Healthy lifestyle and prostate cancer risk in the Million Veteran Program.

BACKGROUND: This study aims to assess the impact of healthy lifestyle on prostate cancer (PCa) risk in a diverse population. METHODS: Data for 281,923 men from the Million Veteran Program (MVP), a nationwide, health system-based cohort study, were analyzed. Self-reported information at enrollment included smoking status, exercise, diet, family history of PCa, and race/ethnicity. Body mass index (BMI) was obtained from clinical records. Genetic risk was assessed via a validated polygenic score. Cox proportional hazards models were used to assess associations with PCa outcomes. RESULTS: After accounting for ancestry, family history, and genetic risk, smoking was associated with an increased risk of metastatic PCa (hazard ratio [HR], 1.83; 95% confidence interval [CI], 1.64-2.02; p < 10-16) and fatal PCa (HR, 2.73; 95% CI, 2.36-3.25; p < 10-16). Exercise was associated with a reduced risk of fatal PCa (HR, 0.86; 95% CI, 0.76-0.98; p = .03). Higher BMI was associated with a slightly reduced risk of fatal PCa, and diet score was not independently associated with any end point. Association with exercise was strongest among those who had nonmetastatic PCa at MVP enrollment. Absolute reductions in the risk of fatal PCa via lifestyle factors were greatest among men of African ancestry (1.7% for nonsmokers vs. 6.1% for smokers) or high genetic risk (1.4% for nonsmokers vs. 4.3% for smokers). CONCLUSIONS: Healthy lifestyle is minimally related to the overall risk of developing PCa but is associated with a substantially reduced risk of dying from PCa. In multivariable analyses, both exercise and not smoking remain independently associated with reduced metastatic and fatal PCa.

Clinical Impact of Contouring Variability for Prostate Cancer Tumor Boost.

PURPOSE: The focal radiation therapy (RT) boost technique was shown in a phase III randomized controlled trial (RCT) to improve prostate cancer outcomes without increasing toxicity. This technique relies on the accurate delineation of prostate tumors on MRI. A recent prospective study evaluated radiation oncologists' accuracy when asked to delineate prostate tumors on MRI and demonstrated high variability in tumor contours. We sought to evaluate the impact of contour variability and inaccuracy on predicted clinical outcomes. We hypothesized that radiation oncologists' contour inaccuracies would yield meaningfully worse clinical outcomes. METHODS AND MATERIALS: Forty-five radiation oncologists and 2 expert radiologists contoured prostate tumors on 30 patient cases. Of these cases, those with CT simulation or diagnostic CT available were selected for analysis. A knowledge-based planning model was developed to generate focal RT boost plans for each contour per the RCT protocol. The probability of biochemical failure (BF) was determined using a model from the RCT. The primary metric evaluated was delta BF (DBF = Participant BF - Expert BF). An absolute increase in BF ≥5% was considered clinically meaningful. RESULTS: Eight patient cases and 394 target volumes for focal RT boost planning were included in this analysis. In general, participant plans were associated with worse predicted clinical outcomes compared to the expert plan, with an average absolute increase in BF of 4.3%. Of participant plans, 37% were noted to have an absolute increase in BF of 5% or more. CONCLUSIONS: Radiation oncologists' attempts to contour tumor targets for focal RT boost are frequently inaccurate enough to yield meaningfully inferior clinical outcomes for patients.

Agent orange exposure and prostate cancer risk in the million veteran program.

BACKGROUND: The US government considers veterans to have been exposed to Agent Orange if they served in Vietnam while the carcinogen was in use, and these veterans are often deemed at high risk of prostate cancer (PCa). Here, we assess whether presumed Agent Orange exposure is independently associated with increased risk of any metastatic or fatal PCa in a diverse Veteran cohort still alive in the modern era (at least 2011), when accounting for race/ethnicity, family history, and genetic risk. PATIENTS AND METHODS: Participants in the Million Veteran Program (MVP; enrollment began in 2011) who were on active duty during the Vietnam War era (August 1964-April 1975) were included (n = 301,470). Agent Orange exposure was determined using the US government definition. Genetic risk was assessed via a validated polygenic hazard score. Associations with age at diagnosis of any PCa, metastatic PCa, and death from PCa were assessed via Cox proportional hazards models. RESULTS AND INTERPRETATION: On univariable analysis, exposure to Agent Orange was not associated with increased PCa (hazard ratio [HR]: 1.02, 95% confidence interval [CI]: 1.00-1.04, p = 0.06), metastatic PCa (HR: 0.98, 95% CI: 0.91-1.05, p = 0.55), or fatal PCa (HR: 0.94, 95% CI: 0.79-1.09, p = 0.41). When accounting for race/ethnicity and family history, Agent Orange exposure was independently associated with slightly increased risk of PCa (HR: 1.06, 95% CI: 1.04-1.09, <10-6) but not with metastatic PCa (HR: 1.07, 95% CI: 0.98-1.15, p = 0.10) or PCa death (HR: 1.02, 95% CI: 0.83-1.23, p = 0.09). Similar results were found when accounting for genetic risk. Agent Orange exposure history may not improve modern PCa risk stratification.

Use of focal radiotherapy boost for prostate cancer: radiation oncologists' perspectives and perceived barriers to implementation.

BACKGROUND: In a recent phase III randomized control trial, delivering a focal radiotherapy (RT) boost to tumors visible on MRI was shown to improve disease-free survival and regional/distant metastasis-free survival for patients with prostate cancer-without increasing toxicity. The aim of this study was to assess how widely this technique is being applied in current practice, as well as physicians' perceived barriers toward its implementation. METHODS: We invited radiation oncologists to complete an online questionnaire assessing their use of intraprostatic focal boost in December 2022 and February 2023. To include perspectives from a broad range of practice settings, the invitation was distributed to radiation oncologists worldwide via email list, group text platform, and social media. RESULTS: 263 radiation oncologist participants responded. The highest-represented countries were the United States (42%), Mexico (13%), and the United Kingdom (8%). The majority of participants worked at an academic medical center (52%) and considered their practice to be at least partially genitourinary (GU)-subspecialized (74%). Overall, 43% of participants reported routinely using intraprostatic focal boost. Complete GU-subspecialists were more likely to implement focal boost, with 61% reporting routine use. In both high-income and low-to-middle-income countries, less than half of participants routinely use focal boost. The most cited barriers were concerns about registration accuracy between MRI and CT (37%), concerns about risk of additional toxicity (35%), and challenges to accessing high-quality MRI (29%). CONCLUSIONS: Two years following publication of a randomized trial of patient benefit without increased toxicity, almost half of the radiation oncologists surveyed are now routinely offering focal RT boost. Further adoption of this technique might be aided by increased access to high-quality MRI, better registration algorithms of MRI to CT simulation images, physician education on benefit-to-harm ratio, and training on contouring prostate lesions on MRI.

The Evolving Landscape of B Cells in Cancer Metastasis.

Metastasis is the leading cause of cancer mortality. Functional and clinical studies have documented diverse B-cell and antibody responses in cancer metastasis. The presence of B cells in tumor microenvironments and metastatic sites has been associated with diverse effects that can promote or inhibit metastasis. Specifically, B cells can contribute to the spread of cancer cells by enhancing tumor cell motility, invasion, angiogenesis, lymphangiogenesis, and extracellular matrix remodeling. Moreover, they can promote metastatic colonization by triggering pathogenic immunoglobulin responses and recruiting immune suppressive cells. Contrastingly, B cells can also exhibit antimetastatic effects. For example, they aid in enhanced antigen presentation, which helps activate immune responses against cancer cells. In addition, B cells play a crucial role in preventing the dissemination of metastatic cells from the primary tumor and secrete antibodies that can aid in tumor recognition. Here, we review the complex roles of B cells in metastasis, delineating the heterogeneity of B-cell activity and subtypes by metastatic site, antibody class, antigen (if known), and molecular phenotype. These important attributes of B cells emphasize the need for a deeper understanding and characterization of B-cell phenotypes to define their effects in metastasis.

Agent Orange exposure and prostate cancer risk in the Million Veteran Program.

Purpose: Exposure to Agent Orange, a known carcinogen, might increase risk of prostate cancer (PCa). We sought to investigate the association of Agent Orange exposure and PCa risk when accounting for race/ethnicity, family history, and genetic risk in a diverse population of US Vietnam War veterans. Methods & Materials: This study utilized the Million Veteran Program (MVP), a national, population-based cohort study of United States military veterans conducted 2011-2021 with 590,750 male participants available for analysis. Agent Orange exposure was obtained using records from the Department of Veterans Affairs (VA) using the US government definition of Agent Orange exposure: active service in Vietnam while Agent Orange was in use. Only veterans who were on active duty (anywhere in the world) during the Vietnam War were included in this analysis (211,180 participants). Genetic risk was assessed via a previously validated polygenic hazard score calculated from genotype data. Age at diagnosis of any PCa, diagnosis of metastatic PCa, and death from PCa were assessed via Cox proportional hazards models. Results: Exposure to Agent Orange was associated with increased PCa diagnosis (HR 1.04, 95% CI 1.01-1.06, p=0.003), primarily among Non-Hispanic White men (HR 1.09, 95% CI 1.06- 1.12, p<0.001). When accounting for race/ethnicity and family history, Agent Orange exposure remained an independent risk factor for PCa diagnosis (HR 1.06, 95% CI 1.04-1.09, p<0.05). Univariable associations of Agent Orange exposure with PCa metastasis (HR 1.08, 95% CI 0.99-1.17) and PCa death (HR 1.02, 95% CI 0.84-1.22) did not reach significance on multivariable analysis. Similar results were found when accounting for polygenic hazard score. Conclusions: Among US Vietnam War veterans, Agent Orange exposure is an independent risk factor for PCa diagnosis, though associations with PCa metastasis or death are unclear when accounting for race/ethnicity, family history, and/or polygenic risk.

ReIGNITE Radiation Therapy Boost: A Prospective, International Study of Radiation Oncologists' Accuracy in Contouring Prostate Tumors for Focal Radiation Therapy Boost on Conventional Magnetic Resonance Imaging Alone or With Assistance of Restriction Spectrum Imaging.

PURPOSE: In a phase III randomized trial, adding a radiation boost to tumor(s) visible on MRI improved prostate cancer (PCa) disease-free and metastasis-free survival without additional toxicity. Radiation oncologists' ability to identify prostate tumors is critical to widely adopting intraprostatic tumor radiotherapy boost for patients. A diffusion MRI biomarker, called the Restriction Spectrum Imaging restriction score (RSIrs), has been shown to improve radiologists' identification of clinically significant PCa. We hypothesized that (1) radiation oncologists would find accurately delineating PCa tumors on conventional MRI challenging and (2) using RSIrs maps would improve radiation oncologists' accuracy for PCa tumor delineation. METHODS AND MATERIALS: In this multi-institutional, international, prospective study, 44 radiation oncologists (participants) and 2 expert radiologists (experts) contoured prostate tumors on 39 total patient cases using conventional MRI with or without RSIrs maps. Participant volumes were compared to the consensus expert volumes. Contouring accuracy metrics included percent overlap with expert volume, Dice coefficient, conformal number, and maximum distance beyond expert volume. RESULTS: 1604 participant volumes were produced. 40 of 44 participants (91%) completely missed ≥1 expert-defined target lesion without RSIrs, compared to 13 of 44 (30%) with RSIrs maps. On conventional MRI alone, 134 of 762 contour attempts (18%) completely missed the target, compared to 18 of 842 (2%) with RSIrs maps. Use of RSIrs maps improved all contour accuracy metrics by approximately 50% or more. Mixed effects modeling confirmed that RSIrs maps were the main variable driving improvement in all metrics. System Usability Scores indicated RSIrs maps significantly improved the contouring experience (72 vs. 58, p < 0.001). CONCLUSIONS: Radiation oncologists struggle with accurately delineating visible PCa tumors on conventional MRI. RSIrs maps improve radiation oncologists' ability to target MRI-visible tumors for prostate tumor boost.

Use of focal radiotherapy boost for prostate cancer and perceived barriers toward its implementation: a survey.

Background: In a recent phase III randomized control trial (FLAME), delivering a focal radiotherapy (RT) boost to tumors visible on MRI was shown to improve outcomes for prostate cancer patients without increasing toxicity. The aim of this study was to assess how widely this technique is being applied in current practice as well as physicians' perceived barriers toward its implementation. Methods: An online survey assessing the use of intraprostatic focal boost was conducted in December 2022 and February 2023. The survey link was distributed to radiation oncologists worldwide via email list, group text platform, and social media. Results: The survey initially collected 205 responses from various countries over a two-week period in December 2022. The survey was then reopened for one week in February 2023 to allow for more participation, leading to a total of 263 responses. The highest-represented countries were the United States (42%), Mexico (13%), and the United Kingdom (8%). The majority of participants worked at an academic medical center (52%) and considered their practice to be at least partially genitourinary (GU)-subspecialized (74%). 57% of participants reported not routinely using intraprostatic focal boost. Even among complete subspecialists, a substantial proportion (39%) do not routinely use focal boost. Less than half of participants in both high-income and low-to-middle-income countries were shown to routinely use focal boost. The most commonly cited barriers were concerns about registration accuracy between MRI and CT (37%), concerns about risk of additional toxicity (35%), and challenges to accessing high-quality MRI (29%). Conclusion: Despite level 1 evidence from the FLAME trial, most radiation oncologists surveyed are not routinely offering focal RT boost. Adoption of this technique might be accelerated by increased access to high-quality MRI, better registration algorithms of MRI to CT simulation images, physician education on benefit-to-harm ratio, and training on contouring prostate lesions on MRI.

Automated Patient-level Prostate Cancer Detection with Quantitative Diffusion Magnetic Resonance Imaging.

Background: Multiparametric magnetic resonance imaging (mpMRI) improves detection of clinically significant prostate cancer (csPCa), but the subjective Prostate Imaging Reporting and Data System (PI-RADS) system and quantitative apparent diffusion coefficient (ADC) are inconsistent. Restriction spectrum imaging (RSI) is an advanced diffusion-weighted MRI technique that yields a quantitative imaging biomarker for csPCa called the RSI restriction score (RSIrs). Objective: To evaluate RSIrs for automated patient-level detection of csPCa. Design setting and participants: We retrospectively studied all patients (n = 151) who underwent 3 T mpMRI and RSI (a 2-min sequence on a clinical scanner) for suspected prostate cancer at University of California San Diego during 2017-2019 and had prostate biopsy within 180 d of MRI. Intervention: We calculated the maximum RSIrs and minimum ADC within the prostate, and obtained PI-RADS v2.1 from medical records. Outcome measurements and statistical analysis: We compared the performance of RSIrs, ADC, and PI-RADS for the detection of csPCa (grade group ≥2) on the best available histopathology (biopsy or prostatectomy) using the area under the curve (AUC) with two-tailed α = 0.05. We also explored whether the combination of PI-RADS and RSIrs might be superior to PI-RADS alone and performed subset analyses within the peripheral and transition zones. Results and limitations: AUC values for ADC, RSIrs, and PI-RADS were 0.48 (95% confidence interval: 0.39, 0.58), 0.78 (0.70, 0.85), and 0.77 (0.70, 0.84), respectively. RSIrs and PI-RADS were each superior to ADC for patient-level detection of csPCa (p < 0.0001). RSIrs alone was comparable with PI-RADS (p = 0.8). The combination of PI-RADS and RSIrs had an AUC of 0.85 (0.78, 0.91) and was superior to either PI-RADS or RSIrs alone (p < 0.05). Similar patterns were seen in the peripheral and transition zones. Conclusions: RSIrs is a promising quantitative marker for patient-level csPCa detection, warranting a prospective study. Patient summary: We evaluated a rapid, advanced prostate magnetic resonance imaging technique called restriction spectrum imaging to see whether it could give an automated score that predicted the presence of clinically significant prostate cancer. The automated score worked about as well as expert radiologists' interpretation. The combination of the radiologists' scores and automated score might be better than either alone.

HER2 and HER3 as Therapeutic Targets in Head and Neck Cancer.

ABSTRACT: Work over the past several decades has identified that aberrations in the ErbB signaling pathways are key drivers of oncogenesis, and concurrent efforts to discover targetable vulnerabilities to counter this aberrant oncogenic signaling offer tremendous promise in treating a host of human cancers. These efforts have been centered primarily on EGFR (also known as HER1), leading to the discovery of the first targeted therapies approved for head and neck cancer. More recently, HER2 and HER3 signaling pathways have been identified as highly dysregulated in head and neck cancer. This review highlights the HER2 and HER3 signaling pathways and clinical efforts to target these receptors and their aberrant signaling to treat head and neck squamous cell carcinomas and other head and neck malignancies, including salivary gland carcinomas. This includes the use of small molecule inhibitors and blocking antibodies, both as single agents or as part of multimodal precision targeted and immunotherapies.

Correcting B0 inhomogeneity-induced distortions in whole-body diffusion MRI of bone.

Diffusion-weighted magnetic resonance imaging (DWI) of the musculoskeletal system has various applications, including visualization of bone tumors. However, DWI acquired with echo-planar imaging is susceptible to distortions due to static magnetic field inhomogeneities. This study aimed to estimate spatial displacements of bone and to examine whether distortion corrected DWI images more accurately reflect underlying anatomy. Whole-body MRI data from 127 prostate cancer patients were analyzed. The reverse polarity gradient (RPG) technique was applied to DWI data to estimate voxel-level distortions and to produce a distortion corrected DWI dataset. First, an anatomic landmark analysis was conducted, in which corresponding vertebral landmarks on DWI and anatomic T2-weighted images were annotated. Changes in distance between DWI- and T2-defined landmarks (i.e., changes in error) after distortion correction were calculated. In secondary analyses, distortion estimates from RPG were used to assess spatial displacements of bone metastases. Lastly, changes in mutual information between DWI and T2-weighted images of bone metastases after distortion correction were calculated. Distortion correction reduced anatomic error of vertebral DWI up to 29 mm. Error reductions were consistent across subjects (Wilcoxon signed-rank p < 10-20). On average (± SD), participants' largest error reduction was 11.8 mm (± 3.6). Mean (95% CI) displacement of bone lesions was 6.0 mm (95% CI 5.0-7.2); maximum displacement was 17.1 mm. Corrected diffusion images were more similar to structural MRI, as evidenced by consistent increases in mutual information (Wilcoxon signed-rank p < 10-12). These findings support the use of distortion correction techniques to improve localization of bone on DWI.

Performance of African-ancestry-specific polygenic hazard score varies according to local ancestry in 8q24.

BACKGROUND: We previously developed an African-ancestry-specific polygenic hazard score (PHS46+African) that substantially improved prostate cancer risk stratification in men with African ancestry. The model consists of 46 SNPs identified in Europeans and 3 SNPs from 8q24 shown to improve model performance in Africans. Herein, we used principal component (PC) analysis to uncover subpopulations of men with African ancestry for whom the utility of PHS46+African may differ. MATERIALS AND METHODS: Genotypic data were obtained from the PRACTICAL consortium for 6253 men with African genetic ancestry. Genetic variation in a window spanning 3 African-specific 8q24 SNPs was estimated using 93 PCs. A Cox proportional hazards framework was used to identify the pair of PCs most strongly associated with the performance of PHS46+African. A calibration factor (CF) was formulated using Cox coefficients to quantify the extent to which the performance of PHS46+African varies with PC. RESULTS: CF of PHS46+African was strongly associated with the first and twentieth PCs. Predicted CF ranged from 0.41 to 2.94, suggesting that PHS46+African may be up to 7 times more beneficial to some African men than others. The explained relative risk for PHS46+African varied from 3.6% to 9.9% for individuals with low and high CF values, respectively. By cross-referencing our data set with 1000 Genomes, we identified significant associations between continental and calibration groupings. CONCLUSION: We identified PCs within 8q24 that were strongly associated with the performance of PHS46+African. Further research to improve the clinical utility of polygenic risk scores (or models) is needed to improve health outcomes for men of African ancestry.

Disrupting interferon-alpha and NF-kappaB crosstalk suppresses IFITM1 expression attenuating triple-negative breast cancer progression.

Overexpression of interferon induced transmembrane protein-1 (IFITM1) enhances tumor progression in multiple cancers, but its role in triple-negative breast cancer (TNBC) is unknown. Here, we explore the functional significance and regulation of IFITM1 in TNBC and strategies to target its expression. Immunohistochemistry staining of a tissue microarray demonstrates that IFITM1 is overexpressed in TNBC samples which is confirmed by TCGA analysis. Targeting IFITM1 by siRNA or CRISPR/Cas9 in TNBC cell lines significantly inhibits proliferation, colony formation, and wound healing in vitro. Orthotopic mammary fat pad and mammary intraductal studies reveal that loss of IFITM1 reduces TNBC tumor growth and invasion in vivo. RNA-seq analysis of IFITM1/KO cells reveals significant downregulation of several genes involved in proliferation, migration, and invasion and functional studies identified NF-κB as an important downstream target of IFITM1. Notably, siRNA knockdown of p65 reduces IFITM1 expression and a drug-repurposing screen of FDA approved compounds identified parthenolide, an NFκB inhibitor, as a cytotoxic agent for TNBC and an inhibitor of IFITM1 in vitro and in vivo. Overall, our findings suggest that targeting IFITM1 by suppressing interferon-alpha/NFκB signaling represents a novel therapeutic strategy for TNBC treatment.

A rare case of group A streptococcal toxic-shock syndrome in a postpartum adolescent leading to multi-organ failure.

Teenage pregnancy is not uncommon, but given the age of the patient, experience, and competency among medical providers varies. While toxic-shock syndrome from group A streptococcus is rare in teenage pregnancy, observed is a gap in care of bridging.

Interaction Between MUC1 and STAT1 Drives IFITM1 Overexpression in Aromatase Inhibitor-Resistant Breast Cancer Cells and Mediates Estrogen-Induced Apoptosis.

The human oncoprotein, mucin 1 (MUC1), drives tumorigenesis in breast carcinomas by promoting epithelial-to-mesenchymal transition (EMT), epigenetic reprogramming, and evasion of immune response. MUC1 interacts with STAT1, through JAK/STAT signaling, and stimulates transcription of IFN-stimulated genes, specifically IFN-induced transmembrane protein 1 (IFITM1). Our laboratory has previously shown that IFITM1 overexpression in aromatase inhibitor (AI)-resistant breast cancer cells promotes aggressiveness. Here, we demonstrate that differential regulation of MUC1 in AI-sensitive (MCF-7 and T-47D) compared with AI-resistant (MCF-7:5C) cells is critical in mediating IFITM1 expression. A tumor microarray of 94 estrogen receptor-positive human breast tumors correlated coexpression of MUC1 and IFITM1 with poor recurrence-free survival, poor overall survival, and AI-resistance. In this study, we investigated the effects of MUC1/IFITM1 on cell survival and proliferation. We knocked down MUC1 levels with siRNA and pharmacologic inhibitors, which abrogated IFITM1 mRNA and protein expression and induced cell death in AI-resistant cells. In vivo, estrogen and ruxolitinib significantly reduced tumor size and decreased expression of MUC1, P-STAT1, and IFITM1. IMPLICATIONS: MUC1 and IFITM1 overexpression drives AI resistance and can be targeted with currently available therapies.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/5/1180/F1.large.jpg.

IFITM1 suppression blocks proliferation and invasion of aromatase inhibitor-resistant breast cancer in vivo by JAK/STAT-mediated induction of p21.

Interferon induced transmembrane protein 1 (IFITM1) belongs to a family of interferon stimulated genes (ISGs) that is associated with tumor progression and DNA damage resistance; however, its role in endocrine resistance is not known. Here, we correlate IFITM1 expression with clinical stage and poor response to endocrine therapy in a tissue microarray consisting of 94 estrogen receptor (ER)-positive breast tumors. IFITM1 overexpression is confirmed in the AI-resistant MCF-7:5C cell line and not found in AI-sensitive MCF-7 cells. In this study, the orthotopic (mammary fat pad) and mouse mammary intraductal (MIND) models of breast cancer are used to assess tumor growth and invasion in vivo. Lentivirus-mediated shRNA knockdown of IFITM1 in AI-resistant MCF-7:5C cells diminished tumor growth and invasion and induced cell death, whereas overexpression of IFITM1 in wild-type MCF-7 cells promoted estrogen-independent growth and enhanced their aggressive phenotype. Mechanistic studies indicated that loss of IFITM1 in MCF-7:5C cells markedly increased p21 transcription, expression and nuclear localization which was mediated by JAK/STAT activation. These findings suggest IFITM1 overexpression contributes to breast cancer progression and that targeting IFITM1 may be therapeutically beneficial to patients with endocrine-resistant disease.

Everolimus downregulates estrogen receptor and induces autophagy in aromatase inhibitor-resistant breast cancer cells.

BACKGROUND: mTOR inhibition of aromatase inhibitor (AI)-resistant breast cancer is currently under evaluation in the clinic. Everolimus/RAD001 (Afinitor®) has had limited efficacy as a solo agent but is projected to become part of combination therapy for AI-resistant breast cancer. This study was conducted to investigate the anti-proliferative and resistance mechanisms of everolimus in AI-resistant breast cancer cells. METHODS: In this study we utilized two AI-resistant breast cancer cell lines, MCF-7:5C and MCF-7:2A, which were clonally derived from estrogen receptor positive (ER+) MCF-7 breast cancer cells following long-term estrogen deprivation. Cell viability assay, colony formation assay, cell cycle analysis and soft agar anchorage-independent growth assay were used to determine the efficacy of everolimus in inhibiting the proliferation and tumor forming potential of MCF-7, MCF-7:5C, MCF-7:2A and MCF10A cells. Confocal microscopy and transmission electron microscopy were used to evaluate LC3-II production and autophagosome formation, while ERE-luciferase reporter, Western blot, and RT-PCR analyses were used to assess ER expression and transcriptional activity. RESULTS: Everolimus inhibited the proliferation of MCF-7:5C and MCF-7:2A cells with relatively equal efficiency to parental MCF-7 breast cancer cells. The inhibitory effect of everolimus was due to G1 arrest as a result of downregulation of cyclin D1 and p21. Everolimus also dramatically reduced estrogen receptor (ER) expression (mRNA and protein) and transcriptional activity in addition to the ER chaperone, heat shock protein 90 protein (HSP90). Everolimus restored 4-hydroxy-tamoxifen (4OHT) sensitivity in MCF-7:5C cells and enhanced 4OHT sensitivity in MCF-7 and MCF-7:2A cells. Notably, we found that autophagy is one method of everolimus insensitivity in MCF-7 breast cancer cell lines. CONCLUSION: This study provides additional insight into the mechanism(s) of action of everolimus that can be used to enhance the utility of mTOR inhibitors as part of combination therapy for AI-resistant breast cancer.

Interferon-induced transmembrane protein 1 (IFITM1) overexpression enhances the aggressive phenotype of SUM149 inflammatory breast cancer cells in a signal transducer and activator of transcription 2 (STAT2)-dependent manner.

BACKGROUND: Inflammatory breast cancer (IBC) is a very aggressive and lethal subtype of breast cancer that accounts for about 4 % of all breast cancers diagnosed in the United States. Despite the efforts of several investigators to identify the molecular factors driving the aggressive phenotype of IBC, a great deal is still unknown about the molecular underpinnings of the disease. In the present study, we investigated the role of interferon-induced transmembrane protein 1 (IFITM1), a well-known interferon-stimulated gene (ISG), in promoting the aggressiveness of SUM149 IBC cells. METHODS: Western blot and real-time polymerase chain reaction analyses were performed to assess the protein and messenger RNA (mRNA) levels of IFITM1 and other ISGs in three IBC cell lines: SUM149, MDA-IBC-3, and SUM190. IFITM1 expression and cellular localization were assessed by using immunofluorescence, while the tumorigenic potential was assessed by performing cell migration, invasion, and colony formation assays. Small interfering RNA and short hairpin RNA knockdowns, enzyme-linked immunosorbent assays, and luciferase assays were performed to determine the functional significance of IFITM1 and signal transducers and activators of transcription 1 and 2 (STAT1/2) in SUM149 cells. RESULTS: We found that IFITM1 was constitutively overexpressed at the mRNA and protein levels in triple-negative SUM149 IBC cells, but that it was not expressed in SUM190 and MDA-IBC-3 IBC cells, and that suppression of IFITM1 or blockade of the IFNα signaling pathway significantly reduced the aggressive phenotype of SUM149 cells. Additionally, we found that knockdown of STAT2 abolished IFITM1 expression and IFITM1 promoter activity in SUM149 cells and that loss of STAT2 significantly inhibited the ability of SUM149 cells to proliferate, migrate, invade, and form 2-D colonies. Notably, we found that STAT2-mediated activation of IFITM1 was particularly dependent on the chromatin remodeler brahma-related gene 1 (BRG1), which was significantly elevated in SUM149 cells compared with SUM190 and MDA-IBC-3 cells. CONCLUSIONS: These findings indicate that overexpression of IFITM1 enhances the aggressive phenotype of triple-negative SUM149 IBC cells and that this effect is dependent on STAT2/BRG1 interaction. Further studies are necessary to explore the potential of IFITM1 as a novel therapeutic target and prognostic marker for some subtypes of IBCs.

Targeting interferon response genes sensitizes aromatase inhibitor resistant breast cancer cells to estrogen-induced cell death.

INTRODUCTION: Estrogen deprivation using aromatase inhibitors (AIs) is currently the standard of care for postmenopausal women with hormone receptor-positive breast cancer. Unfortunately, the majority of patients treated with AIs eventually develop resistance, inevitably resulting in patient relapse and, ultimately, death. The mechanism by which resistance occurs is still not completely known, however, recent studies suggest that impaired/defective interferon signaling might play a role. In the present study, we assessed the functional role of IFITM1 and PLSCR1; two well-known interferon response genes in AI resistance. METHODS: Real-time PCR and Western blot analyses were used to assess mRNA and protein levels of IFITM1, PLSCR1, STAT1, STAT2, and IRF-7 in AI-resistant MCF-7:5C breast cancer cells and AI-sensitive MCF-7 and T47D cells. Immunohistochemistry (IHC) staining was performed on tissue microarrays consisting of normal breast tissues, primary breast tumors, and AI-resistant recurrence tumors. Enzyme-linked immunosorbent assay was used to quantitate intracellular IFNα level. Neutralizing antibody was used to block type 1 interferon receptor IFNAR1 signaling. Small interference RNA (siRNA) was used to knockdown IFITM1, PLSCR1, STAT1, STAT2, IRF-7, and IFNα expression. RESULTS: We found that IFITM1 and PLSCR1 were constitutively overexpressed in AI-resistant MCF-7:5C breast cancer cells and AI-resistant tumors and that siRNA knockdown of IFITM1 significantly inhibited the ability of the resistant cells to proliferate, migrate, and invade. Interestingly, suppression of IFITM1 significantly enhanced estradiol-induced cell death in AI-resistant MCF-7:5C cells and markedly increased expression of p21, Bax, and Noxa in these cells. Significantly elevated level of IFNα was detected in AI-resistant MCF-7:5C cells compared to parental MCF-7 cells and suppression of IFNα dramatically reduced IFITM1, PLSCR1, p-STAT1, and p-STAT2 expression in the resistant cells. Lastly, neutralizing antibody against IFNAR1/2 and knockdown of STAT1/STAT2 completely suppressed IFITM1, PLSCR1, p-STAT1, and p-STAT2 expression in the resistant cells, thus confirming the involvement of the canonical IFNα signaling pathway in driving the overexpression of IFITM1 and other interferon-stimulated genes (ISGs) in the resistant cells. CONCLUSION: Overall, these results demonstrate that constitutive overexpression of ISGs enhances the progression of AI-resistant breast cancer and that suppression of IFITM1 and other ISGs sensitizes AI-resistant cells to estrogen-induced cell death.