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Human coronavirus disease 2019 (COVID-19) is a life-threatening and highly contagious disease caused by coronavirus SARS-CoV-2. Sensitive and specific detection of SARS-CoV-2 viral proteins in tissues and cells of COVID-19 patients will support investigations of the biologic behavior and tissue and cell tropism of this virus. We identified commercially available affinity-purified polyclonal antibodies raised against nucleocapsid and spike proteins of SARS-CoV-2 that provide sensitive and specific detection of the virus by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Two immunohistochemistry protocols are presented that are mutually validated by the matched detection patterns of the two distinct viral antigens in virus-infected cells within autopsy lung tissue of COVID-19 deceased patients. Levels of nucleocapsid protein in the lungs of COVID-19 decedents, as measured by quantitative histo-cytometry of immunohistochemistry images, showed an excellent log–linear relationship with levels of viral nucleocapsid RNA levels, as measured by qRT-PCR. Importantly, since the nucleocapsid protein sequence is conserved across all known viral strains, the nucleocapsid immunohistochemistry protocol is expected to recognize all common variants of SARS-CoV-2. Negative controls include autopsy lung tissues from patients who died from non-COVID-19 respiratory disease and control rabbit immunoglobulin. Sensitive detection of SARS-CoV-2 in human tissues will provide insights into viral tissue and cell distribution and load in patients with active infection, as well as provide insight into the clearance rate of virus in later COVID-19 disease stages. The protocols are also expected to be readily transferable to detect SARS-CoV-2 proteins in tissues of experimental animal models or animals suspected to serve as viral reservoirs.

The chaperone protein and guanine nucleotide exchange factor SmgGDS (RAP1GDS1) is a key promoter of cancer cell proliferation and tumorigenesis. SmgGDS undergoes nucleocytoplasmic shuttling, suggesting that it has both cytoplasmic and nuclear functions that promote cancer. Previous studies indicate that SmgGDS binds cytoplasmic small GTPases and promotes their trafficking to the plasma membrane. In contrast, little is known about the functions of SmgGDS in the nucleus, or how these nuclear functions might benefit cancer cells. Here we show unique nuclear localization and regulation of gene transcription pathways by SmgGDS. Strikingly, SmgGDS depletion significantly reduces expression of over 600 gene products that are targets of the DREAM complex, which is a transcription factor complex that regulates expression of proteins controlling the cell cycle. The cell cycle regulators E2F1, MYC, MYBL2 (B-Myb) and FOXM1 are among the DREAM targets that are diminished by SmgGDS depletion. E2F1 is well known to promote G1 cell cycle progression, and the loss of E2F1 in SmgGDS-depleted cells provides an explanation for previous reports that SmgGDS depletion characteristically causes a G1 cell cycle arrest. We show that SmgGDS localizes in nucleoli, and that RNAi-mediated depletion of SmgGDS in cancer cells disrupts nucleolar morphology, signifying nucleolar stress. We show that nucleolar SmgGDS interacts with the RNA polymerase I transcription factor upstream binding factor (UBF). The RNAi-mediated depletion of UBF diminishes nucleolar localization of SmgGDS and promotes proteasome-mediated degradation of SmgGDS, indicating that nucleolar sequestration of SmgGDS by UBF stabilizes SmgGDS protein. The ability of SmgGDS to interact with UBF and localize in the nucleolus is diminished by expressing DiRas1 or DiRas2, which are small GTPases that bind SmgGDS and act as tumor suppressors. Taken together, our results support a novel nuclear role for SmgGDS in protecting malignant cells from nucleolar stress, thus promoting cell cycle progression and tumorigenesis.

Tumor-associated macrophages (TAMs) are major constituents of the tumor microenvironment in solid tumors and have been implicated as mediators of tumor progression, invasion and metastasis. Correspondingly, accumulation of TAMs is associated with unfavorable clinical outcomes in numerous types of solid tumors. E-selectin is a hallmark of inflammation and a key adhesion molecule that accommodates the initial contact of circulating immune cells with the inflamed vessel surface. Currently, the association between E-selectin and TAMs is not fully elucidated; therefore, the present study investigated the association between vessel inflammation, TAM infiltration, and clinical outcome in breast cancer. A total of 53 procedure-naive invasive breast cancer cases were immunohistochemically analyzed for the presence of cluster of differentiation (CD)68+ TAMs, E-selectin+ vessels and tumor inflammation. The association between CD68 and E-selectin expression, and tumor inflammation as well as overall survival was evaluated using Kaplan-Meier survival curves and multivariable Cox's proportional hazards regression analysis. The abundance of TAMs was identified to be positively associated with tumor inflammation, estrogen receptor and E-selectin expression levels. A greater prevalence of TAMs and tumor inflammation was significantly associated with shorter overall survival times. E-selectin expression levels were significantly higher in tumor vessels among elderly patients, but were not associated with overall survival. The abundance of TAMs was associated with the presence of E-selectin-expressing inflamed tumor vessels and tumor inflammation, as well as overall survival in patients with invasive breast carcinoma.

For nearly a century, the rat has served as a key model for studying the pathophysiology and genetic risk modifiers of breast cancer. Rat mammary tumors that initiate after exposure to carcinogens or estrogens closely resemble the etiological, histopathological, and genomic features of human breast cancer. Recent developments in genome-editing techniques in the rat have also enabled the development of sophisticated models for identifying the genetic modifiers of the nonmalignant tumor microenvironment that contribute to the formation, progression, and outcome of breast cancer. In this protocol review, we discuss the current methodologies for the three genetic mapping techniques in the rat that are widely used for identifying and testing the heritable genetic modifiers of breast cancer.

As of October 2020, there are over 40 million confirmed cases, and more than 1 million confirmed deaths of Covid-19 worldwide. The main cause of death in hospitalized patients is a respiratory failure due to acute respiratory distress syndrome. It has been suggested that the very intense immune response induces diffuse alveolar damage that far exceeds the harm that would have been caused by virus replication per se, resulting in lethal tissue destruction. We present a detailed report of the histopathological findings on cryo transbronchial biopsy in the patient with persistent (3 months) interstitial pneumonitis and severe CD8 positive cell infiltration in the lungs due to SARS-CoV-2 infection. CD8 positive T-lymphocytes have a great potential to damage tissue either through direct cytotoxicity or through cytokines release.

Background: Endocrine therapy provides significant improvement in the long-term outcomes of patients with hormone receptor-positive (HR+) breast cancer (BC). However, metastatic recurrences of endocrine resistant disease develop in about 20-25% of patients and remain a major cause of BC mortality. Up-regulation of the HER2 growth factor receptor represents a common escape strategy used by cancer cells to survive and continue to proliferate in an ER-independent manner. Neoadjuvant endocrine therapy (NET) offers a unique opportunity to identify responsiveness of HR+ BCs and detect tumors that display up-regulation of HER2, an early endocrine resistance mechanism. Methods: A single arm, interventional, exploratory clinical trial evaluating four weeks of NET in early stage HR+/HER2-negative BC patients was conducted at our institution (NCT03219476). The primary objective was to assess changes in HER protein expression (HER1-4) from diagnostic core biopsies to surgically resected tumors treated with NET. Secondary objectives included assessment of other molecular markers, tumor proliferation and volumetric responses. Optimized protocols for immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were used to assess HER2 status in the pre- and post-treatment tumor specimens. Chi-square and t-tests were performed. Linear regression multivariate analysis was performed to evaluate association of covariates with the primary outcome with 80% power at a significance level of 0.05. Here we present the changes in HER2 protein expression with NET in this study. Up-regulation was defined as an increase of ≥ 1 in IHC scores (ordinal 0,1,2,3) or gene amplification by FISH. Results: Thirty-seven patients with cT1-T3, cN0 HR+/HER2-negative BC were enrolled. Median age at diagnosis was 64 yrs (42-81) and median BMI was 28.3 (19.3-55.1). Most patients were post-menopausal (83.8%). Median tumor size clinically was 1.3 cm (0.5-7.7). Most tumors were low (42.1%) or intermediate (47.4%) grade and invasive ductal histology was seen in 70%. Median tumor size at surgery was 1.2 cm (0.09-4). One patient had a complete pathologic response (pCR) at surgery. Seven patients had pN1 disease at surgery (six with 1 lymph node involved, one with pN1mi). There was no significant change in ER-positivity between pre and post-treatment specimens. However, a trend towards decrease in PR-positivity was seen in post-treatment tumors consistent with functional ER pathway disruption (p=0.08). On HER2 protein assessment by IHC, most patients had IHC 0 (37.8%) or IHC 1+ (54%) at diagnosis. Significant up-regulation in HER2 protein was seen in 46% (17/37) of patients (p=0.0004), whereas down-regulation was detected in only 5% (2/37) and no change in the remaining 49% (18/37). Three patients converted to HER2-positive status (IHC 3+ or FISH amplified) at surgery and received adjuvant trastuzumab-based treatment. No significant associations were identified between any clinicopathologic covariates and change in HER2 protein expression. Conclusions: HER2 was up regulated in 46% of tumors after short-term NET in patients diagnosed with early stage HR+/HER2-negative BC. Short-term NET is a promising strategy to identify HR+ tumors that up-regulate HER2 as an early escape pathway and endocrine resistance mechanism. Patients with such HER2 up-regulated tumors after NET may benefit from HER2-directed therapies upon disease recurrence or as adjuvant combination therapy. These findings need to be further explored in larger randomized clinical trials. Citation Format: Lubna N Chaudhary, Julie M Jorns, Yee Chung Cheng, Sailaja Kamaraju, Mary Beth Gonyo, Amanda Kong, Caitlin Patten, Tina Yen, Chandler Cortina, Ebony Carson, Nedra Johnson, Carmen Bergom, Anjishnu Banerjee, Yu Wang, Christopher R Chitambar, Hallgeir Rui. Neoadjuvant endocrine therapy helps identify HER2 up-regulation in patients with hormone receptor-positive HER2-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-07.

Protein marker levels in formalin-fixed, paraffin-embedded tissue sections traditionally have been assayed by chromogenic immunohistochemistry and evaluated visually by pathologists. Pathologist scoring of chromogen staining intensity is subjective and generates low-resolution ordinal or nominal data rather than continuous data. Emerging digital pathology platforms now allow quantification of chromogen or fluorescence signals by computer-assisted image analysis, providing continuous immunohistochemistry values. Fluorescence immunohistochemistry offers greater dynamic signal range than chromogen immunohistochemistry, and combined with image analysis holds the promise of enhanced sensitivity and analytic resolution, and consequently more robust quantification. However, commercial fluorescence scanners and image analysis software differ in features and capabilities, and claims of objective quantitative immunohistochemistry are difficult to validate as pathologist scoring is subjective and there is no accepted gold standard. Here we provide the first side-by-side validation of two technologically distinct commercial fluorescence immunohistochemistry analysis platforms. We document highly consistent results by (1) concordance analysis of fluorescence immunohistochemistry values and (2) agreement in outcome predictions both for objective, data-driven cutpoint dichotomization with Kaplan–Meier analyses or employment of continuous marker values to compute receiver-operating curves. The two platforms examined rely on distinct fluorescence immunohistochemistry imaging hardware, microscopy vs line scanning, and functionally distinct image analysis software. Fluorescence immunohistochemistry values for nuclear-localized and tyrosine-phosphorylated Stat5a/b computed by each platform on a cohort of 323 breast cancer cases revealed high concordance after linear calibration, a finding confirmed on an independent 382 case cohort, with concordance correlation coefficients >0.98. Data-driven optimal cutpoints for outcome prediction by either platform were reciprocally applicable to the data derived by the alternate platform, identifying patients with low Nuc-pYStat5 at ~3.5-fold increased risk of disease progression. Our analyses identified two highly concordant fluorescence immunohistochemistry platforms that may serve as benchmarks for testing of other platforms, and low interoperator variability supports the implementation of objective tumor marker quantification in pathology laboratories.

BACKGROUND CD163 is a scavenger receptor specifically expressed by cells of monocyte/macrophage lineage. It is a biomarker in many clinical conditions, including coronary artery disease, anti-inflammatory response, and cancers. In breast cancer, high numbers of CD163-positive macrophages correlated with unfavorable outcome. Expression of CD163 in breast cancer was also related to early distant recurrence and poor survival. In this study we evaluated whether CD163 expression was associated with aggressive breast cancers from patients enrolled in the Clinical Breast Care Project (CBCP). METHODS Patients were enrolled into the CBCP following IRB-approved, HIPAA-compliant protocols. The study focused on 129 invasive breast cancer samples with CD163 immunohistochemistry (IHC) results. Expression of CD163, ER, PR, HER2, and Ki67 were assayed by IHC. CD163 was positive if IHC>0. ER and PR were positive if there was >5% nuclear staining. HER2 was negative if IHC=0 or 1+ and positive if IHC=3+. For HER2 IHC=2+, HER2 was negative if FISH was 2.2. Ki67 was positive if there was ≥15% nuclear staining. For subtyping, Luminal A (LA) was ER+/HER2-/Ki67-, Luminal B1 (LB1) was ER+/HER2-/Ki67+, Luminal B2 (LB2) was ER+/HER2+, Her2+ was ER-/PR-/HER2+, and triple negative (TN) was ER-/PR-/HER2-. Associations of CD163 IHC score with age, race, and IHC subtype were examined by Wilcoxon rank-sum tests and/or Fisher's exact tests. Prognoses of CD163 in overall survival (OS), disease specific survival (DSS), disease free survival (DFS), and recurrence were studied using univariable and multivariable Cox proportional hazards regression models. CD163 score, age, race, AJCC stage, and subtype were included in the multivariable model. RESULTS CD163 IHC score displayed a significant negative correlation with age (R=-0.20, P=0.022). Patients with a CD163 score of 3+ were significantly younger than those with a score of 0 (P=0.019). CD163 score distributions were not statistically different between white and African American patients. CD163 scores of LA tumors were significantly lower than those of the tumors with all other subtypes except Her2+. Similarly, the CD163 scores of TN tumors were significantly higher than those of the tumors with all other subtypes but LB2. A higher CD163 score predicted worse DSS (HR=3.87 & P=0.0020 in univariable model; HR=4.21 & P=0.033 in multivariable model) and higher risk of recurrence (HR=2.85 & P=0.00016 in univariable model; HR=2.81 & P=0.012 in multivariable model). CONCLUSION Higher CD163 expression in breast cancer was significantly associated with younger age, the TN subtype, worse DSS, and higher risk of recurrence. These results highlight CD163 as a prognostic marker for breast cancer. The views expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S. Government. Citation Format: Ru Y, Hu PT, Kovatich AJ, Hooke JA, Liu J, Kvecher L, Fantacone-Campbell JL, Deyarmin B, Kovatich AW, Cammarata F, Rui H, Davidson-Moncada J, Shriver CD, Hu H. CD163 expression is associated with young age, triple negative subtype, and poor outcome in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-24.

To meaningfully impact cancer disparities, scientists and community need to work differently together. Community-based participatory research (CBPR) offers a strong first step but in practice often falls short of its full potential. The Community Cancer Science Network (CCSN) is an initiative of the Medical College of Wisconsin Cancer Center to address breast and lung cancer disparities statewide. This initiative began with a three-year study and design team process which identified a need for academic medicine and community to come together to address disparities in Wisconsin. A design team made up of basic and population science researchers, clinicians and community-based organization leaders representing communities impacted by disparities convened to gather data and examine trends to better understand root causes. The team identified several hurdles impeding authentic community-academic partnerships including: 1) isolated solutions putting the primary responsibility for change on individuals and communities; 2) significant issues of trust due to racism and other forms of oppression; 3) misunderstandings and misinformation about community priorities/needs and the intersections of science and quality of life; and 4) structures to support working together in authentic ways. Following this work, the initiative moved into the next phase to address these hurdles. In this phase, CCSN uses developmental evaluation to help shape new ways of working together to go beyond CBPR by explicitly addressing mistrust, shared understanding, power and privilege differentials and more. CCSN begins with creating change in academic medicine and places community members at the table as experts. We report on first year learning, including the development of our guiding framework, managing resistance to change, and putting in practice an anti- oppression, co-learning model to support innovation and engage community and researchers as equitable partners. Further, we discuss the challenges of using a developmental evaluation approach in an academic environment. These include: 1) the need for significant shifts within core leadership teams and funders to look inward at their commitment to change internally; 2) differences in beliefs and practice between evaluation and research around planning, credibility of evidence, and use of data and language; 3) multiple levels of leadership models and engagement structures starting at different places and with varying understandings and assumptions of the models and approaches we are trying to use; 4) moving forward within a set of much larger systems that are becoming ready and changing at different paces. Conclusion. Eradicating cancer disparities requires novel ways to facilitate authentic community and academic medicine collaborations. Developmental evaluation supports an iterative process to quickly identify challenges and successes promoting ongoing learning and the ability to pivot and adapt approaches. Citation Format: Laura Pinsoneault, Tobi Cawthra, Beth Brunner, Linda Conlon, Lyle Ignace, Judy Heubel, Sandra Millon-Underwood, Hallgeir Rui, Jose Salazar, Ellen Velie, Alonzo Walker, Melinda Stolley. A framework for transformed community-academic partnerships to reduce cancer disparities [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-041.

Introduction: Triple Negative Breast Cancer (TNBC) represents the most deadly form of invasive disease. It is associated with clinical and pathological features of highly proliferative and rapidly spreading cancer demonstrating higher incidence in younger women, particularly of African-American ethnicity and accounting for a disproportionately high percentage of early development of metastatic disease and breast-cancer-related death. While chemotherapy remains the main treatment option for both primary and metastatic TNBC (mTNBC) there is no optimized therapy for its management due to tumor heterogeneity, leaving the condition with an unmet medical need. Recent preclinical research demonstrated an important role for chemokine receptor type 5 (CCR5) in modulating cell migration and immune microenvironment suggesting a potential new therapeutic target in mTNBC [Jiao X et al, Can Res 2018;78:1657-71]. Leronlimab (PRO 140) is a humanized IgG4,к monoclonal antibody (mAb) to the C-C chemokine receptor type 5 (CCR5). Leronlimab has been administered and generally well tolerated in more than 750 healthy and HIV-1 infected individuals in Phase I/II/III studies. An ongoing Phase Ib/II study is being conducted to assess the safety and clinical outcomes of leronlimab combined with carboplatin in patients with untreated CCR5+ metastatic Triple Negative Breast Cancer (mTNBC). Methods: Eligible patients are required to have confirmed HER2 negative, ER 10% of primary or metastatic cancer cells shows membranous staining and/or high predominance of CCR5+ tumor-infiltrating leukocytes (see representative IHC staining images below) Phase Ib of the CD07_TNBC study is a multicenter, single arm, dose escalation phase with 3 dose levels of leronlimab administered in combination with a fixed dose of carboplatin at AUC 5. The starting dose is 350 mg with escalation to 525 mg and 700 mg in the absence of dose-limiting toxicities. Leronlimab is administered as subcutaneous injection in the abdomen weekly and can be self-administered by subjects at home after proper training by a healthcare professional. Carboplatin is administered at AUC 5 every 3 weeks. The maximum tolerated dose (MTD) of leronlimab determined during the Phase Ib portion will be administered to 30 patients during the Phase II portion of the study. Correlative Data: Blood samples are collected at Day 1 of each treatment cycle (every 21 days) to assess changes in circulating tumor cells (CTCs) and cancer associated macrophage-like cells (CAMLs) after treatment and to perform correlative analysis with CCR5 expression. Conclusions: Leronlimab (PRO 140), a CCR5 antagonist mAb, in combination with carboplatin is currently enrolling newly diagnosed mTNBC. The preliminary analysis shows safety and tolerability of the combination and continue to enroll patients with initial promising clinical activity, potentially suggesting the future availability of a new effective agent in the management of this serious condition. Citation Format: Massimo Cristofanilli, Milana Dolezal, Jay Lalezari, Hallgeir Rui, Bruce Patterson, Cha-Mei Tang, Daniel Adams, Qiang Zhang, Kazem Kazempour, Nader Pourhassan, Natalie Rabb, Kush Dhody. Phase Ib/II study of leronlimab (PRO 140) combined with carboplatin in CCR5+ mTNBC patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT233.

While radiation therapy (RT) can improve cancer outcomes, it can lead to radiation-induced heart dysfunction (RIHD) in patients with thoracic tumors. This study examines the role of adaptive immune cells in RIHD. In Salt-Sensitive (SS) rats, image-guided whole-heart RT increased cardiac T-cell infiltration. We analyzed the functional requirement for these cells in RIHD using a genetic model of T- and B-cell deficiency (interleukin-2 receptor gamma chain knockout (IL2RG&minus, /&minus, )) and observed a complex role for these cells. Surprisingly, while IL2RG deficiency conferred protection from cardiac hypertrophy, it worsened heart function via echocardiogram three months after a large single RT dose, including increased end-systolic volume (ESV) and reduced ejection fraction (EF) and fractional shortening (FS) (p &lt, 0.05). Fractionated RT, however, did not yield similarly increased injury. Our results indicate that T cells are not uniformly required for RIHD in this model, nor do they account for our previously reported differences in cardiac RT sensitivity between SS and SS.BN3 rats. The increasing use of immunotherapies in conjunction with traditional cancer treatments demands better models to study the interactions between immunity and RT for effective therapy. We present a model that reveals complex roles for adaptive immune cells in cardiac injury that vary depending on clinically relevant factors, including RT dose/fractionation, sex, and genetic background.

Chemotherapy is a mainstay of treatment for solid tumors. However, little is known about how therapy-induced immune cell infiltration may affect therapy response. We found substantial CD45+ immune cell density adjacent to E-selectin expressing inflamed vessels in doxorubicin (DOX)-treated residual human breast tumors. While CD45 level was significantly elevated in DOX-treated wildtype mice, it remained unchanged in DOX-treated tumors from E-selectin null mice. Similarly, intravenous administration of anti-E-selectin aptamer (ESTA) resulted in a significant reduction in CD45+ immune cell density in DOX-treated residual tumors, which coincided with a delay in tumor growth and lung metastasis in MMTV-pyMT mice. Additionally, both tumor infiltrating T-lymphocytes and tumor associated-macrophages were skewed towards TH2 in DOX-treated residual breast tumors, however, ESTA suppressed these changes. This study suggests that DOX treatment instigates de novo intratumoral infiltration of immune cells through E-selectin, and functional blockade of E-selectin may reduce residual tumor burden as well as metastasis through suppression of TH2 shift.

Purpose of the study. To define the role of CCR5 in metastasis of breast cancer cells using a humanized monoclonal antibody to CCR5 (Leronlimab). CCR5, is a G protein coupled receptor that is normally expressed on immune cells. CCR5 expression is increase selectively in adult and pediatric malignancies including breast cancer (BCa), prostate cancer, pancreatic cancer, colon cancer, melanoma). In studies of >2,200 patients with breast CCR5 was overexpressed in >50% of human BCa, primarily in triple negative BCa (TNBC), correlating with poor outcome. Small molecule CCR5 inhibitors block breast and prostate cancer metastasis, but have known serious adverse events. Expression of CCR5 has been linked to both tumor immune heterogeneity and canonical check point inhibitor responses. CCR5 is activated by diverse ligands identified in the tumor microenvironment (CXCL13 (BCA-1), CCL3 (MIP1α), CCL3L1, CCL4 (MP-1β), CCL5 (RANTES), CCL8 (MCP2), CCL11 (Eotaxin), CCL13 (MCP-4), CCL16 (HCC-4). CCR5+ BCa epithelial cells have characteristics of cancer stem cells, initiating tumors and metastasizing in mice. CCR5 reintroduction into CCR5 negative BCa cells promotes tumor metastases and induces DNA repair gene expression and activity. The CCR5 inhibitor Leronlimab has been used in treatment of >750 patients with HIV, and met its primary endpoints in a phase III pivotal study for HIV, without significant adverse events reported. A CCR5 targeted cancer clinical trials (phase 1) using a small molecule inhibitor with the check point inhibitor Pembrolizumab opened to accrual in late 2018 NCT03274804. Results. Leronlimab augmented BCa cell killing by DNA damage inducing agents. Leronlimab bound to CCR5 expressed in human breast cancer cell lines, abrogated CCL5 induced Ca+2 flux, blocked 3-d matrigel invasion and blocked metastasis of MDA-MB-231 cells in murine xenografts. At 7 weeks, metastatic BCa tumor burden was reduced >98% by leronlimab. CCR5 was identified on patients' circulating tumor cells (CTC). Based on these studies a phase 1b/2 clinical trial was approved for Leronlimab and carboplatin treatment of metastatic TNBC (NCT03838367) and granted fast track designation in May 2019. The trial monitors CTC in response to leronlimab and progression free survival, Conclusions. Leronlimab binds CCR5 in BCa cells, blocking breast cancer cellular metastasis with a clinical trial currently accruing. As CCR5 augments DNA repair and is expressed selectively on cancerous but not normal breast epithelial cells, Leronlimab may enhance the tumor specific activities of DDR-based treatments, allowing a reduction in dose of standard chemotherapy. Citation Format: Richard G Pestell, Massimo Cristofanilli, Hallgeir Rui, Xuanmao Jiao, Min Wang. Leronlimab, a humanized monoclonal antibody to CCR5, restrains breast cancer metastasis and enhances cell death induced by DNA damaging chemotherapies [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD4-04.

Background Endocrine therapy provides significant disease-free survival (DFS) and overall survival (OS) benefit to patients with early stage hormone receptor positive (HR+) HER2 negative breast cancer. However, metastatic recurrence of endocrine-resistant disease develops in 20-25% of patients and remains a major cause of breast cancer mortality. Several mechanisms play a role in the progression and development of endocrine resistance. Understanding and characterizing the underlying molecular features of resistance for more accurate response prediction and optimal clinical management is of the utmost importance. Methods We designed an interventional study to assess tumor responses, changes in tumor proliferation and molecular biomarkers in early stage HR+ HER2- clinical node-negative breast cancer patients treated with neoadjuvant endocrine therapy for 4 weeks. The primary objective is to assess changes in HER protein expression (HER1-4) with neoadjuvant endocrine therapy; secondary objectives assess other molecular changes such as therapy-resistant CK5+ progenitor cells, iron-related proteins, Bruton’s tyrosine kinase (BTK), PD-L1, PD-L2 protein expressions, association with Ki-67 and tumor responses. Interim data on changes in HER2 protein in the first 16 patients is reported here. Optimized protocols for immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were used to assess HER2 status in the pre and post treatment tumor specimens. This is an ongoing investigator-initiated clinical trial funded by Rock River Foundation and the Medical College of Wisconsin Cancer Center (NCT03219476). Results Median age at diagnosis was 65 (42-81) and median BMI was 28.3 (19.3-50.1). Most patients were post-menopausal (81%). Median tumor size clinically was 1.3 cm (0.5-7.7). Most tumors were low (50%) or intermediate (37.5%) grade and invasive ductal histology (62.5%). Median tumor size at surgery was 1.1 cm (0-3.9). One patient had a complete pathologic response (pCR) at surgery. Four patients had pN1 (1 lymph node involved) and one patient had pN1mic disease at surgery. There was no significant change in ER-positivity between pre and post-treatment specimens. However, a significant decrease in PR-positivity was seen in post-treatment tumors consistent with functional ER pathway disruption. On HER2 protein assessment by IHC, most patients had IHC 0 (37.5%) or IHC 1+ (56%) at diagnosis. Up-regulation in HER2 protein was seen in post-treatment tumors with 36% (n=5) IHC 2+, with one case showing Her2 gene amplification by FISH. Conclusions HER2 was up regulated in 36% of tumors after short-term neoadjuvant endocrine treatment in patients with early stage HR+ HER2- breast cancer. Short-term neoadjuvant endocrine treatment holds potential for identifying emergent endocrine resistance mechanisms and pathways. Targeted therapy with HER2-directed antibodies for such HER2 up-regulated tumors after neoadjuvant endocrine treatment may benefit patients upon disease recurrence or possibly as adjuvant combination therapy in patients with high risk of recurrence. Citation Format: Lubna N Chaudhary, Julie Jorns, MaryBeth Gonyo, Amanda Kong, Amy R Peck, Yunguang Sun, Carmen Bergom, Anjishnu Banarjee, Christopher Chitambar, Hallgeir Rui. HER2 up-regulation with neoadjuvant endocrine therapy in patients with hormone receptor-positive, HER2-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-23.

Significance The deubiquitylase USP22 is frequently overexpressed in cancer and contributes to tumorigenesis by driving cell cycle progression. Current models define USP22 as functional mediator of gene regulation and chromatin modification, working within the SAGA transcriptional cofactor complex. Here we report a catalytic role for USP22 distinct from its well-characterized transcription regulatory capacity. USP22 directly stabilizes the essential G1-cyclin, CCND1, protecting it from proteasome-mediated degradation via deubiquitylation. Our findings reveal a pathway that regulates CCND1, while also raising the possibility that simulteously targeting USP22 may allow the use of less toxic doses of the new wave of cancer therapies that target the cyclin/CDK complex. Finally, these results provide a mechanistic explanation for the effects of USP22 in cancer cell cycle control., Overexpression of the deubiquitylase ubiquitin-specific peptidase 22 (USP22) is a marker of aggressive cancer phenotypes like metastasis, therapy resistance, and poor survival. Functionally, this overexpression of USP22 actively contributes to tumorigenesis, as USP22 depletion blocks cancer cell cycle progression in vitro, and inhibits tumor progression in animal models of lung, breast, bladder, ovarian, and liver cancer, among others. Current models suggest that USP22 mediates these biological effects via its role in epigenetic regulation as a subunit of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional cofactor complex. Challenging the dogma, we report here a nontranscriptional role for USP22 via a direct effect on the core cell cycle machinery: that is, the deubiquitylation of the G1 cyclin D1 (CCND1). Deubiquitylation by USP22 protects CCND1 from proteasome-mediated degradation and occurs separately from the canonical phosphorylation/ubiquitylation mechanism previously shown to regulate CCND1 stability. We demonstrate that control of CCND1 is a key mechanism by which USP22 mediates its known role in cell cycle progression. Finally, USP22 and CCND1 levels correlate in patient lung and colorectal cancer samples and our preclinical studies indicate that targeting USP22 in combination with CDK inhibitors may offer an approach for treating cancer patients whose tumors exhibit elevated CCND1.

The functional significance of the chemokine receptor CCR5 in human breast cancer epithelial cells is poorly understood. Here, we report that CCR5 expression in human breast cancer correlates with poor outcome. CCR5+ breast cancer epithelial cells formed mammospheres and initiated tumors with >60-fold greater efficiency in mice. Reintroduction of CCR5 expression into CCR5-negative breast cancer cells promoted tumor metastases and induced DNA repair gene expression and activity. CCR5 antagonists Maraviroc and Vicriviroc dramatically enhanced cell killing mediated by DNA-damaging chemotherapeutic agents. Single-cell analysis revealed CCR5 governs PI3K/Akt, ribosomal biogenesis, and cell survival signaling. As CCR5 augments DNA repair and is reexpressed selectively on cancerous, but not normal breast epithelial cells, CCR5 inhibitors may enhance the tumor-specific activities of DNA damage response–based treatments, allowing a dose reduction of standard chemotherapy and radiation. Significance: This study offers a preclinical rationale to reposition CCR5 inhibitors to improve the treatment of breast cancer, based on their ability to enhance the tumor-specific activities of DNA-damaging chemotherapies administered in that disease. Cancer Res; 78(7); 1657–71. ©2018 AACR.

Previously, it has been shown that pancreatic ductal adenocarcinoma (PDA) tumors exhibit high levels of hypoxia, characterized by low oxygen pressure (pO(2)) and decreased O(2) intracellular perfusion. Chronic hypoxia is strongly associated with resistance to cytotoxic chemotherapy and chemoradiation in an understudied phenomenon known as hypoxia-induced chemoresistance. The hypoxia-inducible, pro-oncogenic, serine-threonine kinase PIM1 (Proviral Integration site for Moloney murine leukemia virus 1) has emerged as a key regulator of hypoxia-induced chemoresistance in PDA and other cancers. Although its role in therapeutic resistance has been described previously, the molecular mechanism behind PIM1 overexpression in PDA is unknown. Here, we demonstrate that cis-acting AU-rich elements (ARE) present within a 38-base pair region of the PIM1 mRNA 3’-untranslated region mediate a regulatory interaction with the mRNA stability factor HuR (Hu antigen R) in the context of tumor hypoxia. Predominantly expressed in the nucleus in PDA cells, HuR translocates to the cytoplasm in response to hypoxic stress and stabilizes the PIM1 mRNA transcript, resulting in PIM1 protein overexpression. A reverse-phase protein array revealed that HuR-mediated regulation of PIM1 protects cells from hypoxic stress through phosphorylation and inactivation of the apoptotic effector BAD and activation of MEK1/2. Importantly, pharmacological inhibition of HuR by MS-444 inhibits HuR homodimerization and its cytoplasmic translocation, abrogates hypoxia-induced PIM1 overexpression and markedly enhances PDA cell sensitivity to oxaliplatin and 5-fluorouracil under physiologic low oxygen conditions. Taken together, these results support the notion that HuR has prosurvival properties in PDA cells by enabling them with growth advantages in stressful tumor microenvironment niches. Accordingly, these studies provide evidence that therapeutic disruption of HuR’s regulation of PIM1 may be a key strategy in breaking an elusive chemotherapeutic resistance mechanism acquired by PDA cells that reside in hypoxic PDA microenvironments.

Active Stat5a/b predicts early recurrence and disease-specific death in prostate cancer (PC), which both typically are caused by development of metastatic disease. Herein, we demonstrate that Stat5a/b induces epithelial-to-mesenchymal transition (EMT) of PC cells, as shown by Stat5a/b regulation of EMT marker expression (Twist1, E-cadherin, N-cadherin, vimentin, and fibronectin) in PC cell lines, xenograft tumors in vivo, and patient-derived PCs ex vivo using organ explant cultures. Jak2-Stat5a/b signaling induced functional end points of EMT as well, indicated by disruption of epithelial cell monolayers and increased migration and adhesion of PC cells to fibronectin. Knockdown of Twist1 suppressed Jak2-Stat5a/b-induced EMT properties of PC cells, which were rescued by re-introduction of Twist1, indicating that Twist1 mediates Stat5a/b-induced EMT in PC cells. While promoting EMT, Jak2-Stat5a/b signaling induced stem-like properties in PC cells, such as sphere formation and expression of cancer stem cell markers, including BMI1. Mechanistically, both Twist1 and BMI1 were critical for Stat5a/b induction of stem-like features, because genetic knockdown of Twist1 suppressed Stat5a/b-induced BMI1 expression and sphere formation in stem cell culture conditions, which were rescued by re-introduction of BMI1. By using human prolactin knock-in mice, we demonstrate that prolactin-Stat5a/b signaling promoted metastases formation of PC cells in vivo. In conclusion, our data support the concept that Jak2-Stat5a/b signaling promotes metastatic progression of PC by inducing EMT and stem cell properties in PC cells.

BACKGROUND U.S. military beneficiaries differ from the U.S. general population with regards to access to health care as care is provided at no or much lower cost in the military health system. Other differences also exist. Many of these differences are known factors affecting invasive breast cancer outcomes. Thus it is desirable to conduct a comparative analysis of breast cancer patient outcomes between these two populations to find out whether there is any outcome difference, and if yes what the contributing factors are. METHODS We compared overall survival (OS), disease-specific survival (DSS), and 5-year OS and DSS rates in breast cancers between 399 patients from the Clinical Breast Care Project at the Walter Reed National Military Medical Center (CBCP-WR) and 1,000 sets of 1596 matched patients from the Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute. All patients were diagnosed between 2001 and 2010. Each CBCP-WR patient was randomly matched to four SEER patients on six demographic and clinicopathologic variables (age at diagnosis in 5-year groups, race, diagnosis year, estrogen receptor (ER), progesterone receptor, and AJCC stage). RESULTS The CBCP-WR cohort had better survival than the SEER population. At the whole cohort level, the mean hazard ratios (HRs) from 1,000 matched comparisons for OS and DSS were 0.774 and 0.708, with mean log-rank P-values of 0.124 and 0.125. The numbers of 175 and 141 comparisons showing a log-rank P-value CONCLUSION Overall, these results suggested that breast cancer patients, especially older patients seen in the CBCP-WR, carried more favorable outcomes than those from the general population. The findings warrant further analyses of the contributing factors, such as health care access, treatments, population characteristics, additional pathologic characteristics, and socioeconomic statuses, to this outcome disparity. The views expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S. Government. Citation Format: Yuanbin Ru, Jianfang Liu, Jamie Leigh Campbell, Kangmin Zhu, Albert J Kovatich, Jeffrey A Hooke, Leonid Kvecher, Brenda Deyarmin, Audrey W Kovatich, Frank Cammarata, Hallgeir Rui, Richard J Mural, Craig D Shriver, Hai Hu. Survival comparative analysis of patients with invasive breast cancer treated by a military medical center and matched patients of the US general population [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-07-20.

Motivation The Cancer Genome Atlas (TCGA) has greatly advanced cancer research by generating, curating and publicly releasing deeply measured molecular data from thousands of tumor samples. In particular, gene expression measures, both within and across cancer types, have been used to determine the genes and proteins that are active in tumor cells. Results To more thoroughly investigate the behavior of gene expression in TCGA tumor samples, we introduce a statistical framework for partitioning the variation in gene expression due to a variety of molecular variables including somatic mutations, transcription factors (TFs), microRNAs, copy number alternations, methylation and germ-line genetic variation. As proof-of-principle, we identify and validate specific TFs that influence the expression of PTPN14 in breast cancer cells. Availability and implementation We provide a freely available, user-friendly, browseable interactive web-based application for exploring the results of our transcriptome-wide analyses across 17 different cancers in TCGA at http://ls-shiny-prod.uwm.edu/edge_in_tcga. All TCGA Open Access tier data are available at the Broad Institute GDAC Firehose and were downloaded using the TCGA2STAT R package. TCGA Controlled Access tier data are available via controlled access through the Genomic Data Commons (GDC). R scripts used to download, format and analyze the data and produce the interactive R/Shiny web app have been made available on GitHub at https://github.com/andreamrau/EDGE-in-TCGA.

Many differences between U.S. military beneficiaries and the U.S. general population, including differences in health care access, are known factors affecting invasive breast cancer outcomes. Thus, comparing the two populations for any outcome differences and their contributing factors may provide insights to breast cancer prognosis.Using a marginal Cox proportional hazards regression model, we compared disease-specific survival (DSS) and 5-year DSS rates between 418 patients from the Clinical Breast Care Project at the Walter Reed National Military Medical Center (CBCP-WR) and a set of 1:5 randomly matched patients from the Surveillance, Epidemiology, and End Results program. Patients were compared in the "demographic model" (adjusted by diagnosis year, age, and race) and the "overall model" (further adjusted by estrogen receptor, progesterone receptor, stage, and grade).In the "overall model," CBCP-WR patients were less likely overall to die from breast cancer (hazard ratio [HR] = 0.631, 95% confidence interval [CI] = 0.437-0.911; p = 0.014). This increase in survival was also significant in African American patients (HR = 0.524, 95% CI = 0.277-0.992; p = 0.047) and patients older than 50 (HR = 0.511, 95% CI = 0.306-0.854; p = 0.010). The advantage in 5-year DSS rate for CBCP-WR patients was 5.3% (93.1% vs. 87.8%; p0.001) in the "demographic model" and 3.4% (91.3% vs. 87.9%; p = 0.018) in the "overall model."CBCP-WR patients demonstrated significantly better DSS over matched SEER patients. Although a portion of the outcome disparity, i.e., 36% of the 5.3% DSS rate difference, could be explained by differences in tumor characteristics, the cause(s) behind the majority of the disparity has yet to be identified. Identification and further analysis of contributing factors to survival differences have the potential to improve clinical practice and outcomes for invasive breast cancer patients.

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that drives cell autonomous cell cycle progression and proliferation. Herein we show cyclin D1 abundance is increased >30-fold in the stromal fibroblasts of patients with invasive breast cancer, associated with poor outcome. Cyclin D1 transformed hTERT human fibroblast to a cancer-associated fibroblast phenotype. Stromal fibroblast expression of cyclin D1 (cyclin D1Stroma) in vivo, enhanced breast epithelial cancer tumor growth, restrained apoptosis, and increased autophagy. Cyclin D1Stroma had profound effects on the breast tumor microenvironment increasing the recruitment of F4/80+ and CD11b+ macrophages and increasing angiogenesis. Cyclin D1Stroma induced secretion of factors that promoted expansion of stem cells (breast stem-like cells, embryonic stem cells and bone marrow derived stem cells). Cyclin D1Stroma resulted in increased secretion of proinflammatory cytokines (CCL2, CCL7, CCL11, CXCL1, CXCL5, CXCL9, CXCL12), CSF (CSF1, GM-CSF1) and osteopontin (OPN) (30-fold). OPN was induced by cyclin D1 in fibroblasts, breast epithelial cells and in the murine transgenic mammary gland and OPN was sufficient to induce stem cell expansion. These results demonstrate that cyclin D1Stroma drives tumor microenvironment heterocellular signaling, promoting several key hallmarks of cancer. Published version

Refractoriness of solid tumors, including colorectal cancers (CRCs), to immunotherapies is attributed to the immunosuppressive tumor microenvironment that protects malignant cells from cytotoxic T lymphocytes (CTLs). We found that downregulation of the type I interferon receptor chain IFNAR1 occurs in human CRC and mouse models of CRC. Downregulation of IFNAR1 in tumor stroma stimulated CRC development and growth, played a key role in formation of the immune-privileged niche, and predicted poor prognosis in human CRC patients. Genetic stabilization of IFNAR1 improved CTL survival and increased the efficacy of the chimeric antigen receptor T cell transfer and PD-1 inhibition. Likewise, pharmacologic stabilization of IFNAR1 suppressed tumor growth providing the rationale for upregulating IFNAR1 to improve anti-cancer therapies.

Type 1 interferons (IFN) protect the host against viruses by engaging a cognate receptor (consisting of IFNAR1/IFNAR2 chains) and inducing downstream signaling and gene expression. However, inflammatory stimuli can trigger IFNAR1 ubiquitination and downregulation thereby attenuating IFN effects in vitro . The significance of this paradoxical regulation is unknown. Presented here results demonstrate that inability to stimulate IFNAR1 ubiquitination in the Ifnar1SA knock‐in mice renders them highly susceptible to numerous inflammatory syndromes including acute and chronic pancreatitis, and autoimmune and toxic hepatitis. Ifnar1SA mice (or their bone marrow‐receiving wild type animals) display persistent immune infiltration of inflamed tissues, extensive damage and gravely inadequate tissue regeneration. Pharmacologic stimulation of IFNAR1 ubiquitination is protective against from toxic hepatitis and fulminant generalized inflammation in wild type but not Ifnar1SA mice. These results suggest that endogenous mechanisms that trigger IFNAR1 ubiquitination for limiting the inflammation‐induced tissue damage can be purposely mimicked for therapeutic benefits. ![][1] Inflammatory process in response to injury starts by inducing tissue damage followed by resolution of inflammation. Here, endogenous or induced IFNAR1 ubiquitination and degradation are shown to limit the extent of tissue damage and accelerate healing. EMBO Mol Med (2014) 6, 384–397 [1]: /embed/graphic-1.gif

We investigated the importance of the insulin-like growth factor-1 receptor (IGF-1R) in hepatic metastases of uveal melanoma. The expression pattern of IGF-1R in archival tissue samples of hepatic metastasis from 24 patients was analyzed by immunohistochemistry. All the samples of hepatic metastases stained positive for IGF-1R. To investigate the biological role of IGF-1R on the growth of metastatic uveal melanoma, a long-term cell line obtained from a hepatic metastasis (TJU-UM001) was evaluated. TJU-UM001 expressed cell surface IGF-1R (>90%) and proliferated in response to exogenous and endogenous insulin-like growth factor-1 (IGF-1). Correlatively, anti-IGF-1R antibody completely blocked IGF-1-induced growth of TJU-UM001 cells. IGF-1 preferentially induced phosphorylation of Akt (S473) in quiescent TJU-UM001 cells, and this was blocked by anti-IGF-1R antibody. This study suggests that autocrine and paracrine mechanisms underlie IGF-1-induced growth of metastatic uveal melanoma and underscore the potential benefit of IGF-1 or IGF-1R antagonism in treatment for metastatic uveal melanoma.

Tumor-derived extracellular vesicles (TEV) "educate" healthy cells to promote metastases. We found that melanoma TEV downregulated type I interferon (IFN) receptor and expression of IFN-inducible cholesterol 25-hydroxylase (CH25H). CH25H produces 25-hydroxycholesterol, which inhibited TEV uptake. Low CH25H levels in leukocytes from melanoma patients correlated with poor prognosis. Mice incapable of downregulating the IFN receptor and Ch25h were resistant to TEV uptake, TEV-induced pre-metastatic niche, and melanoma lung metastases; however, ablation of Ch25h reversed these phenotypes. An anti-hypertensive drug, reserpine, suppressed TEV uptake and disrupted TEV-induced formation of the pre-metastatic niche and melanoma lung metastases. These results suggest the importance of CH25H in defense against education of normal cells by TEV and argue for the use of reserpine in adjuvant melanoma therapy.

BACKGROUND It has been reported that women diagnosed with invasive breast cancer (IBC) in their 50s or 60s have better survival than either younger or older women. Young women with IBCs often have a more aggressive phenotype which contributes to worse survival. But there is supporting evidence in the literature for both sides whether age alone is an intrinsic driver of the poor outcome in young women. The availability of large-scale IBC data provided a good opportunity to address this issue. METHODS Clinicopathologic and gene expression data from 2 public datasets, including METABRIC from the European Genome-Phenome Archive (n=1992) and The Cancer Genome Atlas-Breast Cancer project (TCGA-BC, n=980) from National Cancer Institute, were used in this study. PAM50 was used to derive intrinsic subtypes based on microarray and RNA-Seq data. For a given phenotype, Fisher’s exact test was used for its association with age, and 2-sample test for equality of proportions with continuity correction between young and older patients. Kolmogorov-Smirnov’s test was used for equality of age distributions between phenotypes. Disease-specific survival (DSS) was examined for relationship with age, adjusted for race, AJCC stage, nodal metastasis, tumor size, grade, and subtype where applicable. Kaplan-Meier estimate and log-rank test were used to generate and compare survival curves, respectively. Cox proportional hazards model was used for univariate and multivariate analyses and to calculate hazard ratios (HRs). RESULTS Firstly, we confirmed that more aggressive IBCs were enriched in young patients. Younger patients ( CONCLUSION Our results supported that age was not an independent predictor of DSS for patients diagnosed at age The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Defense, or U.S. Government. Citation Format: Yuanbin Ru, Richard J Mural, Patricia S Steeg, Hallgeir Rui, Craig D Shriver, Hai Hu. Age independently predicts worse disease-specific survival in patients diagnosed with invasive breast cancers at 50 years of age or older but not younger [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-08-24.

Resistance to anti-estrogen therapy remains a significant problem in patients diagnosed with estrogen receptor-α (ERα) positive breast cancer. Recent progress has defined a "lumino-basal" subclass of ERα-positive breast cancer characterized by mosaic presence of a minor population of ERα-negative cells expressing the basal cytokeratin-5 (CK5). The CK5-positive cells are therapy-resistant and have increased tumorigenic potential. Initial studies have suggested that progestins but not other steroids expand this CK5+ cell population. Unexpectedly, we discovered that at least two 3-ketosteroids other than progestins, glucocorticoids and mineralocorticoids, are capable of inducing the CK5+/ERα- cell population. CK5+ cells induced by glucocorticoid or aldosterone showed increased clonogenicity in soft agar, expressed the stem cell marker CD44, showed loss of ERα and PR expression, and demonstrated therapy-resistance with reduced apoptosis in response to chemotherapy, and were further enriched following adjuvant antiestrogen or chemotherapies. Induction of CK5+ cells by 3-ketosteroids was consistently preceded by induction of Bcl6, a transcriptional repressor implicated in breast cancer progression. Suppression of Bcl6 by shRNA or the Bcl6 suppressor, prolactin, abolished 3-ketosteroid-induction of CK5+ cells. Prolactin also blocked 3-ketosteroid-induced colony formation in vitro and suppressed progestin-induction of the CK5+ cell population in T47D xenograft tumors in vivo. Survival analyses with recursive partitioning revealed that CK5 and Bcl6 transcripts or protein levels in ERα+ breast cancer identify patients at high or low risk for tumor in two independent cohorts. The observations provide a mechanism by which stress-related or pharmacologic elevation of glucocorticoids may adversely affect patients with ERα+/CK5+ breast cancer, and may justify further exploring of inhibitors to 3-ketosteroid receptors or Bcl6 for therapeutic benefit. Citation Format: Hallgeir Rui, Takahiro Sato, Amy Peck, Melanie A Girondo, Chengbao Liu, Albert J Kovatich, Jeffery A Hooke, Craig D Shriver, Edith Mitchell, Terry Hyslop, Chelain Goodman. Glucocorticoid and aldosterone mimic progestin-induction of a therapy-resistant cytokeratin-5 positive cell population in estrogen receptor-positive breast cancer through a Bcl6-dependent mechanism [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-10.

Breast cancer is a leading form of cancer in the world. Initially cloned as a dominant inhibitor of the hyperactive EGFR, Ellipse, in Drosophila, the mammalian DACH1 regulates expression of target genes in part through interacting with DNA-binding transcription factors (c-Jun, Smads, Six, ERα), and in part through intrinsic DNA-sequence specific binding to Forkhead binding sites. The Drosophila dac gene is a key member of the retinal determination gene network (RDGN), which also includes eyes absent (eya), ey, twin of eyeless (toy), teashirt (tsh) and sin oculis (so), that specifies eye tissue identity. Several lines of evidence suggest DACH1 may function as a tumor suppressor. Clinical studies have demonstrated a correlation between poor prognosis and reduced expression of the cell-fate determination factor DACH1 in breast cancer, and loss of DACH1 expression has been observed in prostate and endometrial cancer. DACH1 inhibits breast cancer tumor metastasis and reduces breast cancer stem cell expansion via Sox2/Nanog. Although these studies suggest DACH1 may function as a tumor suppressor, the molecular mechanisms remain poorly defined. Herein, endogenous DACH1 co-localized with p53 in a nuclear, extranucleolar compartment and bound to p53 in human breast cancer cell lines, p53 and DACH1 bound common genes in ChIP-Seq. Full inhibition of breast cancer contact-independent growth by DACH1 required p53. The p53 breast cancer mutants R248Q and R273H, evaded DACH1 binding. DACH1 phosphorylation at serine residue (S439) inhibited p53 binding and phosphorylation at p53 amino-terminal sites (S15, S20) enhanced DACH1 binding. DACH1 binding to p53 was inhibited by NAD-dependent deacetylation via DACH1 K628. DACH1 repressed p21CIP1 and induced RAD51, an association found in basal breast cancer. DACH1 inhibits breast cancer cellular growth in an NAD and p53 dependent manner through direct protein-protein association. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-11-04.

Previous analyses of three breast cancer cohorts revealed that loss of phospho-Stat5 in breast cancer is associated with significantly elevated risk of hormone therapy failure (1, 2). Nuclear localized tyrosine phosphorylated Stat5 (Nuc-pYStat5) may therefore have clinical value as a predictive marker. Analysis of two of the three previously reported anti-estrogen treated patient cohorts used pathologist scoring of diaminobenzidine (DAB) chromogen-stained Stat5. However the third cohort, analyzed by quantitative immunofluorescence analysis (QIF) on the Genoptix/HistoRx AQUA platform, revealed a greater hazard ratio than the cohorts analyzed by pathologist DAB-scoring. To extend and validate these observations, we applied the Nuc-pYStat5 cutpoint derived in our previous study (2) to an independent cohort of anti-estrogen-treated breast cancer patients using two distinct QIF software platforms, AQUA and Definiens Tissue Studio. Tissue Studio relies on supervised machine learning and multiparametric features of a high-resolution whole slide image to identify cancer cell regions, while AQUA software relies on costaining of a tumor marker to identify cancer cell regions. The two QIF platforms produced highly concordant Nuc-pYStat5 levels (R2 linear = 0.96, P References: 1) Yamashita et al. Stat5 expression predicts response to endocrine therapy and improves survival in estrogen receptor-positive breast cancer. Endocr Relat Cancer. 2006;13:885-93. 2) Peck et al. Loss of nuclear localized and tyrosine phosphorylated Stat5 in breast cancer predicts poor clinical outcome and increased risk of antiestrogen therapy failure. J Clin Oncol. 2011;29:2448-58. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-20.

Human breast cancer is typically characterized by an extensive stromal compartment enriched in fibroblasts. In contrast, xenotransplants of human breast cancer cell lines are typically epithelial-rich and characterized by sparse in-growth of murine fibroblast stroma. Stromal fibroblasts are important for structural integrity of normal tissue and constitute a major element of the stromal microenvironment of invasive cancer. Cancer associated fibroblasts (CAFs) frequently undergo activation as they co-evolve with cancer cells, serving to promote tumor growth and angiogenesis through secretion of multiple paracrine factors. Presence of activated CAFs in solid malignancies is generally associated with higher grade tumors and poor prognosis. Activated CAFs are resistant to apoptosis and display a myofibroblastic phenotype, including expression of a-smooth muscle actin (α-SMA). To more accurately model human tumor-stroma interactions in human breast cancer xenograft lines in mice, we aimed to develop immortalized activated human mammary fibroblasts for admixture xenografting with human breast cancer cells. Human mammary fibroblasts (HMFs) were isolated from multiple surgically excised tissues of reduction mammoplasties or mastectomies. Five isolated primary HMF lines were screened for α-SMA expression. HMF1 expressed the highest levels of α-SMA and was immortalized by stable lentiviral-delivered hTERT. Quantitative real-time PCR, anchorage-independent growth assay, and in vivo studies were used to further characterize selected HMFs. hTERT-HMF1 and HMF2, despite lower level of α-SMA expression in HMF2, displayed significantly elevated levels of mRNA of proteins commonly associated with activated fibroblasts, including SDF-α, SDF-β, HGF, IL-6, VEGF and podoplanin, as well as promoted a 10-fold increase in anchorage-independent growth of MCF7 breast cancer cells in vitro. To evaluate the ability of these HMF lines to promote tumor growth in vivo, MCF7 human breast cancer cells were orthotopically injected into murine mammary fat pads in the presence or absence of hTERT-HMF1 or HMF2. Both HMF lines facilitated in vivo MCF7-xenograft growth and induced histological changes, including higher grade and greater stromal development, when compared to pure MCF7 cell xenografts. IHC for SMA and collagen confirmed the presence of activated fibroblasts in all tumors; however HMF/MCF7 xenografts showed a distinct distribution of fibroblast stroma throughout the tumor in contrast to sparser stroma between larger epithelial aggregates in tumors grafted from MCF7 cells alone. hTERT-HFM1 cells promoted tumor growth in vivo more robustly than HFM2 cells. By 60 days post xenografting, hTERT-HMF1/MCF7 tumors were significantly larger (∼4-fold) than control MCF7 tumors. In addition, admixture with hTERT-HMF1 cells promoted increased tumor angiogenesis and cancer cell proliferation as measured by Ki67 expression. hTERT-HMF1 represents a novel mammary myofibroblast line that may be useful for improved preclinical xenotransplant modeling and tumor drug response testing of human breast cancer in vivo. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-06-10.

// Ke Chen 1 , Kongming Wu 1 , Michael Gormley 1 , Adam Ertel 1 , Jing Wang 1 ,Wei Zhang 1 , Jie Zhou 1 , Gabriele DiSante 1 , Zhiping Li 1 , Hallgeir Rui 1 , Andrew A. Quong 1 , Steven B. McMahon 1,2 , Haiteng Deng 3 , Michael P. Lisanti 2 , Chenguang Wang 1,2 , Richard G. Pestell 1,2 1 Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA, USA 2 Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA 3 Proteomics Resource Center, Rockefeller University, New York, NY, USA Correspondence: Richard G. Pestell, email: // Kongming Wu, email: // Keywords : p53, breast cancer, cell fate, stem cells, dach Received : June 13, 2013 Accepted : June 21, 2013 Published : June 21, 2013 Abstract Breast cancer is a leading form of cancer in the world. The Drosophila Dac gene was cloned as an inhibitor of the hyperactive epidermal growth factor (EGFR), ellipse . Herein, endogenous DACH1 co-localized with p53 in a nuclear, extranucleolar compartment and bound to p53 in human breast cancer cell lines, p53 and DACH1 bound common genes in Chip-Seq. Full inhibition of breast cancer contact-independent growth by DACH1 required p53. The p53 breast cancer mutants R248Q and R273H, evaded DACH1 binding. DACH1 phosphorylation at serine residue (S439) inhibited p53 binding and phosphorylation at p53 amino-terminal sites (S15, S20) enhanced DACH1 binding. DACH1 binding to p53 was inhibited by NAD-dependent deacetylation via DACH1 K628. DACH1 repressed p21 CIP1 and induced RAD51, an association found in basal breast cancer. DACH1 inhibits breast cancer cellular growth in an NAD and p53-dependent manner through direct protein-protein association.

The molecular mechanisms underlying progression of prostate cancer (PCa) to castrate-resistant (CR) and metastatic disease are poorly understood. Our previous mechanistic work shows that inhibition of transcription factor Stat5 by multiple alternative methods induces extensive rapid apoptotic death of Stat5-positive PCa cells in vitro and inhibits PCa xenograft tumor growth in nude mice. Furthermore, STAT5A/B induces invasive behavior of PCa cells in vitro and in vivo, suggesting involvement of STAT5A/B in PCa progression. Nuclear STAT5A/B protein levels are increased in high-grade PCas, CR PCas, and distant metastases, and high nuclear STAT5A/B expression predicts early disease recurrence and PCa-specific death in clinical PCas. Based on these findings, STAT5A/B represents a therapeutic target protein for advanced PCa. The mechanisms underlying increased Stat5 protein levels in PCa are unclear. Herein, we demonstrate amplification at the STAT5A/B gene locus in a significant fraction of clinical PCa specimens. STAT5A/B gene amplification was more frequently found in PCas of high histologic grades and in CR distant metastases. Quantitative in situ analysis revealed that STAT5A/B gene amplification was associated with increased STAT5A/B protein expression in PCa. Functional studies showed that increased STAT5A/B copy numbers conferred growth advantage in PCa cells in vitro and as xenograft tumors in vivo. The work presented herein provides the first evidence of somatic STAT5A/B gene amplification in clinical PCas.

Prolactin controls the development and function of milk-producing breast epithelia but also supports growth and differentiation of breast cancer, especially luminal subtypes. A principal signaling mediator of prolactin, Stat5, promotes cellular differentiation of breast cancer cells in vitro, and loss of active Stat5 in tumors is associated with anti-estrogen therapy failure in patients. In luminal breast cancer progesterone induces a cytokeratin-5 (CK5)-positive basal cell-like population. This population possesses characteristics of tumor stem cells including quiescence, therapy-resistance, and tumor-initiating capacity. Here we report that prolactin counteracts induction of the CK5-positive population by the synthetic progestin R5020 in luminal breast cancer cells both in vitro and in vivo. CK5-positive cells were chemoresistant as determined by four-fold reduced rate of apoptosis following docetaxel exposure. Progestin-induction of CK5 was preceded by marked up-regulation of BCL6, an oncogene and transcriptional repressor critical for the maintenance of leukemia-initiating cells. Knockdown of BCL6 prevented induction of CK5-positive cell population by progestin. Prolactin suppressed progestin-induced BCL6 through Jak2-Stat5 but not Erk- or Akt-dependent pathways. In premenopausal but not postmenopausal patients with hormone receptor-positive breast cancer, tumor protein levels of CK5 correlated positively with BCL6, and high BCL6 or CK5 protein levels were associated with unfavorable clinical outcome. Suppression of progestin-induction of CK5-positive cells represents a novel pro-differentiation effect of prolactin in breast cancer. The present progress may have direct implications for breast cancer progression and therapy since loss of prolactin receptor-Stat5 signaling occurs frequently and BCL6 inhibitors currently being evaluated for lymphomas may have value for breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive disease subtype that has a poor prognosis. Extensive epidemiological evidence demonstrates clear socioeconomic and demographic associations with increased likelihood of TNBC in both poorer and minority populations. Thus, biological aggressiveness with few known therapeutic directions generates disparities in breast cancer outcomes for vulnerable populations. Emerging molecular evidence of potential targets in triple-negative subpopulations offers great potential for future clinical trial directions. However, trials must appropriately consider populations at risk for aggressive subtypes of disease in order to address this disparity most completely. New US FDA draft guidance documents provide both flexible outcomes for accelerated approvals as well as flexibility in design with adaptive trials. Careful planning with design, potential patient population and choices of molecular targets informed by biomarkers will be critical to address TNBC clinical care.

E-selectin is an adhesion molecule expressed on the luminal surface of inflamed blood vessels that mediates hematogenous metastasis by assisting shear-resistant adhesion of circulating tumor cells to the vessel surface under dynamic blood flow. Previously, we developed an E-selectin antagonistic thioaptamer (ESTA) for the prevention of hematogenous metastasis through the blockade of CD44high breast cancer cells (BCa) adhesion to E-selectin-expressing premetastatic endothelial niche. The current study focuses on developing a PEGylated E-selectin targeting thioaptamer with improved pharmaceutical properties. A serial deletion of stem-loops reveled that loop-1 and -2 (ESTA7) are the minimally effective backbone structure necessary to obtain inhibition of the E-selectin/CD44 interaction and shear resistant adhesion of CD44high BCa to E-selectin-expressing human endothelial cells (HMVECs) at a level equal to ESTA. Chemical conjugation of methoxy-polyethylene-glycol (PEG) at the sizes of 5 and 10 kDa did not interfere with ESTA7-mediated shear-resistant adhesion. However, in vivo study demonstrated that only 10 kDa PEG-conjugated ESTA7 (ESTA7-p10) retains the activity to inhibit metastases at a level equal to parental ESTA. Additionally, a single intravenous injection of ESTA7-p10 inhibited the development of lung, brain, and bone metastases of MDA-MB-231, through the blockade of E-selectin. Moreover, PEGylation led to an extension of elimination half-life and increase of AUC, resulting in superior inhibition of metastasis development compared to parental ESTA with a longer interval between dosing in a spontaneous metastasis model. Lastly, repeated intravenous administration of ESTA7-p10 was tolerated in mice, highlighting the potential prophylactic application of ESTA7-p10 for metastasis prevention.

Phototherapy with ultraviolet (UV) light is a standard treatment for psoriasis, yet the mechanisms underlying the therapeutic effects are not well understood. Studies in human and mouse keratinocytes and in the skin tissues from human patients and mice showed that UV treatment triggers ubiquitination and downregulation of the type I interferon (IFN) receptor chain IFNAR1, leading to suppression of IFN signaling and an ensuing decrease in the expression of inflammatory cytokines and chemokines. The severity of imiquimod-induced psoriasiform inflammation was greatly exacerbated in skin of mice deficient in IFNAR1 ubiquitination (Ifnar1SA). Furthermore, these mice did not benefit from UV phototherapy. Pharmacologic induction of IFNAR1 ubiquitination and degradation by an antiprotozoal agent halofuginone also relieved psoriasiform inflammation in wild type but not in Ifnar1SA mice. These data identify downregulation of IFNAR1 by UV as a major mechanism of the UV therapeutic effects against the psoriatic inflammation and provide a proof of principle for future development of agents capable of inducing IFNAR1 ubiquitination and downregulation for the treatment of psoriasis.

Background Distant metastasis resulting from vascular dissemination of cancer cells is the primary cause of mortality from breast cancer. We have previously reported that E-selectin expression on the endothelial cell surface mediates shear-resistant adhesion and migration of circulating cancer cells via interaction with CD44. As a result of shedding, soluble E-selectin (sE-selectin) from the activated endothelium is present in the serum. In this study, we aimed to understand the role of sE-selectin in tumor progression and metastasis. Methods We investigated the effect of sE-selectin on shear-resistant adhesion and migration of metastatic breast cancer cells and leukocytes in vitro and in vivo. Results We found that sE-selectin promoted migration and shear-resistant adhesion of CD44+/high breast cancer cell lines (MDA-MB-231 and MDA-MB-468) to non-activated human microvessel endothelial cells (ES-HMVECs), but not of CD44-/low breast cancer cell lines (MCF-7 and T-47D). This endothelial E-selectin independent, sE-selectin-mediated shear-resistant adhesion was also observed in a leukocyte cell line (HL-60) as well as human peripheral blood mononuclear cells (PBMCs). Additionally, the incubation of MDA-MB-231 cells with sE-selectin triggered FAK phosphorylation and shear-resistant adhesion of sE-selectin-treated cells resulted in increased endothelial permeabilization. However, CD44 knockdown in MDA-MB-231 and HL-60 cells resulted in a significant reduction of sE-selectin-mediated shear-resistant adhesion to non-activated HMVECs, suggesting the involvement of CD44/FAK. Moreover, functional blockade of ICAM-1 in non-activated HMVECs resulted in a marked reduction of sE-selectin-mediated shear-resistant adhesion. Finally, the pre-incubation of CD44+ 4 T1 murine breast cancer cells with sE-selectin augmented infiltration into the lung in E-selectin K/O mice and infusion of human PBMCs pre-incubated with sE-selectin stimulated MDA-MB-231 xenografted breast tumor growth in NSG mice. Conclusions Our data suggest that circulating sE-selectin stimulates a broad range of circulating cells via CD44 and mediates pleiotropic effects that promote migration and shear-resistant adhesion in an endothelial E-selectin independent fashion, in turn accelerating tissue infiltration of leukocytes and cancer cells. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2366-2) contains supplementary material, which is available to authorized users.

Background: Invasive breast cancer (IBC) has been classified into four major subtypes based on gene expression profiling. The luminal A subtype (LA) has the best prognosis, when compared to luminal B (LB), HER2+, and basal-like (Basal). Ki67 by gene expression or immunohistochemistry (IHC) is commonly used as a proliferation index. The function of Ki67 in proliferation remains unknown. AURKA (STK15) is known to play an important role in mitosis, and is a component of the 21-gene recurrence score of the Oncotype Dx. With multiple platforms of molecular data available from hundreds of IBC tissues in The Cancer Genome Atlas project (TCGA), we sought to study the association of AURKA with different IBC subtypes and explore its use as a proliferation marker in IBCs. Methods: Gene expression (Agilent, log2 transformed), relative DNA copy number (CN, Affymetrix SNP 6.0), and exome sequence mutation (Illumina) data for 459 IBC cases were downloaded from the TCGA data portal. PAM50 classification results of all samples were obtained from the TCGA breast cancer AWG group and included 203 LA, 113 LB, 51 HER2+, 84 Basal-like, and 8 Normal-like which were not used in this study due to the low numbers. Kruskal-Wallis tests were used to evaluate the differences among four subtypes on AURKA expression and CN, followed by Wilcoxon Mann-Whitney test with Bonferroni adjustment for pairwise analyses. Pearson's Correlation Coefficient was used for correlation analyses. All statistical analyses were performed using SAS and R, and two-sided, p values Results: There was a significant difference among IBC subtypes, in gene expression as well as in CN (p values < 0.0001). AURKA mRNA levels were significantly lower in LA (mean±SD, −2.61±0.63) compared to LB (−1.45±0.78), HER2+ (−1.38±0.61), and Basal (−1.26±0.62) subtypes (p values all < 0.0001). No significant difference was detected between other subtype pairs. In CN analysis, Basal (0.09±0.22) was lower than HER2+ (0.32±0.308, p < 0.0002) and LB (0.33±0.41, p < 0.0001), and LA (0.14±0.28) is lower than HER2 (p < 0.0016) and LB (p < 0.0001), but no other significant CN difference between the subtypes were found. The means and SDs are provided for reference only. No correlation of p53 mutation status and AURKA expression were observed. However, AURKA gene expression level is correlated with MKI67 gene expression (R = 0.69, p < 2.2e−16), and its correlation with PAM50 proliferation score is even higher (R = 0.80, p < 2.2e−16). Discussion: Using the TCGA data we observed that the mean gene expression level of AURKA is significantly lower in LA than the other IBC subtypes, by more than 50% (note the log2 transformation). This differential expression is not completely due to CN changes (especially for the Basal subtype). There is a strong association with other tumor cell proliferation markers such as the MKI67 gene and the PAM50 proliferation score. We are using computational and laboratorial studies to better understand the role of AURKA in the etiology of invasive breast cancers. The views expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S. Government. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-05-21.

Eighty percent of newly diagnosed breast cancer represents estrogen receptor(ER)-positive luminal subtypes. Many patients with luminal breast cancer develop antiestrogen resistant disease. It has historically been particularly difficult to establish ER-positive breast cancer lines from primary breast cancer in the laboratory or in mice. Murine and bovine prolactins, the major lactogens in current laboratory experimental in vivo and in vitro conditions, fail to activate human prolactin receptors because of species incompatibility. In fact, murine prolactin is a potent antagonist for human prolactin receptors. Because ER-positive, luminal breast cancers also express prolactin receptors, we hypothesized that lack of human lactogenic activity under experimental conditions selected against establishment of ER-positive breast cancer in the laboratory. We therefore genetically engineered mice to express physiological levels of human prolactin in place of mouse prolactin and backcrossed the mice for ten generations into the immunodeficient NSG strain. The resulting hPRL.NSG mice have a greatly improved take rate for ER positive, luminal type of breast cancer, suggesting key tumor-promoting roles for prolactin in luminal breast cancer. A panel of novel transplantable human breast cancer lines has been established in hPrl.NSG mice, the majority of which are ER-positive. The transplantable lines maintain key histopathological characteristics and expression of major marker proteins of the primary patient tumors. Intriguingly, initial tumor establishment and growth rates of breast cancer xenografts were consistently greater in the hPrl.NSG mice than in wildtype NSG mice. Furthermore, tumors grown in hPrl.NSG were more responsive to tamoxifen than size-matched tumors grown in wildtype NSG mice. At least two new tumor lines examined so far develop spontaneous distant metastases in hPrl.NSG mice, with evidence of prolactin-dependent progression of ER-positive disease. Collectively, these observations validate the hPrl.NSG mice as an improved recipient for preclinical modeling of human breast cancer in vivo, both for therapeutic targeting of prolactin-pathways and other growth and survival pathways, as well as overcoming anti-estrogen resistance. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr S1-8.

Background: The aim of this study was to investigate the value of the cyclin D1 isoforms D1a and D1b as prognostic factors and their relevance as predictors of response to adjuvant chemotherapy with 5-fluorouracil and levamisole (5-FU/LEV) in colorectal cancer (CRC). Methods: Protein expression of nuclear cyclin D1a and D1b was assessed by immunohistochemistry in 335 CRC patients treated with surgery alone or with adjuvant therapy using 5-FU/LEV. The prognostic and predictive value of these two molecular markers and clinicopathological factors were evaluated statistically in univariate and multivariate survival analyses. Results: Neither cyclin D1a nor D1b showed any prognostic value in CRC or colon cancer patients. However, high cyclin D1a predicted benefit from adjuvant therapy measured in 5-year relapse-free survival (RFS) and CRC-specific survival (CSS) compared to surgery alone in colon cancer (P=0.012 and P=0.038, respectively) and especially in colon cancer stage III patients (P=0.005 and P=0.019, respectively) in univariate analyses. An interaction between treatment group and cyclin D1a could be shown for RFS (P=0.004) and CSS (P=0.025) in multivariate analysis. Conclusion: Our study identifies high cyclin D1a protein expression as a positive predictive factor for the benefit of adjuvant 5-FU/LEV treatment in colon cancer, particularly in stage III colon cancer.

Cyclin D1 regulates cell proliferation and is a candidate molecular target for breast cancer therapy. This study addresses whether Cyclin D1 is indispensable for ErbB2-associated mammary tumor initiation and progression using a breast cancer model in which this cell-cycle regulator can be genetically ablated prior to or after neoplastic transformation. Deficiency in Cyclin D1 delayed tumor onset but did not prevent the occurrence of mammary cancer in mice overexpressing wild-type ErbB2. The lack of Cyclin D1 was associated with a compensatory upregulation of Cyclin D3, which explains why the targeted downregulation of Cyclin D1 in established mammary tumors had no effect on cancer cell proliferation. Cyclin D1 and D3 are overexpressed in human breast cancer cell lines and primary invasive breast cancers, and Cyclin D3 frequently exceeded the expression of Cyclin D1 in ErbB2-positive cases. The simultaneous inhibition of both cyclins in mammary tumor cells reduced cancer cell proliferation in vitro and decreased the tumor burden in vivo. Collectively, the results of this study suggest that only the combined inhibition of Cyclin D1 and D3 might be a suitable strategy for breast cancer prevention and therapy. Cancer Res; 71(24); 7513–24. ©2011 AACR.

Nuclear-localized and tyrosine-phosphorylated Stat5 has been reported as a favorable prognostic marker and predictor of response to antiestrogen therapy in breast cancer. Phospho-Stat5 antibodies do not distinguish between phosphorylated Stat5a and the closely related Stat5b, but Stat5a is considered more critical for normal mammary development than Stat5b. The purpose of this study was to determine whether levels of nuclear-localized Stat5a protein (Nuc-Stat5a) were prognostic of clinical outcome or predictive of antiestrogen response. Stat5a was detected by traditional diaminobenzidine-chromogen immunohistochemistry (IHC) and pathologist scoring or by quantitative immunofluorescence in five archival cohorts of breast cancer. Levels of nuclear-localized Stat5a (Nuc-Stat5a) were evaluated by pathologist scoring of whole tissue sections detected by IHC or automated quantitative analysis (AQUA) of immunofluorescently-labeled tissue microarrays. Levels of Nuc-Stat5a were reduced in invasive breast cancer tissues and lymph node metastases compared to normal tissue and ductal carcinoma in situ when quantified by AQUA (Material I; n=180). Tissues from patients not treated with adjuvant therapy or treated with antiestrogen monotherapy were analyzed according to Nuc-Stat5a status for breast cancer-specific survival (CSS) and time to recurrence (TTR) using univariate and multivariate statistical models, adjusting for clinical features including tumor grade, size, lymph node and ER, PR and Her2 status. In two prognostic cohorts of node-negative breast cancer patients, low expression of Nuc-Stat5a, detected by standard IHC (Material II; n=223) or quantitative analysis (Material III; n=198), was prognostic of poor breast cancer outcome as measured by univariate and multivariate CSS (Material II/III) and TTR (Material II). CSS and TTR analysis of two independent materials of tumors from patients treated with antiestrogen monotherapy and analyzed by standard IHC (Material IV; n=73) or quantitative immunofluorescence (Material V; n=97) indicated that patients whose tumors expressed low levels of Nuc-Stat5a were at a greater than 4-fold risk of antiestrogen therapy failure when adjusted for hormone receptor status and clinical features (multivariate CSS: Material IV HR=4.3 (1.2,15.6), p=0.03; Material V HR=5.0 (1.87,13.06), p=0.001). In conclusion, loss of Nuc-Stat5a is a promising independent marker of poor breast cancer prognosis in node-negative, non-adjuvant treated breast cancer patients. Additionally, Nuc-Stat5a may be a useful clinical tool to predict tumor response to antiestrogen therapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-24.

Introduction: Bayesian Belief Networks have been used in medicine to evaluate clinical data and develop predictive and prognostic models. As classification models, they allow us to represent pattern complexity beyond what can be accomplished with traditional Kaplan-Meier or regression models. We sought to evaluate the use of machine-learned Bayesian Belief Networks (ml-BBNs) to develop mortality models in breast cancer and to evaluate classification performance for this method. Methods: A set of 2,300 breast bancer cases from a tumor registry at Thomas Jefferson University were used to train ml-BBNs. The registry set was broken into cohorts for modeling by follow-up times of 1 (n=2,202), 2 (n=2,183), 3 (n=2,157), and 5 (n=2,027) years. Each cohort was then used to train a ***m1-BBN and each model was evaluated for structure. Variables were recoded into categories: biomarkers (ER, PR, Ki67, HER2, p53) as positive or negative; grading, staging, and size were broken in categories; while race was recoded into Caucasian or African-American. Income and poverty level by census tract were also included. Models were evaluated for ability to classify mortality (yes/no) within the follow-up period using 10-fold cross-validation and Receiver Operating Characteristic curves. Results: Area Under the Curve (AUC), Positive Predictive Value (PPV), and Negative Predictive Value (NPV) were calculated for each set of cohort training models and mean values and 95% confidence intervals were calculated for mortality (yes/no) within the follow-up period. AUCs (and CIs) for 1, 2, 3, and 5 years were: 0.81 (0.70 — 0.91), 0.74 (0.69 — 0.79), 0.81 (0.77 — 0.86), 0.77 (0.74 — 0.80). PPVs for 1, 2, 3, and 5 years were: 12.3% (7.5% — 17.1%), 18.8% (15.4% — 22.1%), 18.0% (15.1% — 20.9%), 28.2% (24.7% — 31.7%). NPVs for 1, 2, 3, and 5 years were: 99.2% (98.8% — 99.7%), 97.4% (96.9% — 97.8%), 96.4% (95.1% — 97.7%), 91.7% (89.1% — 94.3%). Predictors of mortality at 1 year were Tumor Stage, at 2 years were Estrogen Receptor and Tumor Stage, and at 3 and 5 years were Diagnosis Age, Tumor Stage, Estrogen Receptor status, and Ki-67 receptor status. Discussion / Conclusion: We were able to successfully train ***m1-BBNs to estimate mortality using breast cancer registry cohorts. Cross-validation showed the models to be robust. The structure of the models can inform us how different data elements contribute to the estimate of mortality. These models can be used to calculate individual probabilities for prognostic guidance given age, staging criteria, and biomarkers. Overall 5-year mortality in the study set is 15.2%, however we can derive subject-specific mortality estimates. For example, a 43-year old Stage 3, ER-Negative, Ki-67 Negative subject has a 19.9% probability of 5-year mortality, while the same subject with positive Ki-67 has a 37.8% probability of mortality. Meanwhile, the same probabilities for a 70 year old woman are 67.0% and 59.0%, respectively. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-14-12.

Interferon alpha (IFNα) is widely used for treatment of melanoma and certain other malignancies. This cytokine as well as the related IFNβ exerts potent anti-tumorigenic effects; however, their efficacy in patients is often suboptimal. Here, we report that inflammatory signaling impedes the effects of IFNα/β. Melanoma cells can secrete pro-inflammatory cytokines that inhibit cellular responses to IFNα/β via activating the ligand-independent pathway for the phosphorylation and subsequent ubiquitination and accelerated degradation of the IFNAR1 chain of type I IFN receptor. Catalytic activity of the p38 protein kinase was required for IFNAR1 downregulation and inhibition of IFNα/β signaling induced by proinflammatory cytokines such as interleukin 1 (IL-1). Activation of p38 kinase inversely correlated with protein levels of IFNAR1 in clinical melanoma specimens. Inhibition of p38 kinase augmented the inhibitory effects of IFNα/β on cell viability and growth in vitro and in vivo. The roles of inflammation and p38 protein kinase in regulating cellular responses to IFNα/β in normal and tumor cells are discussed.

Basal levels of nuclear localized, tyrosine phosphorylated Stat5 are present in healthy human breast epithelia. In contrast, Stat5 phosphorylation is frequently lost during breast cancer progression, a finding that correlates with loss of histological differentiation and poor patient prognosis. Identifying the mechanisms underlying loss of Stat5 phosphorylation could provide novel targets for breast cancer therapy. Pervanadate, a general tyrosine phosphatase inhibitor, revealed marked phosphatase regulation of Stat5 activity in breast cancer cells. Lentiviral-mediated shRNA allowed specific examination of the regulatory role of five tyrosine phosphatases (PTP1B, TC-PTP, SHP1, SHP2, and VHR), previously implicated in Stat5 regulation in various systems. Enhanced and sustained prolactin-induced Stat5 tyrosine phosphorylation was observed in T47D and MCF7 breast cancer cells selectively in response to PTP1B depletion. Conversely, PTP1B overexpression suppressed prolactin-induced Stat5 tyrosine phosphorylation. Furthermore, PTP1B knockdown increased Stat5 reporter gene activity. Mechanistically, PTP1B suppression of Stat5 phosphorylation was mediated, at least in part, through inhibitory dephosphorylation of the Stat5 tyrosine kinase, Jak2. PTP1B knockdown enhanced sensitivity of T47D cells to prolactin phosphorylation of Stat5 by reducing the EC(50) from 7.2 nmol/L to 2.5 nmol/L. Immunohistochemical analyses of two independent clinical breast cancer materials revealed significant negative correlations between levels of active Stat5 and PTP1B, but not TC-PTP. Collectively, our data implicate PTP1B as an important negative regulator of Stat5 phosphorylation in invasive breast cancer.

Transcription factor Stat5a/b is critical for prostate cancer cell survival and for prostate xenograft tumor growth. In addition, the Stat5a/b signaling pathway may contribute to progression of organ-confined prostate cancer to castration-resistant and/or metastatic disease. Expression of nuclear Stat5a/b is clustered to high grade human prostate cancers, and nuclear Stat5a/b in primary prostate cancer predicts early disease recurrence after initial treatment. Here, we show by Western blotting and electromobility shift assay that Stat5a/b protein in human prostate cancer is N-terminally truncated. This short form of Stat5a/b is generated post-translationally in vivo in prostate cancer cells and is the predominant form of Stat5a/b that binds to DNA. We further demonstrate by mutagenesis and co-immunoprecipitations that the N-domain of Stat5a/b is required for binding to PIAS3, and that PIAS3 inhibits transcriptional activity of Stat5a/b in breast cancer cells but not in prostate cancer cells. Thus, the proteolytic cleavage of the N-terminus of Stat5a/b may be a mechanism by which Stat5 evades the transcriptional repression by PIAS3 in prostate cancer cells, and results in increased Stat5-driven gene expression and prostate cancer progression.