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Because the incidence of breast cancer increases decades after ionizing radiation exposure, aging has been implicated in the evolution of the tumor microenvironment and tumor progression. Here, we investigated radiation-induced carcinogenesis using a model in which the mammary glands of 10-month-old BALB/c mice were transplanted with Trp53-null mammary tissue 3 days after exposure to low doses of sparsely ionizing γ-radiation or densely ionizing particle radiation. Mammary transplants in aged, irradiated hosts gave rise to significantly more tumors that grew more rapidly than those in sham-irradiated mice, with the most pronounced effects seen in mice irradiated with densely ionizing particle radiation. Tumor transcriptomes identified a characteristic immune signature of these aggressive cancers. Consistent with this, fast-growing tumors exhibited an immunosuppressive tumor microenvironment with few infiltrating lymphocytes, abundant immunosuppressive myeloid cells, and high COX-2 and TGFβ. Only irradiated hosts gave rise to tumors lacking cytotoxic CD8+ lymphocytes (defined here as immune desert), which also occurred in younger irradiated hosts. These data suggest that host irradiation may promote immunosuppression. To test this, young chimera mice were fed chow containing a honeybee-derived compound with anti-inflammatory and immunomodulatory properties, caffeic acid phenethyl ester (CAPE). CAPE prevented the detrimental effects of host irradiation on tumor growth rate, immune signature, and immunosuppression. These data indicated that low-dose radiation, particularly densely ionizing exposure of aged mice, promoted more aggressive cancers by suppressing antitumor immunity. Dietary intervention with a nontoxic immunomodulatory agent could prevent systemic effects of radiation that fuel carcinogenesis, supporting the potential of this strategy for cancer prevention.

Supplemental data tables and analysis

Supplementary Figure 6. Representative images of immune cells infiltration and proliferating cells.

Purpose: High-throughput profiling experiments have linked altered expression of microRNAs (miRNA) to different types of cancer. Tumor tissues are a heterogeneous mixture of not only cancer cells, but also supportive and reactive tumor microenvironment elements. To clarify the clinical significance of altered miRNA expression in solid tumors, we developed a sensitive fluorescence-based in situ hybridization (ISH) method to visualize miRNA accumulation within individual cells in formalin-fixed, paraffin-embedded tissue specimens. This ISH method was implemented to be compatible with routine clinical immunohistochemical (IHC) assays to enable the detection of miRNAs and protein markers in the same tissue section for colocalization and functional studies.Experimental Design: We used this combined ISH/IHC assay to study a subset of cancer-associated miRNAs, including miRNAs frequently detected at low (miR-34a and miR-126) and high (miR-21 and miR-155) levels, in a panel of breast, colorectal, lung, pancreas, and prostate carcinomas.Results: Despite the distinct histopathologic alterations of each particular cancer type, general trends emerged that pinpointed distinct source cells of altered miRNA expression. Although altered expressions of miR-21 and miR-34a were manifested within cancer cells, those of miR-126 and miR-155 were predominantly confined to endothelial cells and immune cells, respectively. These results suggest a heterogeneous participation of miRNAs in carcinogenesis by intrinsically affecting cancer cell biology or by modulating stromal, vascular, and immune responses.Conclusions: We described a rapid and sensitive multicolor ISH/IHC assay and showed that it could be broadly applied as an investigational tool to better understand the etiologic relevance of altered miRNA expression in cancer. Clin Cancer Res; 16(16); 4246–55. ©2010 AACR.

Supplementary Figures S1-S7 and Supplementary Tables S1-S3.

Supplementary tables 1-3

Purpose:Women treated with radiotherapy before 30 years of age have increased risk of developing breast cancer at an early age. Here, we sought to investigate mechanisms by which radiation promotes aggressive cancer.Experimental Design:The tumor microenvironment (TME) of breast cancers arising in women treated with radiotherapy for Hodgkin lymphoma was compared with that of sporadic breast cancers. To investigate radiation effects on carcinogenesis, we analyzed tumors arising from Trp53-null mammary transplants after irradiation of the target epithelium or host using immunocompetent and incompetent mice, some of which were treated with aspirin.Results:Compared with age-matched specimens of sporadic breast cancer, radiation-preceded breast cancers (RP-BC) were characterized by TME rich in TGFβ, cyclooxygenase 2, and myeloid cells, indicative of greater immunosuppression, even when matched for triple-negative status. The mechanism by which radiation impacts TME construction was investigated in carcinomas arising in mice bearing Trp53-null mammary transplants. Immunosuppressive TMEs (iTME) were recapitulated in mice irradiated before transplantation, which implicated systemic immune effects. In nu/nu mice lacking adaptive immunity irradiated before Trp53-null mammary transplantation, cancers also established an iTME, which pointed to a critical role for myeloid cells. Consistent with this, irradiated mammary glands contained more macrophages and human cells cocultured with polarized macrophages underwent dysplastic morphogenesis mediated by IFNγ. Treating mice with low-dose aspirin for 6 months postirradiation prevented establishment of an iTME and resulted in less aggressive tumors.Conclusions:These data show that radiation acts via nonmutational mechanisms to promote markedly immunosuppressive features of aggressive, RP-BCs.

Age and physiologic status, such as menopause, are risk factors for breast cancer. Less clear is what factors influence the diversity of breast cancer. In this study, we investigated the effect of host age on the distribution of tumor subtypes in mouse mammary chimera consisting of wild-type hosts and Trp53 nullizygous epithelium, which undergoes a high rate of neoplastic transformation. Wild-type mammary glands cleared of endogenous epithelium at 3 weeks of age were subsequently transplanted during puberty (5 weeks) or at maturation (10 weeks) with syngeneic Trp53-null mammary tissue fragments and monitored for one year. Tumors arose sooner from adult hosts (AH) compared with juvenile hosts (JH). However, compared with AH tumors, JH tumors grew several times faster, were more perfused, exhibited a two-fold higher mitotic index, and were more highly positive for insulin-like growth factor receptor phosphorylation. Most tumors in each setting were estrogen receptor (ER)-positive (80% JH vs. 70% AH), but JH tumors were significantly more ER-immunoreactive (P = 0.0001) than AH tumors. A differential expression signature (JvA) of juvenile versus adult tumors revealed a luminal transcriptional program. Centroids of the human homologs of JvA genes showed that JH tumors were more like luminal A tumors and AH tumors were more like luminal B tumors. Hierarchical clustering with the JvA human ortholog gene list segregated luminal A and luminal B breast cancers across datasets. These data support the notion that age-associated host physiology greatly influences the intrinsic subtype of breast cancer. Cancer Res; 74(23); 7149–58. ©2014 AACR.

Human orthologs of JvA 243 profile. Proliferation-related genes removed.

Genes modulated by at least 1.5-fold in tumors arising in juvenile hosts relative to those from adult hosts.

Supplemental Table 1 and Figures S1-6. Table S1: tumor features. Fig. S1. Tumor growth curves from different irradiation group. Fig. S2. Host irradiation promotes ER-negative tumor growth. Fig. S3. Unsupervised hierarchical clustering of mouse tumors based on SD of 1.0. Fig. S4. Highly expressed genes in mammary stem cell are preferentially underexpressed in K18 compared to K14/18 tumor. Fig. S5. K14/18-IHPsi is negatively associated with MaSC signature. Fig. S6. Host irradiation exerts more robust effects on gene expression profile in ER-negative(ER-neg) than ER-positive(ER-pos) tumors.

Histopathology and features of JH and AH tumors.

Purpose: Women treated with radiotherapy before 30 years of age have increased risk of developing breast cancer at an early age. Here, we sought to investigate mechanisms by which radiation promotes aggressive cancer. Experimental Design: The tumor microenvironment (TME) of breast cancers arising in women treated with radiotherapy for Hodgkin lymphoma was compared with that of sporadic breast cancers. To investigate radiation effects on carcinogenesis, we analyzed tumors arising from Trp53-null mammary transplants after irradiation of the target epithelium or host using immunocompetent and incompetent mice, some of which were treated with aspirin. Results: Compared with age-matched specimens of sporadic breast cancer, radiation-preceded breast cancers (RP-BC) were characterized by TME rich in TGFβ, cyclooxygenase 2, and myeloid cells, indicative of greater immunosuppression, even when matched for triple-negative status. The mechanism by which radiation impacts TME construction was investigated in carcinomas arising in mice bearing Trp53-null mammary transplants. Immunosuppressive TMEs (iTME) were recapitulated in mice irradiated before transplantation, which implicated systemic immune effects. In nu/nu mice lacking adaptive immunity irradiated before Trp53-null mammary transplantation, cancers also established an iTME, which pointed to a critical role for myeloid cells. Consistent with this, irradiated mammary glands contained more macrophages and human cells cocultured with polarized macrophages underwent dysplastic morphogenesis mediated by IFNγ. Treating mice with low-dose aspirin for 6 months postirradiation prevented establishment of an iTME and resulted in less aggressive tumors. Conclusions: These data show that radiation acts via nonmutational mechanisms to promote markedly immunosuppressive features of aggressive, RP-BCs.

Because the incidence of breast cancer increases decades after ionizing radiation exposure, aging has been implicated in the evolution of the tumor microenvironment and tumor progression. Here, we investigated radiation-induced carcinogenesis using a model in which the mammary glands of 10-month-old BALB/c mice were transplanted with Trp53-null mammary tissue 3 days after exposure to low doses of sparsely ionizing γ-radiation or densely ionizing particle radiation. Mammary transplants in aged, irradiated hosts gave rise to significantly more tumors that grew more rapidly than those in sham-irradiated mice, with the most pronounced effects seen in mice irradiated with densely ionizing particle radiation. Tumor transcriptomes identified a characteristic immune signature of these aggressive cancers. Consistent with this, fast-growing tumors exhibited an immunosuppressive tumor microenvironment with few infiltrating lymphocytes, abundant immunosuppressive myeloid cells, and high COX-2 and TGFβ. Only irradiated hosts gave rise to tumors lacking cytotoxic CD8+ lymphocytes (defined here as immune desert), which also occurred in younger irradiated hosts. These data suggest that host irradiation may promote immunosuppression. To test this, young chimera mice were fed chow containing a honeybee-derived compound with anti-inflammatory and immunomodulatory properties, caffeic acid phenethyl ester (CAPE). CAPE prevented the detrimental effects of host irradiation on tumor growth rate, immune signature, and immunosuppression. These data indicated that low-dose radiation, particularly densely ionizing exposure of aged mice, promoted more aggressive cancers by suppressing antitumor immunity. Dietary intervention with a nontoxic immunomodulatory agent could prevent systemic effects of radiation that fuel carcinogenesis, supporting the potential of this strategy for cancer prevention.

Age and physiologic status, such as menopause, are risk factors for breast cancer. Less clear is what factors influence the diversity of breast cancer. In this study, we investigated the effect of host age on the distribution of tumor subtypes in mouse mammary chimera consisting of wild-type hosts and Trp53 nullizygous epithelium, which undergoes a high rate of neoplastic transformation. Wild-type mammary glands cleared of endogenous epithelium at 3 weeks of age were subsequently transplanted during puberty (5 weeks) or at maturation (10 weeks) with syngeneic Trp53-null mammary tissue fragments and monitored for one year. Tumors arose sooner from adult hosts (AH) compared with juvenile hosts (JH). However, compared with AH tumors, JH tumors grew several times faster, were more perfused, exhibited a two-fold higher mitotic index, and were more highly positive for insulin-like growth factor receptor phosphorylation. Most tumors in each setting were estrogen receptor (ER)-positive (80% JH vs. 70% AH), but JH tumors were significantly more ER-immunoreactive (P = 0.0001) than AH tumors. A differential expression signature (JvA) of juvenile versus adult tumors revealed a luminal transcriptional program. Centroids of the human homologs of JvA genes showed that JH tumors were more like luminal A tumors and AH tumors were more like luminal B tumors. Hierarchical clustering with the JvA human ortholog gene list segregated luminal A and luminal B breast cancers across datasets. These data support the notion that age-associated host physiology greatly influences the intrinsic subtype of breast cancer. Cancer Res; 74(23); 7149–58. ©2014 AACR.

Background Multiple risk factors have been linked to venous thromboembolism (VTE). It is well established that elevated level of homocysteine correlated with increased risk of VTE. Some studies showed that vitamin B12 deficiency may be the cause for homocysteinemia. Therefore, it is recommended by some groups that vitamin B12 should be supplemented in the patient with high homocysteine to prevent VTE. However, more recent study has shown that there is correlation between the high serum Vitamin B12 level and risk of VTE in cancer patients.[1] provoked VTE after orthopedic surgery was also shown to be associated with elevated vitamin B12 serum levels in elderly patients. The role of vitamin B12 in VTE patients is not fully understood. Our study is to see whether there is correlation between serum Vitamin B12 level and risk of VTE in general population. Methods 177 Patients admitted to Kingsbrook Jewish Medical Center between January 1st, 2015 to December 31st, 2018 with the diagnosis of VTE were identified. 95 cases had measured serum Vitamin B12 results within 3 months. 97 patients without any history of VTE who were seen in the hematology clinic during the same period time were used as control in this study. Demographic information and Vitamin B12 level were collected. T test and chi-square were used for statistical analysis. Results VTE patients had a median age of 70, which is significantly higher than the median age of 57 in the control group. The mean level of vitamin B12 in control group was 620 pg/mL, which was lower than the average vitamin B12 level (770 pg/mL, p=0.0344) in the VTE group. When we divided the patients into 3 groups based on Vitamin B12 level: Low B12 (800 pg/mL). We observed 41% of VTE patients have B12 level more than 800 pg/mL while only 20% of control patients have high B12 level (chi-square, p=0.006). Discussion Our study showed that vitamin B12 level is significantly elevated in patients diagnosed with VTE and more than 40% of those patients actually have vitamin b12 level more than 800 pg/mL. It is known that elevated vitamin B12 level is associated with inflammation and increased mortality.[2] An association between elevated B12 levels and cancer has been reported in several studies [3]. It is unclear elevated vitamin B12 levels predict undiagnosed cancer which is a known risk factor for VTE or vitamin B12 directly contribute hypercoagulation. Nevertheless, we should be cautious when supplementing vitamin B12 and the dosage may need to be titrated closely. More clinical studies are definitely warranted. References 1. Arendt JFH, el al. Elevated plasma vitamin B12 levels and risk of venous thromboembolism among cancer patients: A population-based cohort study. PubMed ID: 26724465 2. Grossfeld A1, et al. Symptomatic venous thromboembolism in elderly patients following major orthopedic surgery of the lower limb is associated with elevated vitamin B12 serum levels. PubMed ID:23000313 3. Johan F.H. Arendt, Henrik T. Sørensen, Laura J. Horsfall and Irene Petersen. Elevated Vitamin B12 Levels and Cancer Risk in UK Primary Care: A THIN Database Cohort Study. EPI-17-1136 Published April 2019 Disclosures No relevant conflicts of interest to declare.

Background Rivaroxaban and apixaban are two direct oral anticoagulants (DOACs) targeting Factor Xa. Each DOAC was separately proven effective and safe when compared to standard treatment (heparin followed by warfarin) in patients diagnosed with venous thromboembolism (VTE). Several retrospective cohort analyses suggest apixaban may be superior to rivaroxaban due to less bleeding rates. One recent study showed the safety of apixaban and rivaroxaban for acute VTE were comparable. Also, long-term anticoagulation with low molecular weight heparin (LMWH), has never been directly compared with Factor Xa inhibitors. Given the patient population at our facility includes a significant percentage of elderly from nursing homes with multiple comorbidities and significantly higher bleeding risks than the general population, we aimed to evaluate the local real-world DOACs and heparin use with particular focus on safety. Methods A retrospective study was conducted at Kingsbrook Jewish Medical Center. Demographics, relevant laboratory/ imaging studies for patients admitted from 1/2016 to 12/2018 with the diagnosis of VTE based on the ICD 9/10 codes were collected from the IT dept. VTE patients who had bleeding events during the same admission for VTE or were admitted for relevant bleeding events based on ICD 9/10 codes within 6 months from the diagnosis of VTE were identified. Major bleeding events were defined as requiring hospitalization, blood transfusion or a significant drop in hemoglobin (more than 2 g/dl). The rest of the bleeding events were classified as minor. Demographics and clinical characteristics were summarized with means/median for continuous variables and with proportions for categorical variables. The differences in covaries were assessed with chi-square, Fisher exact test or t-tests. Results A total of 177 acute VTE patients were identified in the study. 37.9% (n=67) and 32.8% (n=58) patients were started on rivaroxaban and apixaban, respectively, as monotherapy. 29.3% (n=52) patients were given unfractionated heparin (23%, n=12) or LMWH (77%, n=40) based on the renal function. The bleeding rate in the apixaban group (4/58, 6.9%) was slightly higher than that in the rivaroxaban group (3/67, 4.5%), however, there was no statistical significance. Compared to patients received heparin (11/52, 21.2%), patients started on rivaroxaban (p=0.008) or apixaban (p=0.049) had a significantly lower rate of bleeding. Of note, majority of the bleeding event (16/18) was captured during the same admission when VTE was diagnosed. VTE patients in the apixaban group were older (p = 0.007) and had a longer length of stay (LOS, p= 0.024) compared to the ones in the rivaroxaban group. We then combined rivaroxaban and apixaban into DOAC group. Compared to Heparin group (n=11, 21.1%), the bleeding events in DOAC group (n=7, 5.6%) were significantly less (P=0.0045). Heparin group (n=21, 40.4%) included more patients with cancer than the DOAC group (n=9, 7.2%, p We then looked at the bleeding risk in non-cancer patients. Similarly, we didn't observe any superiority between rivaroxaban (n=2, 3.1%) and apixaban (n=3, 5.8%) regarding bleeding events in non-cancer patients with acute VTE. The average LOS in the apixaban group was significantly longer than that in the rivaroxaban group with non-cancer patients. It may be associated with relatively older age in the apixaban group compared to that in the rivaroxaban group. Most importantly, in patients without active malignancy, we found that the bleeding rate in the DOAC group was only 4.3% while the heparin group had a much high bleeding rate of 16.1% (p=0.035). Discussion Our study suggested that the safety of apixaban and rivaroxaban are comparable in VTE patients. In contrast, heparin including LMWH had much higher bleeding risk compared to either DOAC, especially in the beginning. For patients who are hospitalized for acute VTE, heparin intravenously or subcutaneously are usually initiated while the decision for oral anticoagulants are still pending. However, the benefits of such "bridging with heparin" strategy are not warranted, given the high bleeding risk associated with heparin treatment as shown in our study. Except for hemodynamically instability, excessive burden or clots, or impeding procedures, we recommend that DOACs should be used as monotherapy in VTE patients. Disclosures No relevant conflicts of interest to declare.

Densely ionizing radiation, which is present in the space radiation environment and used in radiation oncology, has potentially greater carcinogenic effect compared with sparsely ionizing radiation that is prevalent on earth. Here, we used a radiation chimera in which mice were exposed to densely ionizing 350 MeV/amu Si-particles, γ-radiation, or sham-irradiated and transplanted 3 days later with syngeneic Trp53-null mammary fragments. Trp53-null tumors arising in mice irradiated with Si-particles had a shorter median time to appearance and grew faster once detected compared with those in sham-irradiated or γ-irradiated mice. Tumors were further classified by markers keratin 8/18 (K18, KRT18), keratin 14 (K14, KRT14) and estrogen receptor (ER, ESR1), and expression profiling. Most tumors arising in sham-irradiated hosts were comprised of both K18- and K14-positive cells (K14/18) while those tumors arising in irradiated hosts were mostly K18. Keratin staining was significantly associated with ER status: K14/18 tumors were predominantly ER-positive, whereas K18 tumors were predominantly ER-negative. Genes differentially expressed in K18 tumors compared with K14/18 tumor were associated with ERBB2 and KRAS, metastasis, and loss of E-cadherin. Consistent with this, K18 tumors tended to grow faster and be more metastatic than K14/18 tumors, however, K18 tumors in particle-irradiated mice grew significantly larger and were more metastatic compared with sham-irradiated mice. An expression profile that distinguished K18 tumors arising in particle-irradiated mice compared with sham-irradiated mice was enriched in mammary stem cell, stroma, and Notch signaling genes. These data suggest that carcinogenic effects of densely ionizing radiation are mediated by the microenvironment, which elicits more aggressive tumors compared with similar tumors arising in sham-irradiated hosts. Cancer Res; 74(23); 7137–48. ©2014 AACR.

This review focuses on recent experimental and modeling studies that attempt to define the physiological context in which high linear energy transfer (LET) radiation increases epithelial cancer risk and the efficiency with which it does so. Radiation carcinogenesis is a two-compartment problem: ionizing radiation can alter genomic sequence as a result of damage due to targeted effects (TE) from the interaction of energy and DNA; it can also alter phenotype and multicellular interactions that contribute to cancer by poorly understood non-targeted effects (NTE). Rather than being secondary to DNA damage and mutations that can initiate cancer, radiation NTE create the critical context in which to promote cancer. Systems biology modeling using comprehensive experimental data that integrates different levels of biological organization and time-scales is a means of identifying the key processes underlying the carcinogenic potential of high-LET radiation. We hypothesize that inflammation is a key process, and thus cancer susceptibility will depend on specific genetic predisposition to the type and duration of this response. Systems genetics using novel mouse models can be used to identify such determinants of susceptibility to cancer in radiation sensitive tissues following high-LET radiation. Improved understanding of radiation carcinogenesis achieved by defining the relative contribution of NTE carcinogenic effects and identifying the genetic determinants of the high-LET cancer susceptibility will help reduce uncertainties in radiation risk assessment.

MicroRNAs (miRNAs) regulate gene expression. It has been suggested that obtaining miRNA expression profiles can improve classification, diagnostic, and prognostic information in oncology. Here, we sought to comprehensively identify the miRNAs that are overexpressed in lung cancer by conducting miRNA microarray expression profiling on normal lung versus adjacent lung cancers from transgenic mice. We found that miR-136, miR-376a, and miR-31 were each prominently overexpressed in murine lung cancers. Real-time RT-PCR and in situ hybridization (ISH) assays confirmed these miRNA expression profiles in paired normal-malignant lung tissues from mice and humans. Engineered knockdown of miR-31, but not other highlighted miRNAs, substantially repressed lung cancer cell growth and tumorigenicity in a dose-dependent manner. Using a bioinformatics approach, we identified miR-31 target mRNAs and independently confirmed them as direct targets in human and mouse lung cancer cell lines. These targets included the tumor-suppressive genes large tumor suppressor 2 (LATS2) and PP2A regulatory subunit B alpha isoform (PPP2R2A), and expression of each was augmented by miR-31 knockdown. Their engineered repression antagonized miR-31-mediated growth inhibition. Notably, miR-31 and these target mRNAs were inversely expressed in mouse and human lung cancers, underscoring their biologic relevance. The clinical relevance of miR-31 expression was further independently and comprehensively validated using an array containing normal and malignant human lung tissues. Together, these findings revealed that miR-31 acts as an oncogenic miRNA (oncomir) in lung cancer by targeting specific tumor suppressors for repression.

Purpose: High-throughput profiling experiments have linked altered expression of microRNAs (miRNA) to different types of cancer. Tumor tissues are a heterogeneous mixture of not only cancer cells, but also supportive and reactive tumor microenvironment elements. To clarify the clinical significance of altered miRNA expression in solid tumors, we developed a sensitive fluorescence-based in situ hybridization (ISH) method to visualize miRNA accumulation within individual cells in formalin-fixed, paraffin-embedded tissue specimens. This ISH method was implemented to be compatible with routine clinical immunohistochemical (IHC) assays to enable the detection of miRNAs and protein markers in the same tissue section for colocalization and functional studies. Experimental Design: We used this combined ISH/IHC assay to study a subset of cancer-associated miRNAs, including miRNAs frequently detected at low (miR-34a and miR-126) and high (miR-21 and miR-155) levels, in a panel of breast, colorectal, lung, pancreas, and prostate carcinomas. Results: Despite the distinct histopathologic alterations of each particular cancer type, general trends emerged that pinpointed distinct source cells of altered miRNA expression. Although altered expressions of miR-21 and miR-34a were manifested within cancer cells, those of miR-126 and miR-155 were predominantly confined to endothelial cells and immune cells, respectively. These results suggest a heterogeneous participation of miRNAs in carcinogenesis by intrinsically affecting cancer cell biology or by modulating stromal, vascular, and immune responses. Conclusions: We described a rapid and sensitive multicolor ISH/IHC assay and showed that it could be broadly applied as an investigational tool to better understand the etiologic relevance of altered miRNA expression in cancer. Clin Cancer Res; 16(16); 4246–55. ©2010 AACR.

Pancreatic ductal carcinoma (PDAC) is the fourth leading cause of cancer-related death in Unites States. Due to its aggressive local invasion and metastatic nature, the median survival for patients with PDAC is only 6-month. There is therefore an urgent need to improve our understanding of the pathobiology of this malignancy. In this regard, microRNAs (miRs) have emerged as potentially important regulators in PDAC progression by acting on multiple signaling pathways involved in proliferation, apoptosis, invasiveness and metastasis. MiR-10b is an important RNA implicated in the facilitation of cancer cell invasion and metastasis in several cancers. We have previously shown that miR-10b is upregulated in pancreatic cancer cells, and that high levels of miR-10b are associated with decreased time to metastasis following therapy, enhanced resistance to gemcitabine, and decreased overall survival. In the present study, we have sought to characterize the mechanisms of action of miR-10b in PDAC. We found that overexpression of miR-10b promoted migration and invasion of PANC-1 cells in vitro, without enhancing their proliferation. Microarray analysis revealed that TIP30, a tumor and metastasis suppressor, was downregulated by miR-10b overexpression. Validation experiments using luciferase readout assays showed that TIP30 is a direct target of miR-10b. Taken together, these observations suggest that miR-10b plays an important role in PDAC and may be a novel therapeutic target in this deadly malignancy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 177. doi:1538-7445.AM2012-177

MicroRNAs (miRNAs) encode small RNAs that regulate gene expression. The expression profiles of miRNAs have proven useful to improve classification, diagnostic and prognostic information for human cancers. We sought to uncover miRNAs preferentially over-expressed in lung cancers versus adjacent normal lung tissues. We comprehensively interrogated miRNA array expression profiles conducted on lung cancers versus adjacent normal lung tissues isolated from murine transgenic cyclin E models that were engineered. Notably, miR-136, miR-376a and miR-31 were each significantly over-expressed in murine lung cancers as compared to adjacent normal lung tissues. Real-time RT-PCR and in situ hybridization assays independently confirmed these miRNA expression profiles in paired normal-malignant lung tissues from transgenic mice as well as from a well characterized panel of paired human normal-malignant lung tissues. Knock-down of miR-31, but not other highlighted miRNAs, using a synthetic antagonist significantly repressed lung cancer cell growth and in vivo tumorigenicity. Bioinformatically predicted miR-31 target genes were identified and studied. These included two tumor suppressors: large tumor suppressor 2 (LATS2) and PP2A regulatory subunit B alpha isoform (PPP2R2A) that were each substantially increased by miR-31 knock-down and repressed by miR-31 over-expression. Engineered repression of either LATS2 or PPP2R2A significantly antagonized miR-31-mediated growth inhibition. Luciferase binding assays confirmed that miR-31 directly suppressed LATS2 and PPP2R2A through binding to their respective 3′-untranslated regions (UTRs). Interestingly, miR-31 was inversely expressed relative to these identified target genes in both murine and human lung cancers. Clinical relevance was uncovered using miR-31 in situ hybridization and cyclin E immunohistochemistry assays performed on tissue arrays from the population-based New Hampshire Lung Cancer Registry. As expected, cyclin E expression was significantly associated with miR-31 levels. Real-time RT-PCR assays validated LATS2 and PPP2R2A repression in lung cancers versus adjacent normal lung tissues. Thus, these findings implicate miR-31 as an oncogenic miRNA (oncomir) that exerts its effects by repressing specific tumor suppressors. Taken together, miR-31 is proposed as a novel molecular target for lung cancer therapy and chemoprevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5698.

MicroRNAs (miRNAs) are a class of short non-coding regulatory RNA genes, which have been implicated in the control of developmental, physiological and pathological processes. High-throughput profiling experiments have linked altered expression of miRNAs to different types of cancer. To clarify the clinical significance of these expression changes, we developed a sensitive fluorescence-based in situ hybridization (ISH) method to visualize miRNA accumulation within individual cells in formalin-fixed paraffin-embedded tissues. This ISH method was implemented to be compatible with routine automated clinical immunohistochemical (IHC) assays to enable co-detection with protein markers on the same tissue section for co-localization and functional studies. Here, we utilized this robust and rapid combined ISH/IHC assay to study a subset of commonly highlighted miRNAs including tumor suppressive miRNAs (let-7 and miR-34) and oncogenic miRNAs (miR-21 and miR-155) in a panel of breast, colon, lung, pancreas and prostate carcinomas, which collectively are the leading causes of cancer-related mortality in the United States. Despite the idiosyncrasies of each particular cancer type, general trends emerged that pinpointed to distinct cellular sources of altered miRNA expression. While altered expression of miR-21 and miR-34 was manifested within cancer cells, that of miR-126 and miR-155 was predominantly confined to endothelial cells and immune cells, respectively. These findings suggest a heterogeneous and complex participation of miRNAs in carcinogenesis occurring through cancer cell-autonomous effects as well as through the modulation of stromal, vascular and immune responses. We discuss potential applications of miRNA-based ISH/IHC assays as novel diagnostic and prognostic tools, as well as clinical implications of our findings for miRNA-based therapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-360.