biomarkers for distinguishing these types of IBTR using primary tumors and matched IBTRs from 24 breast cancer patients, half of which were identified as putative TRs and half as NPs using a previously reported clinical algorithm. Intratumoral lymphocyte populations (CD3, CD8, CD4, CD25, FOXP3, TIA1, CD20) and macrophages (CD68) were quantified by immunohistochemistry in each tumor. Compared to matched primaries, TRs showed significant trends towards increased CD3(+) and CD8(+) TIL, while these populations were often diminished in NPs. Comparison of IBTRs showed that TRs had significantly higher levels of CD3(+) (P = 0.0136), CD8(+) (P = 0.0092), and CD25(+) (P = 0.0159) TIL than NPs. We conclude that TIL may be a novel diagnostic biomarker to distinguish NP from TR IBTRs.

Structural and functional characterization of a triple mutant form of S100A7 defective for Jab1 binding.

S100A7 (psoriasin) is a calcium- and zinc-binding protein implicated in breast cancer. We have shown previously that S100A7 enhances survival mechanisms in breast cells through an interaction with c-jun activation domain binding protein 1 (Jab1), and an engineered S100A7 triple mutant (Asp(56)Gly, Leu(78)Met, and Gln(88)Lys-S100A7(3)) ablates Jab1 binding. We extend these results to include defined breast cancer cell lines and demonstrate a disrupted S100A7(3)/Jab1 phenotype is maintained. To establish the basis for the abrogated Jab1 binding, we have recombinantly produced S100A7(3), demonstrated that it retains the ability to form an exceptionally thermostable dimer, and solved the three dimensional crystal structure to 1.6 A. Despite being positioned at the dimer interface, the Leu(78)Met mutation is easily accommodated and contributes to a methionine-rich pocket formed by Met(12), Met(15), and Met(34). In addition to altering the surface charge, the Gln(88)Lys mutation results in a nearby rotameric shift in Tyr(85), leading to a substantially reorganized surface cavity and may influence zinc binding. The final mutation of Asp(56) to Gly results in the largest structural perturbation shortening helix IV by one full turn. It is noteworthy that position 56 lies in one of two divergent clusters between S100A7 and the functionally distinct yet highly homologous S100A15. The structure of S100A7(3) provides a unique perspective from which to characterize the S100A7-Jab1 interaction and better understand the distinct functions between S100A7, and it is closely related paralog S100A15.

Identification and characterization of binding sites on S100A7, a participant in cancer and inflammation pathways.

S100A7 (psoriasin) is a member of the S100 family of signaling proteins. It is implicated in and considered a therapeutic target for inflammation and cancer, yet no small molecule ligands for S100A7 have been identified. To begin the development of specific small molecule inhibitors of S100A7 function, we have used a series of surface binding fluorescent dyes to probe the surface hydrophobic sites. Two naphthalene-based dyes (2,6-ANS and 1,8-ANS) were found to bind S100A7 in a distinct cleft. We characterized the binding interaction by determining both the structure of S100A7 bound to 2,6-ANS and the structure of S100A7 bound to 1,8-ANS to 1.6 A. In both cases, two molecules of dye were docked such that the naphthalene groups were positioned in two symmetry-related grooves that are formed by the N-terminal helices of each monomer. We observed that Met12 acts as a gatekeeper to the binding cleft, adopting an "open" conformation for the more elongated 2,6-ANS while remaining in a "closed" conformation for the more compact 1,8-ANS. Steady-state fluorescence experiments revealed that S100A7 binds two copies of 2,6-ANS, each with a K(d) of 125 muM. Time-resolved fluorescence lifetime measurements indicated that the two molecules of 2,6-ANS bind in two independent binding sites with different fluorescence lifetimes, suggesting that the S100A7 homodimer is not perfectly symmetric in solution. Isothermal titration calorimetry studies demonstrate that S100A7 has a higher affinity for 2,6-ANS than 1,8-ANS. Yeast two-hybrid studies were also used to probe contributions of individual residues of an S100A7 triple mutant with respect to Jab1 binding. Mutation of Leu78, which forms part of the Met12 cleft occupied by 2,6-ANS, reduced the level of Jab1 binding, suggesting a potentially important role for the Met12 hydrophobic pocket in defining a Jab1 interface. Additional Y2H studies also delineate contributions of Gln88 and in particular Asp56 that shows the most significant abrogated binding to Jab1. Collectively, these data suggest a complex interaction between S100A7 and the much larger Jab1. These studies form the basis for the development of small molecule reporters and modifiers of S100A7 form and function.

Jab1 is a target of EGFR signaling in ERalpha-negative breast cancer.

INTRODUCTION: c-Jun activation domain-binding protein-1 (Jab1) is a multifunctional signaling protein that previously has been shown to be a master regulator of a poor prognostic gene signature in invasive breast cancer and to mediate the action of S100A7. Since epidermal growth factor receptor (EGFR), like S100A7, is often expressed in estrogen receptor-alpha-negative (ERalpha-) breast cancer, we set out to investigate the role of Jab1 in mediating EGFR signaling, another facet of the ERalpha- phenotype. METHODS: MDA-MB-231 and MDA-MB-468 ERalpha-/EGFR+ cell lines were assessed for localization of Jab1 and levels of downstream genes by immunofluorescence and nuclear protein extract assay following treatment with epidermal growth factor (EGF) and extracellular signal-regulated kinase (ERK) pathway inhibitor. A cohort of 424 human breast tumors was also assessed by immunohistochemistry. RESULTS: EGF treatment of cell lines resulted in increased Jab1 nuclear expression. This effect was inhibited by the ERK pathway inhibitor, PD98059. EGF treatment was also associated with colocalization of pERK (phosphorylated ERK) and Jab1 as well as regulation of the Jab1 downstream target gene, p27. When Jab1 activity was knocked down, p27 levels were restored to pre-EGF treatment level. Analysis of EGFR and Jab1 expression in a cohort of invasive breast tumors by tissue microarray and immunohistochemistry confirmed a relationship between EGFR and increased nuclear Jab1 within the ERalpha- subset (n = 154, P = 0.019). The same association was also confirmed for S100A7 and Jab1 (P = 0.036), and high Jab1 nuclear expression was most frequent in tumors that were positive for both EGFR and S100A7 (P = 0.004). CONCLUSION: Jab1 is a target of EGFR signaling in ERalpha- cell lines and breast tumors and therefore may be a common central factor and potential therapeutic target for important cell signaling pathways in ERalpha- breast cancer.