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Figure S4 Quantification of Western blot analysis (related to Figure 4) (B-G). Data are presented as mean fold increases ({plus minus}SD) in treated groups over basal values from three independent experiments. B, *p

Figure S6 The comparison of expression levels of phospho-ERK and phospho-Smad4 between lean and overweight/obesity patients, Related to Figure 7. Staining was analyzed by using a system based on the percentage of positively stained cells and the staining intensity. Integrated optical density of all the positive staining in each image was determined, and its ratio to total area of each photograph was calculated as density.

Obesity increases the risk of distant metastatic recurrence and reduces breast cancer survival. However, the mechanisms behind this pathology and identification of relevant therapeutic targets are poorly defined. Plasma free fatty acids (FFA) levels are elevated in obese individuals. Here we report that TGFβ transiently activates ERK and subsequently phosphorylates SMAD4 at Thr277, which facilitates a SMAD4–USP9x interaction, SMAD4 nuclear retention, and stimulates TGFβ/SMAD3–mediated transcription of Twist and Snail. USP9x inhibited the E3 ubiquitin-protein ligase TIF1γ from binding and monoubiquitinating SMAD4, hence maintaining the SMAD4 nuclear retention. FFA further facilitated TGFβ-induced ERK activation, SMAD4 phosphorylation, and nuclear retention, promoting TGFβ-dependent cancer progression. Inhibition of ERK and USP9x suppressed obesity-induced metastasis. In addition, clinical data indicated that phospho-ERK and -SMAD4 levels correlate with activated TGFβ signaling and metastasis in overweight/obese patient breast cancer specimens. Altogether, we demonstrate the vital interaction of USP9x and SMAD4 for governing TGFβ signaling and dyslipidemia-induced aberrant TGFβ activation during breast cancer metastasis. Cancer Res; 77(6); 1383–94. ©2017 AACR.

Figure S1 Quantification of Western blot analysis (related to Figure 1) (A-H). Data are presented as mean fold increases ({plus minus}SD) in treated groups over basal values from three independent experiments. *p < 0.01 versus controls; #p < 0.01 versus their corresponding DMSO controls. FFA facilitates TGF-beta-induced ERK activation and SAMD4 nuclear retention, Related to Figure 1. I, Nuclear extracts were made from MDA-MB-231 cells that were treated with 3 ng/ml of TGF-beta for the time points shown. They were analyzed by Western blotting with antibodies against Smad4 or phosphor-ERK and ERK. H3 was used as a loading control for nuclear fractions. We evaluated the cytoplasmic contamination of the nuclear fractions by blotting for α-vinculin. J, Nuclear extracts were made from MDA-MB-231 cells treated with 3 ng/ml of TGF-beta in the presence of palmitate (PA, 400 microM) for the time points shown. Phosphor-ERK and ERK and nuclear SMAD4 were analyzed by Western blotting.

Figure S3 FFA promotes TGF-beta-induced migration by activating ERK and USP9x, Related to Figure 3. A, BT549 cells were plated in Ibidi culture insert dishes (Madison, WI). Cells were cultured in complete medium to 80% confluence, inserts were removed to create a gap and cells were then incubated with BSA, PA, PA plus AZD6244 or WP1130 in the absence or presence of TGF-beta1. Migrating cells were photographed under a phase contrast microscope. B, The percentage of the wound closed was quantified from three independent replicates and is expressed as mean {plus minus} SD. *p

Intro: Hematopoietic stem-cell transplant (HSCT) recipients are considered to be at high risk for poor outcomes following COVID-19 infection given their co-morbidities and immunosuppression. Sharma et al published the CIBMTR observational report data that showed that recipients of allogeneic HSCT who contract COVID-19 have poor overall survival with a 30-day mortality of 32%. That being said, there have been relatively few studies that look into the effect of COVID-19 on HSCT recipients in the setting of in vivo T cell depletion protocols. With the increased use of post-transplant cyclophosphamide (PTCy) based GVHD prophylaxis regimens for our match related and match unrelated HSCT recipients since 2018 at our institution, we are interested to see if our COVID-19 outcomes differ from those published in the CIBMTR report. Methods: This is a single institution retrospective analysis evaluating outcomes of HSCT recipients who were diagnosed with COVID-19 between March 2020 and April 2021. Patients 18 years or older who underwent HSCT and subsequently contracted COVID-19 were included in the data collection. Demographic data including age, type of hematologic malignancy, conditioning regimen, GVHD prophylaxis, date of COVID-19 infection, with pre- and post-COVID-19 infection labs were obtained. Our primary endpoint in this retrospective analysis was non-relapse mortality within 30 days of COVID-19 diagnosis. Results: There were 21 patients at our institution who had undergone HSCT and subsequently contracted COVID-19. The most common primary disease types were acute lymphoblastic leukemia (33.3%), acute myeloid leukemia (23.8%), and myelodysplastic syndrome (19.0%). The median age of our patient population was 53 years (range, 24-66). 6 of the patients received match related allografts. 7 received cells from match unrelated donors. 7 received cells from haploidentical donors. 1 patient had received an autologous stem cell transplant. Of the remaining 20 allo-HSCT recipients, 14 of them (70.0%) received myeloablative conditioning regimens, whereas 6 (30.0%) received reduced intensity or non-myeloablative regimens. Our GVHD prophylaxis regimens were PTCy/Tacro/MMF (12 pts, 60.0%) and Tacro/MTX (8 pts, 40.0%). Patient demographics and outcomes are found on Tables 1 and 2. Our patients were diagnosed with COVID-19 a median 469 days post-transplant, with 8 patients (38.1%) diagnosed with COVID-19 within 1 year of transplant. 11 of the patients (52.4%) received steroids following their diagnosis with COVID-19. Of the 20 allo-HSCT recipients with confirmed COVID-19 infection, 1 passed away 20 days after the diagnosis was made. This gives us a 5.0% case fatality rate attributable to COVID-19 in our population in our allo-HSCT population. 16 of the 20 patients were symptomatic at the time of diagnosis (80.0%). 7 of the 20 patients (35.0%) were hospitalized for a median of 7 days (range, 5-17 days), with 2 requiring ICU level of care. The one patient who passed away tested positive for COVID-19 177 days post-transplant and was hospitalized approximately 7 days after diagnosis, where he was intubated on hospital day 4 and ultimately passed away on hospital day 13. The patient had received Tacro/MTX for GVHD prophylaxis. Discussion: Although this is a small sample size, our data suggests that our allo-HSCT recipients who contracted COVID-19 have had generally good short-term outcomes. Our study is limited by the small number of patients who got infected with COVID-19, particularly those within 1-year post-transplant. Furthermore, we acknowledge it is difficult to claim that the PTCy based GVHD prophylaxis regimens for our HSCT recipients were solely responsible for their improved outcomes since 40% of allo-HSCT recipients did not get PTCy for GVHD prophylaxis. However, we believe it would be valuable to evaluate in a prospective analysis. We are currently evaluating if a COVID-19 diagnosis has any effect on long term transplant related complications and outcomes in this population. Figure 1 Figure 1. Disclosures Chaudhary: Angeles Therapeutics: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties: Cell therapy ; Celldex: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company; Pancella: Consultancy; Oncotartis: Consultancy; Athelas: Consultancy, Current holder of stock options in a privately-held company; TCR2: Current equity holder in publicly-traded company; Allogene: Current equity holder in publicly-traded company. Yaghmour: Novartis: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Alexion: Speakers Bureau; Astellas: Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Jazz: Speakers Bureau; Agios: Consultancy, Speakers Bureau.

Intro: Acute and chronic graft versus host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplant (allo-HSCT). Post-transplant cyclophosphamide (PTCy), alongside mycophenolate mofetil (MMF) and tacrolimus (TAC), is known to be effective in reducing cGVHD in haploidentical (HI) transplant recipients. Results of the prospective randomized HOVON-96 trial in recipients of matched related (MRD) and unrelated donors (MUD) showed that PTCy leads to a reduction in risk of severe cGVHD. However, the role of PTCy in HSCT with acute lymphoblastic leukemia (ALL) remains controversial. The purpose of this study is to evaluate outcomes using PTCy, TAC, and MMF for GVHD prophylaxis in MRD and MUD stem cell recipients who have ALL. Methods: This study is a retrospective analysis of adult patients (aged 19 and up) with ALL who received MRD, MUD, and HI HSCT at USC Norris Cancer Hospital from 2018 to 2020. The primary end-points were to compare incidence of aGVHD and cGVHD within the first year post-transplant after receiving MRD, MUD, and HI donor transplants. Secondary end-points were day +100 mortality, 1-year overall survival (OS), relapse free survival (RFS), transplant-related mortality (TRM), and GRFS within the same subgroups. Kaplan-Meier curves and Log Rank tests were used to evaluate a difference in outcomes. Results: A total of 46 patients with ALL who received HSCTs were identified and separated based on type of GVHD prophylaxis received: 7 (15.1%), 16 (34.8%), and 23 (50.0%) in the PT-Cy, HI and standard groups respectively. Demographic data can be found on Table 1 attached. Baseline characteristics among all groups were similar with the exception of the more Human Leukocyte Antigen (HLA) mismatch (p Discussion: In this single institution analysis, we observed that patients with ALL who underwent HI transplant had the best outcomes. Moreover, patients receiving MUD/MRD HSCT with use of PTCy had a non-significant reduced incidence of aGVHD and cGVHD, though had significantly worse 1-year OS, RFS, and GRFS when compared to standard GVHD prophylaxis or patients receiving HI transplants. We believe this is likely a multifactorial process. However, the increased percentage of mismatched antigens in the MUD transplants (p=0.19) and higher proportion of patients receiving less than 5.0 x 10^6 CD34+ cells/kg (p=0.02) in the PTCy group is important to acknowledge and can also be contributing to their poorer outcomes. We pose the idea that the use of PTCy may also lead to a reduced graft vs. leukemia effect in this population and thus a higher incidence of relapse and worse outcomes overall. A larger, prospective study should be considered to take into account these factors to better determine if patients with ALL could benefit from PTCy for GVHD prophylaxis. Figure 1 Figure 1. Disclosures Chaudhary: Celldex: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company; Pancella: Consultancy; Oncotartis: Consultancy; Athelas: Consultancy, Current holder of stock options in a privately-held company; TCR2: Current equity holder in publicly-traded company; Allogene: Current equity holder in publicly-traded company; Angeles Therapeutics: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties: Cell therapy . Yaghmour: Takeda: Consultancy, Speakers Bureau; Astellas: Speakers Bureau; Alexion: Speakers Bureau; BMS: Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Agios: Consultancy, Speakers Bureau; Jazz: Speakers Bureau.

Background: Cytomegalovirus (CMV) is a major cause of morbidity in allogeneic hematopoietic stem cell transplant (HSCT) patients. Marty et al. 2017 showed that letermovir is effective in preventing CMV reactivation in high-risk HSCT patients, though only 16% were haplo-identical. Recently, Karam et al. 2019 showed decreased rates of CMV reactivation in haplo-identical HSCT using letermovir in unselected high-risk patients. The effects of letermovir on other transplant-related outcomes including overall survival (OS), relapse free survival (RFS), and Graft-versus-host-disease (GVHD)-free-/relapse-free survival (GRFS), however, are not as well-known. With the increased use of post-transplant cyclophosphamide (PTCy) as GVHD prophylaxis in all transplant types, letermovir use may need to be broadened as those that are T-cell depleted are also at increased risk of CMV reactivation. Methods: We retrospectively analyzed adult patients at USC Norris Cancer Hospital (age ≥ 18) who received allo-HSCT from 2018 to 2020. Recipients who were CMV positive, received T-cell depleting therapies such as PTCy for GVHD prophylaxis and/or ATG in the conditioning regimen, and those who fulfilled the criteria in Marty et al. 2017 were categorized as high-risk. Patients were considered to have CMV reactivation if they had clinically significant serum CMV viremia or organ involvement by day+100. Letermovir was initiated on day+21 for high-risk patients. The primary end-point assessed was day+100 CMV reactivation. Secondary end-points included 1-year OS, 1-year RFS, 1-year transplant-related mortality (TRM), and 1-year GRFS, defined as grade 3-4 acute GVHD, systemic therapy-requiring chronic GVHD, relapse, or death in the 1-year post-HSCT period. Results: A total of 116 adult HSCT recipients were reviewed. 51% were male and 49% were female. The donor sources comprised of 27% match related, 28% match-unrelated, and 49% haplo-identical. Most common diseases included AML (38%), ALL (38%) and MDS (8%). 64% received myeloablative conditioning regimens while 36% received reduced intensity regimens. Furthermore, 92% of patients received peripheral blood with 8% receiving bone marrow. 70% of patients at time of transplant were in CR1 or had stable disease. 61% of patients received letermovir prophylaxis (n=71), all high-risk, and 39% did not (n=45). 13 high-risk patients did not receive letermovir due to insurance limitations and were included in the non-letermovir group. 85% (n =60) received T-cell depleting therapies in the letermovir group compared to 51% (n=23). 90% (n=64) were CMV positive in the letermovir group and 64% (n= 29) were positive in the non-letermovir group. Both groups were similar in regard to the incidence of GVHD. The only factors significantly associated with CMV reactivation were haplo-HSCT, CMV recipient positivity, use of PTCy as GVHD prophylaxis and/or ATG in conditioning regimen, risk status, and GVHD (p = 0.054, p = .017, p = 0.003, p = 0.050, and p = 0.024 respectively). In a subset analysis of all high-risk patients, CMV reactivation was 32% in the letermovir group and 69% in the non-letermovir group (p = 0.03). All other factors were not significantly associated with outcomes. CMV reactivation in the letermovir group was (32%) compared to the non-letermovir group (29%). 1-year OS, 1-year TRM, 1-year RFS, and 1-year GRFS were 85%, 13.2%, 87%, and 60% respectively in the letermovir group compared with 88%, 10%, 90%, and 56% in the non-letermovir group, though not statistically significant. Discussion: Our study confirms improved CMV reactivation outcomes with use of letermovir in high-risk patients. Furthermore, all end-points were similar between both groups, despite increased usage of myeloablative conditioning overall and more high-risk patients in the letermovir group, indicating improved 1-year OS, TRM, and GRFS compared with historical outcomes. Although the primary and secondary end-points were not statistically significant, it may be clinically meaningful, as our center has demonstrated similar outcomes amongst those who were high-risk and received letermovir and those who were low-risk and did not. This suggests broadened use of letermovir may be needed in the prevention of CMV reactivation and the improvement in overall outcomes. Further studies using letermovir in low risk patients may be necessary to investigate improved outcomes in the allo-HSCT setting. Disclosures Yaghmour: Jazz: Consultancy, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Consultancy, Speakers Bureau; Alexion: Consultancy, Speakers Bureau.

Context Hematopoietic stem cell transplant (HSCT) recipients are generally considered high risk for poor outcomes following COVID-19 infection. The CIBMTR observational report published by Sharma et al. found that recipients of allogeneic HSCT who contracted COVID-19 had poor overall survival with a 30-day mortality of 32%. However, there are relatively few other studies that evaluate the effect of COVID-19 on HSCT recipients, especially in the setting of in vivo T-cell depletion protocol. Objective This is a single-institution, retrospective analysis evaluating outcomes of HSCT recipients who were diagnosed with COVID-19 between March 2020 and April 2021. Patients There were 21 patients at our institution who had undergone HSCT and subsequently contracted COVID-19. Their median age was 53 years at the time of diagnosis. Thirteen of the patients were Hispanic (61.9%). Six of the patients received match-related stem cells (28.6%), 7 received cells from match unrelated donors (33.3%), and 7 received cells from haploidentical donors (33.3%). Fifteen of the patients (71.4%) received myeloablative conditioning regimens, whereas 5 (23.8%) received reduced intensity or non-myeloablative regimens. One patient had received an autologous stem cell transplant. Our most common GVHD prophylaxis regimens were PTCy/Tacro/MMF (12 pts, 57.1%) and Tacro/MTX (6 pts, 28.5%). Results Our primary endpoint in this retrospective analysis was non-relapse mortality within 60 days of COVID-19 diagnosis. Of the 21 patients with confirmed COVID-19 infection, 1 passed away 20 days after diagnosis, giving us a 4.8% case fatality rate. The median time of COVID-19 diagnosis post-transplant was 469 days. Sixteen of the 21 patients were symptomatic at the time of diagnosis (76.2%). Eight patients received steroids for treatment of COVID-19 (38.1%). Seven of the 21 patients (33.3%) were hospitalized for a median of 7 days (range, 5–17 days), with 2 requiring ICU level of care (9.5%). One patient was intubated and ultimately passed away. Conclusion Our data suggest that our HSCT recipients who contracted COVID-19 have had generally good short-term outcomes. Longer follow-up is required to determine if a COVID-19 diagnosis has any effect on long-term transplant related complications and outcomes in this population.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide. The malignant CRC that undergoes metastasis in the advanced stage is usually refractory to existing chemotherapy and shows a poor prognosis. However, to date, efficient targeted-therapy for metastatic CRC is ill-defined. We tested the hypothesis that combined treatment of flavonoid morin and telomerase inhibitor MST-312 may reduce the cancer stem cell (CSC) traits. To characterize CSC phenotype, we performed the CD133/CD44 subpopulation profiling, tumorsphere formation assay, cell invasion assay and wound healing assay. We have examined the augmenting effects of the combined treatment of morin and MST-312 for 5-FU (5-fluorouracil) efficacy in human colorectal cancer. Morin and MST-312 combined treatment reduced CD133 (+) and CD44 (+) subpopulations in human colorectal and breast cancer cells, respectively. Tumorsphere formation and cell invasiveness were decreased with the morin and MST-312 combination treatment. Consistent with these data, morin and MST-312 treatment decreased the wound healing capacity of human breast cancer cells. Stress and apoptosis antibody arrays revealed that there were specific upregulated and downregulated proteins resulting from different treatments. Phosphorylation levels of BAD, p53 and Chk1 were enhanced upon morin/MST-312 treatments in HT-29 cells, whereas caspase-3 cleavage level and expression of IκBα were down-regulated by combined morin/MST-312 treatment in SW620 cells. Finally, morin and MST-312 co-treatment further augmented the 5-FU efficacy, chemosensitizing the 5-FU resistant human colorectal cancer cells. Taken together, our study suggests that novel targeted-therapy can be implemented by using flavonoid morin and telomerase inhibitor MST-312 for improved cancer prognosis.

Hepatic angiosarcoma is a rare and heterogeneous neoplasm that carries with it an especially poor prognosis. Diagnosis is challenging given that patients often present with vague, nonspecific complaints, and early multiorgan dysfunction has the potential to produce a number of unique clinical syndromes. Here, we present a case of a patient with hepatic angiosarcoma who developed cryptogenic cirrhosis, Kasabach-Merritt Syndrome (consumptive thrombocytopenia), and bone marrow fibrosis as a result of her malignant lesions. While parenchymal biopsy ultimately remains the gold standard for diagnosis, careful review of enhancement patterns on imaging may suggest malignant features in need of further investigation.

Purpose The aim of this study is to investigate the mechanisms of interactions between TGF-β and Wnt/β-catenin pathways that induce and regulate EMT and promote breast cancer cells to become resistant to treatment. Methods The effect of TGF-β on Wnt/β-catenin signaling pathway was examined by using a human Wnt/β-catenin-regulated cDNA plate array and western blot analysis. The interaction of Twist at promoter of Wnt3 was examined by chromatin immunoprecipitation (ChIP) assay. Secreted Wnt3 level was determined by ELISA assay. Results HER2-overexpressing breast cancer cells treated with TGF-β have a reduced response to trastuzumab and exhibited EMT-like phenotype. The TGF-β-induced EMT in HER2-cells was concordant with upregulation of Wnt3 and β-catenin pathways. The TGF-β-induced induction of Wnt3 during EMT was found to be Smad3-dependent. ChIP analysis identified occupancy of Twist at promoter region of Wnt3. Knock-down of Twist by shRNA confirmed the significance of Twist in response to TGF-β regulating Wnt3 during EMT. Subsequently, TGF-β-induced matrix metalloproteinases, MMP1, MMP7, MMP9, MMP26, Vascular endothelial growth factors (VEGF), and activation of Wnt/β-catenin signaling were repressed by the shRNA treatment. TGF-βR1 ALK5 kinase inhibitor, A83-01 can effectively prevent the TGF-β-induced Twist and Wnt3. Co-treating A83-01 and trastuzumab inhibited TGF-β-induced cell invasion significantly in both trastuzumab responsive and resistant cells. Conclusions Our data demonstrated an important interdependence between TGF-β and Wnt/β-catenin pathways inducing EMT in HER2-overexpressing breast cancer cells. Twist served as a linkage between the two pathways during TGF-β-induced EMT. A83-01 could inhibit the TGF-β-initiated pathway interactions and enhance HER2-cells response to trastuzumab treatment. Electronic supplementary material The online version of this article (doi:10.1007/s10549-017-4211-y) contains supplementary material, which is available to authorized users.

Objectives/Hypothesis To evaluate the prevalence, characteristics, and associated risk factors of tinnitus in U.S. adolescents. Study Design Cross-sectional analyses of U.S. representative demographic and audiometric data, the National Health and Nutrition Examination Survey (NHANES) 2005 to 2008. Methods The study population consisted of 3,520 individuals aged 12 to 19 years with complete tinnitus-related data. Tinnitus was defined as the presence of ringing or buzzing in the ears lasting for at least 5 minutes during the preceding 12 months. In addition, we defined a chronic tinnitus subgroup as being bothered by tinnitus for more than 3 months. Demographic and other data regarding tinnitus, smoking, body mass index (BMI), anemia, hypertension, history of ear infections, tympanostomy tube placement, otoscopy, tympanometry and hearing thresholds, history of firearm use, and recreational and occupational exposure to noise were extracted and analyzed. Results Overall, tinnitus lasting 5 minutes or more in the preceding 12 months was reported by 7.5% of the 12- to 19-year-old population. This represents about 2.5 million adolescents in the United States. The prevalence of chronic tinnitus was 4.7%, corresponding to about 1.6 million adolescents in the United States. Multivariable-adjusted analysis revealed that both overall and chronic tinnitus were associated with female gender, low income, exposure to passive smoking, type A tympanogram, and occupational and recreational noise exposure. History of ≥3 ear infections and history of tympanostomy tube placement were associated only with overall tinnitus. Conclusions Tinnitus afflicts a substantial portion of the youth population. Further investigation of the association between tinnitus and the identified risk factors is warranted. Level of Evidence N/A. Laryngoscope, 123:2001-2008, 2013 © 2013 The American Laryngological, Rhinological and Otological Society, Inc.

Obesity increases the risk of distant metastatic recurrence and reduces breast cancer survival. However, the mechanisms behind this pathology and identification of relevant therapeutic targets are poorly defined. Plasma free fatty acids (FFA) levels are elevated in obese individuals. Here we report that TGFβ transiently activates ERK and subsequently phosphorylates SMAD4 at Thr277, which facilitates a SMAD4–USP9x interaction, SMAD4 nuclear retention, and stimulates TGFβ/SMAD3–mediated transcription of Twist and Snail. USP9x inhibited the E3 ubiquitin-protein ligase TIF1γ from binding and monoubiquitinating SMAD4, hence maintaining the SMAD4 nuclear retention. FFA further facilitated TGFβ-induced ERK activation, SMAD4 phosphorylation, and nuclear retention, promoting TGFβ-dependent cancer progression. Inhibition of ERK and USP9x suppressed obesity-induced metastasis. In addition, clinical data indicated that phospho-ERK and -SMAD4 levels correlate with activated TGFβ signaling and metastasis in overweight/obese patient breast cancer specimens. Altogether, we demonstrate the vital interaction of USP9x and SMAD4 for governing TGFβ signaling and dyslipidemia-induced aberrant TGFβ activation during breast cancer metastasis. Cancer Res; 77(6); 1383–94. ©2017 AACR.

Colorectal cancer (CRC) is one of the three leading causes for cancer mortality. CRC kills over 600,000 people annually worldwide. The most common cause of death from CRC is the metastasis to distant organs. However, biomarkers for CRC metastasis remain ill-defined. We compared primary and metastatic CRC cell lines for their angiogenesis-protein profiles and intracellular signaling profiles to identify novel biomarkers for CRC metastasis. To this end, we used primary and metastatic CRC cell lines as a model system and normal human colon cell line as a control. The angiogenesis profiles two isogenic CRC cell lines, SW480 and SW620, and HT-29 and T84 revealed that VEGF was upregulated in both SW620 and T84 whereas coagulation factor III, IGFBP-3, DPP IV, PDGF AA/AB, endothelin I and CXCL16 were downregulated specifically in metastatic cell lines. Furthermore, we found that TIMP-1, amphiregulin, endostatin, angiogenin were upregulated in SW620 whereas downregulated in T84. Angiogenin was downregulated in T84 and GM-CSF was also downregulated in SW620. To induce CRC cell metastasis, we treated cells with pro-inflammatory cytokine IL-6. Upon IL-6 treatment, epithelial-mesenchymal transition was induced in CRC cells. When DLD-1 and HT-29 cells were treated with IL-6; Akt, STAT3, AMPKα and Bad phosphorylation levels were increased. Interestingly, SW620 showed the same signal activation pattern with IL-6 treatment of HT-29 and DLD-1. Our data suggest that Akt, STAT3, AMPKα and Bad activation can be biomarkers for metastatic colorectal cancer. IL-6 treatment specifically reduced phosphorylation levels of EGFR, HER2 receptor, Insulin R and IGF-1R in receptor tyrosine kinase array study with HT-29. Taken together, we have identified novel biomarkers for metastatic CRC through the angiogenesis-antibody array and intracellular signaling array studies. Present study suggests that those novel biomarkers can be used as CRC prognosis biomarkers, and as potential targets for the metastatic CRC therapy.

Obesity increases the risk of developing distant metastatic recurrence, and diminishes overall and breast cancer-specific survival; however, the mechanisms behind this correlation and relevant therapeutic targets are poorly defined. Plasma free fatty acids (FFA) levels are usually elevated in obese individuals. Here, using human breast cancer cell lines, we found that TGF-β transiently activates the extracellular signal-regulated kinase (ERK) and subsequently marks SMAD4 for phosphorylation, which facilitates SMAD4-ubiquitin specific peptidase 9, X-linked (USP9x) interaction, SMAD4 nuclear retention, SMAD3-SMAD4 complex nuclear accumulation, and stimulated TGF-β/SMAD3–mediated transcriptional activity of Twist and Snail. USP9x competitively inhibited the E3 ubiquitin-protein ligase transcriptional intermediary factor 1 γ (TIF1γ) from binding and monoubiquitinating SMAD4. This process maintained SMAD4 nuclear retention and stabilized the SMAD3/SMAD4 complex in the nucleus. Inhibition of ERK resulted in SMAD4 de-phosphorylation, USP9x-SMAD4 disassociation and SMAD4-TIF1γ interaction, which ultimately marks SMAD4 for mono-ubiquitination, leading to SMAD4 nuclear export and termination of the transcription of Twist and Snail. In addition, loss of USP9x abolished TGF-β–induced SMAD4 nuclear retention and SMAD3/SMAD4 formation, leading to inhibition of Twist and Snail expression. We also found that ERK-induced SMAD4 T277 phosphorylation and SMAD4-USP9x interaction was required for TGF-β–induced breast cancer invasion, which was attributed to SMAD3/SMAD4-dependent up-regulation of the transcription factors, Twist and Snail. Importantly, FFA further facilitates TGF-β–induced ERK activation, SMAD4 phosphorylation, SMAD4 nuclear retention and SMAD3/SMAD4 formation, thus promoting TGF-β–dependent cancer progression. Inhibition of ERK and USP9x suppressed obesity-induced metastasis. Additionally, phospho-ERK and -SMAD4 levels correlated with activated TGF-β signaling and metastasis in obese patient breast cancer specimens. Altogether, we demonstrated that USP9x promotes breast cancer invasion by promoting nuclear retention of SMAD4 and identified crosstalk between USP9x and TGF-β/ERK/SMAD4/SMAD3 pathways. Our study highlights the vital interaction of USP9x and SMAD4 for governing TGF-β signaling and dyslipidemia-induced aberrant TGF-β activation during breast cancer metastasis. Citation Format: Yong Wu, Xiaoting Yu, Kruttika Bhat, Sami Dwabe, Mohammad Atefi, Jay Vadgama. Persistent activation of ERK and SMAD4 nuclear retention by high free fatty acids promote breast cancer metastasis. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B33.

To evaluate the prevalence, characteristics, and associated risk factors of tinnitus in U.S. adolescents.Cross-sectional analyses of U.S. representative demographic and audiometric data, the National Health and Nutrition Examination Survey (NHANES) 2005 to 2008.The study population consisted of 3,520 individuals aged 12 to 19 years with complete tinnitus-related data. Tinnitus was defined as the presence of ringing or buzzing in the ears lasting for at least 5 minutes during the preceding 12 months. In addition, we defined a chronic tinnitus subgroup as being bothered by tinnitus for more than 3 months. Demographic and other data regarding tinnitus, smoking, body mass index (BMI), anemia, hypertension, history of ear infections, tympanostomy tube placement, otoscopy, tympanometry and hearing thresholds, history of firearm use, and recreational and occupational exposure to noise were extracted and analyzed.Overall, tinnitus lasting 5 minutes or more in the preceding 12 months was reported by 7.5% of the 12- to 19-year-old population. This represents about 2.5 million adolescents in the United States. The prevalence of chronic tinnitus was 4.7%, corresponding to about 1.6 million adolescents in the United States. Multivariable-adjusted analysis revealed that both overall and chronic tinnitus were associated with female gender, low income, exposure to passive smoking, type A tympanogram, and occupational and recreational noise exposure. History of ≥ 3 ear infections and history of tympanostomy tube placement were associated only with overall tinnitus.Tinnitus afflicts a substantial portion of the youth population. Further investigation of the association between tinnitus and the identified risk factors is warranted.

Hearing loss (HL) is the most common sensory impairment and is caused by a broad range of inherited to environmental causes. Inherited HL consists 50–60% of all HL cases. The inherited form of HL is further classified to different categories. More than 300 syndromes and 40 genes have been identified to result in different levels of HL. Although several diagnostic or screening tests have been developed, yet there are controversies around their use.

Introduction: Many studies have shown that the ALDH1 and Ki67 markers can accurately identify different types of cells in the breast. ALDH1 has proven to be a biomarker of breast cancer stem-like cells, while Ki67 has considered a proliferative marker. However, although Ki67 has been correlated with breast cancer grading, little attention has been given to the relationship between ALDH1 and histological grading. On the other hand, although ALDH1 has been correlated with intrinsic molecular subtype of breast cancer, there is an absence of literature between Ki67 and breast cancer subtypes. Thus, the goals of this study are to address the relationship between ALDH, Ki67 and histological grading in breast cancer. Methods and Material: Immunohistochemistry staining (IHC) for ALDH1 was performed on tissue micro array (TMA) of 200 patient samples with different histological grades of breast cancer. Similarly, IHC was used to identify and assess the expression of Ki67 on TMA of 66 patient samples of various breast cancer subtypes (40 Luminal and 26 TNBC). Both biomarkers were compared to respective normal breast tissue using t-test statistical analysis. Results: results showed that 51/129 (39.5%) of cancerous breast tissues were ALDH1 positive. ALDH1 positivity differed significantly between different grades cancerous breast tissues and normal tissue (p=0.0007). In addition, a direct relationship exists between increasing grade and increasing ALDH1 positivity, with grade 3 having the highest frequency and intensity of ALDH1 positivity (p=0.005). In normal tissue, 6/25 (24%) cores on the tissue microarray showed presence of Ki67. The average percentage of cells containing in Ki67 positive cores for normal tissue was 2.54%. In luminal subtypes, 30/38 (78.9%) patients on the tissue microarray showed presence of Ki67. The average percentage of cells containing Ki67 in luminal breast cancer, for patients with Ki67 positive cores was 3.93%, with a median of 2.5%. Ki67 was also present in 24/26 (92.3%) patients with triple negative breast cancer. Among the 24 patients with Ki67 present, an average of 6.91% of cells exhibited Ki67 in each core, with a median of 4.25%. A one-tailed student's T-test was used to determine statistical significance of the difference in Ki67 expression (including the patients with no Ki67 expression) between Luminal and Triple Negative subtypes of breast cancer with a p-value of 0.026 Conclusion: Our findings suggest that higher grades of breast cancer may contain higher quantities of breast cancer stem like cells. Given the role of Ki67 in cell proliferation, the increased presence of Ki67 in triple negative breast cancer, when compared to Luminal subtypes of breast cancer, may suggest a correlation with a worse prognosis. Identification of Ki67 positivity in breast cancer cells may aid in the development of prognosis and novel treatment techniques. Citation Format: Yahya I. Elshimali, Pierre Tamer, Sami Dwabe, Yanyuan Wu, Marianna Sarkissyan, Jaydutt V. Vadgama. The association between expression of ALDH1, KI67 and grading system in breast adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3887. doi:10.1158/1538-7445.AM2014-3887