Distinct Lipid Phenotype of Cancer-Associated Fibroblasts (CAFs) Isolated From Overweight/Obese Endometrial Cancer Patients as Assessed Using Raman Spectroscopy.

Obesity is strongly linked with increased risk and poorer prognosis of endometrial cancer (EC). Cancer-associated fibroblasts (CAFs) are activated fibroblasts that form a large component of the tumor microenvironment and undergo metabolic reprogramming to provide critical metabolites for tumor growth. However, it is still unknown how obesity, characterized by a surplus of free fatty acids drives the modifications of CAFs lipid metabolism which may provide the mechanistic link between obesity and EC progression. The present study aims to evaluate the utility of Raman spectroscopy, an emerging nondestructive analytical tool to detect signature changes in lipid metabolites of CAFs from EC patients with varying body mass index. We established primary cultures of fibroblasts from human EC tissues, and CAFs of overweight/obese and nonobese women using antibody-conjugated magnetic beads isolation. These homogeneous fibroblast cultures expressed fibroblast markers, including α-smooth muscle actin and vimentin. Analysis was made in the Raman spectra region best associated with cancer progression biochemical changes in lipids (600-1800 cm-1 and 2800-3200 cm-1). Direct band analysis and ratiometric analysis were conducted to extract information from the Raman spectrum. Present results demonstrated minor shifts in the CH2 symmetric stretch of lipids at 2879 cm-1 and CH3 asymmetric stretching from protein at 2932 cm-1 in the overweight/obese CAFS compared to nonobese CAFs, indicating increased lipid content and a higher degree of lipid saturation. Principal component analysis showed that CAFs from overweight/obese and nonobese EC patients can be clearly distinguished indicating the capability of Raman spectroscopy to detect changes in biochemical components. Our results suggest Raman spectroscopy supported by chemometric analysis is a reliable technique for characterizing metabolic changes in clinical samples, providing an insight into obesity-driven alteration in CAFs, a critical stromal component during EC tumorigenesis.

Aberrant upregulation of CDK1 contributes to medroxyprogesterone acetate (MPA) resistance in cancer-associated fibroblasts of the endometrium.

The response to medroxyprogesterone acetate (MPA) decreases as endometrial disease progresses from the benign to malignancy. In a mouse model, progesterone receptor (PR) expression in normal fibroblasts is accountable for the MPA's inhibitory effects in cancer cells. However, it is still unclear, if and how, fibroblasts from human tumors respond to MPA. In this study, three benign-associated fibroblasts (BAFs) and four cancer-associated fibroblasts (CAFs) were isolated from human benign and cancerous endometrial tissues, respectively, to examine MPA activation on PR signaling. PR-B protein expression were heterogeneously expressed in both CAFs and BAFs, despite a lower mRNA expression in the former. In a luciferase reporter assay, MPA treatment stimulated some PR DNA-binding activity in BAFs but not in CAFs. Yet, activation of PR target gene was generally more pronounced in MPA-treated CAFs compared to BAFs. Cyclin-dependent kinase 1 (CDK1) was exclusively upregulated by 10 nM MPA in CAFs (5.1-fold vs. 1.1-fold in BAFs, P < 0.05), leading to a higher CDK1 protein expression. Subsequently in a dose-response study, CAFs showed an average of ∼20% higher cell viability when compared to BAFs, indicative of drug resistance to MPA. MPA resistance was also observed in EC-CAFs co-culture, when MPA-treated cells showed greater tumor spheroid formation than in EC-BAFs co-culture (2-fold, P < 0.01). The increased cell viability observed in CAFs was reversed with mifepristone (RU486), a PR antagonist which suppressed MPA-induced CDK1 expression. This indicates that MPA-induced abnormal upregulation of CDK1 may contribute to the enhanced CAFs cell proliferation, suggesting a new mechanism of MPA resistance within endometrial cancer microenvironment.

MPA alters metabolic phenotype of endometrial cancer-associated fibroblasts from obese women via IRS2 signaling.

Obese women have a higher risk of developing endometrial cancer (EC) than lean women. Besides affecting EC progression, obesity also affects sensitivity of patients to treatment including medroxprogesterone acetate (MPA). Obese women have a lower response to MPA with an increased risk for tumor recurrence. While MPA inhibits the growth of normal fibroblasts, human endometrial cancer-associated fibroblasts (CAFs) were reported to be less responsive to MPA. However, it is still unknown how CAFs from obese women respond to progesterone. CAFs from the EC tissues of obese (CO) and non-obese (CN) women were established as primary cell models. MPA increased cell proliferation and downregulated stromal differentiation genes, including BMP2 in CO than in CN. Induction of IRS2 (a BMP2 regulator) mRNA expression by MPA led to activation of glucose metabolism in CO, with evidence of greater mRNA levels of GLUT6, GAPDH, PKM2, LDHA, and increased in GAPDH enzymatic activity. Concomitantly, MPA increased the mRNA expression of a fatty acid transporter, CD36 and lipid droplet formation in CO. MPA-mediated increase in glucose metabolism genes in CO was reversed with a progesterone receptor inhibitor, mifepristone (RU486), leading to a decreased proliferation. Our data suggests that PR signaling is aberrantly activated by MPA in CAFs isolated from endometrial tissues of obese women, leading to activation of IRS2 and glucose metabolism, which may lead to lower response and sensitivity to progesterone in obese women.

Oncologist-led BRCA counselling improves access to cancer genetic testing in middle-income Asian country, with no significant impact on psychosocial outcomes.

BACKGROUND: Identifying patients with BRCA mutations is clinically important to inform on the potential response to treatment and for risk management of patients and their relatives. However, traditional referral routes may not meet clinical needs, and therefore, mainstreaming cancer genetics has been shown to be effective in some high-income and high health-literacy settings. To date, no study has reported on the feasibility of mainstreaming in low-income and middle-income settings, where the service considerations and health literacy could detrimentally affect the feasibility of mainstreaming. METHODS: The Mainstreaming Genetic Counselling for Ovarian Cancer Patients (MaGiC) study is a prospective, two-arm observational study comparing oncologist-led and genetics-led counselling. This study included 790 multiethnic patients with ovarian cancer from 23 sites in Malaysia. We compared the impact of different method of delivery of genetic counselling on the uptake of genetic testing and assessed the feasibility, knowledge and satisfaction of patients with ovarian cancer. RESULTS: Oncologists were satisfied with the mainstreaming experience, with 95% indicating a desire to incorporate testing into their clinical practice. The uptake of genetic testing was similar in the mainstreaming and genetics arm (80% and 79%, respectively). Patient satisfaction was high, whereas decision conflict and psychological impact were low in both arms of the study. Notably, decisional conflict, although lower than threshold, was higher for the mainstreaming group compared with the genetics arm. Overall, 13.5% of patients had a pathogenic variant in BRCA1 or BRCA2, and there was no difference between psychosocial measures for carriers in both arms. CONCLUSION: The MaGiC study demonstrates that mainstreaming cancer genetics is feasible in low-resource and middle-resource Asian setting and increased coverage for genetic testing.

High throughput silencing identifies novel genes in endometrioid endometrial cancer.

OBJECTIVE: To validate the gene expression profile obtained from the previous microarray analysis and to further study the biological functions of these genes in endometrial cancer. From our previous study, we identified 621 differentially expressed genes in laser-captured microdissected endometrioid endometrial cancer as compared to normal endometrial cells. Among these genes, 146 were significantly up-regulated in endometrial cancer. MATERIALS AND METHODS: A total of 20 genes were selected from the list of up-regulated genes for the validation assay. The qPCR confirmed that 19 out of the 20 genes were up-regulated in endometrial cancer compared with normal endometrium. RNA interference (RNAi) was used to knockdown the expression of the upregulated genes in ECC-1 and HEC-1A endometrial cancer cell lines and its effect on proliferation, migration and invasion were examined. RESULTS: Knockdown of MIF, SOD2, HIF1A and SLC7A5 by RNAi significantly decreased the proliferation of ECC-1 cells (p < 0.05). Our results also showed that the knockdown of MIF, SOD2 and SLC7A5 by RNAi significantly decreased the proliferation and migration abilities of HEC-1A cells (p < 0.05). Moreover, the knockdown of SLC38A1 and HIF1A by RNAi resulted in a significant decrease in the proliferation of HEC1A cells (p < 0.05). CONCLUSION: We have identified the biological roles of SLC38A1, MIF, SOD2, HIF1A and SLC7A5 in endometrial cancer, which opens up the possibility of using the RNAi silencing approach to design therapeutic strategies for treatment of endometrial cancer.

Impact of elemental iron on human spermatozoa and mouse embryonic development in a defined synthetic culture medium.

There is a paucity of studies on effect of iron in embryo culture procedures. This study aims to ascertain the optimal, tolerance and toxic levels of iron in a protein-free embryo culture medium (PFM) to determine the effect of iron on embryonic development. The application of PFM in assisted reproductive technologies (ART) is intended to eliminate disease transmission and improve ART treatment outcome. The optimal, tolerance and toxic levels of iron on human spermatozoa and mouse embryos were determined by challenging them with different levels of iron (ferric iron; Fe+3). Human normozoospermic semen samples (n=24) and days 1-4 Quakenbush Special (Qs) mouse embryos (n=1160) were incubated in PFM supplemented with different concentrations of Fe+3 over different periods of time. 2.0µg/mL (35.8µM) of Fe+3 was the optimal level of Fe+3 for human spermatozoa with a tolerance range of 0.5-2µg/mL; whereas a level of 0.11µg/mL (2µM) of Fe+3 was the optimum for day 2 embryos. Levels of ferric iron at 0.11 to 2.8µg/mL appear to enhance spermatozoa motility, preserve its DNA integrity and possibly increase percentage of blastocysts developed but levels of ferric iron >16µg/mL is hazardous for both spermatozoa and embryos. In spite of beneficial effects of iron it is premature to recommend its supplementation in embryo culture media because of the known deleterious nature of iron and the paucity of toxicological data. Toxicological studies must be performed following which it can be decided whether it is safe to consider iron as a supplement in human embryo and spermatozoa culture media.

Cancer-associated fibroblasts promote endometrial cancer growth via activation of interleukin-6/STAT-3/c-Myc pathway.

Cancer-associated fibroblasts (CAFs) secrete various pro-tumorigenic cytokines, yet the role of these cytokines in the progression of endometrial cancer remains unclear. We found that CAFs isolated from human endometrial cancer (EC) tissues secreted high levels of interleukin-6 (IL-6), which promotes EC cell proliferation in vitro. Neutralizing IL-6 in CAF-conditioned media reduced (47% inhibition) while IL-6 recombinant protein increased cell proliferation (~2.4 fold) of both EC cell lines and primary cultures. IL-6 receptors (IL-6R and gp130) were expressed only in EC epithelial cells but not in CAF, indicating a one-way paracrine signaling. In the presence of CAF-conditioned media, Janus kinase/signal transducers and activators of transcription (JAK/STAT3) pathway was activated in EC cells. Treatment with JAK and STAT3 specific inhibitors, AD412 and STATTIC, respectively, significantly abrogated CAF-mediated cell proliferation, indicating the role of IL-6 activation in EC cell proliferation. We further showed that one of STAT-3 target genes, c-Myc, was highly induced in EC cells after exposure to CAF-conditioned medium at both mRNA (>105-fold vs. control) and protein level (>2-fold vs. control). EC cell proliferation was dependent on c-Myc expression, as RNAi-mediated c-Myc down-regulation led to a significant 46% reduction in cell viability when compared with scrambled control. Interestingly, CAF-conditioned media failed to promote proliferation in EC cells with reduced c-Myc expression, suggesting that CAF-mediated cell proliferation was also dependent on c-Myc expression. Subcutaneous tumor xenograft model showed that EC cells grew at least 1.4 times larger when co-injected with CAF, when compared to those injected with EC cells alone. Mice injected with EC cells with down-regulated c-Myc expression, however, showed at least 2.5 times smaller tumor compared to those in control group. Notably, there was no increase of tumor size when co-injected with CAFs. Further immunohistochemical staining on human tissues showed positive expression of IL-6 receptors, phosphorylated-STAT3 and c-Myc in human EC tissues with less signals in benign endometrium. Taken together, our data suggests that IL-6 secreted by CAF induces c-Myc expression to promote EC proliferation in vitro and in vivo. IL-6 pathway can be a potential target to disrupt tumor-stroma interaction in endometrial cancer progression.

Cancer-associated fibroblasts promote proliferation of endometrial cancer cells.

Endometrial cancer is the most commonly diagnosed gynecologic malignancy worldwide; yet the tumor microenvironment, especially the fibroblast cells surrounding the cancer cells, is poorly understood. We established four primary cultures of fibroblasts from human endometrial cancer tissues (cancer-associated fibroblasts, CAFs) using antibody-conjugated magnetic bead isolation. These relatively homogenous fibroblast cultures expressed fibroblast markers (CD90, vimentin and alpha-smooth muscle actin) and hormonal (estrogen and progesterone) receptors. Conditioned media collected from CAFs induced a dose-dependent proliferation of both primary cultures and cell lines of endometrial cancer in vitro (175%) when compared to non-treated cells, in contrast to those from normal endometrial fibroblast cell line (51%) (P<0.0001). These effects were not observed in fibroblast culture derived from benign endometrial hyperplasia tissues, indicating the specificity of CAFs in affecting endometrial cancer cell proliferation. To determine the mechanism underlying the differential fibroblast effects, we compared the activation of PI3K/Akt and MAPK/Erk pathways in endometrial cancer cells following treatment with normal fibroblasts- and CAFs-conditioned media. Western blot analysis showed that the expression of both phosphorylated forms of Akt and Erk were significantly down-regulated in normal fibroblasts-treated cells, but were up-regulated/maintained in CAFs-treated cells. Treatment with specific inhibitors LY294002 and U0126 reversed the CAFs-mediated cell proliferation (P<0.0001), suggesting for a role of these pathways in modulating endometrial cancer cell proliferation. Rapamycin, which targets a downstream molecule in PI3K pathway (mTOR), also suppressed CAFs-induced cell proliferation by inducing apoptosis. Cytokine profiling analysis revealed that CAFs secrete higher levels of macrophage chemoattractant protein (MCP)-1, interleukin (IL)-6, IL-8, RANTES and vascular endothelial growth factor (VEGF) than normal fibroblasts. Our data suggests that in contrast to normal fibroblasts, CAFs may exhibit a pro-tumorigenic effect in the progression of endometrial cancer, and PI3K/Akt and MAPK/Erk signaling may represent critical regulators in how endometrial cancer cells respond to their microenvironment.