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Brain metastasis is a significant cause of morbidity and mortality in multiple cancer types and represents an unmet clinical need. The mechanisms that mediate metastatic cancer growth in the brain parenchyma are largely unknown. Melanoma, which has the highest rate of brain metastasis among common cancer types, is an ideal model to study how cancer cells adapt to the brain parenchyma. Our unbiased proteomics analysis of melanoma short-term cultures revealed that proteins implicated in neurodegenerative pathologies are differentially expressed in melanoma cells explanted from brain metastases compared with those derived from extracranial metastases. We showed that melanoma cells require amyloid beta (Aβ) for growth and survival in the brain parenchyma. Melanoma-secreted Aβ activates surrounding astrocytes to a prometastatic, anti-inflammatory phenotype and prevents phagocytosis of melanoma by microglia. Finally, we demonstrate that pharmacologic inhibition of Aβ decreases brain metastatic burden.Significance:Our results reveal a novel mechanistic connection between brain metastasis and Alzheimer's disease, two previously unrelated pathologies; establish Aβ as a promising therapeutic target for brain metastasis; and demonstrate suppression of neuroinflammation as a critical feature of metastatic adaptation to the brain parenchyma.This article is highlighted in the In This Issue feature, p. 1171

Supplementary Table from Melanoma-Secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis

Supplementary Data from Melanoma-Secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis

Supplementary Figure from Melanoma-Secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis

There is considerable interest in the clinical development of inhibitors of mTOR complexes mTORC1 and 2. Because mTORC1 and its downstream mRNA translation effectors may protect against genotoxic DNA damage, we investigated the inhibition of mTORC1 and mTORC1/2 in the ability to reverse platinum resistance in tissue culture and in animal tumor models of serous ovarian cancer. Cell survival, tumor growth, PI3K–AKT–mTOR pathway signaling, DNA damage and repair response (DDR) gene expression, and translational control were all investigated. We show that platinum-resistant OVCAR-3 ovarian cancer cells are resensitized to low levels of carboplatin in culture by mTOR inhibition, demonstrating reduced survival after treatment with either mTORC1 inhibitor everolimus or mTORC1/2 inhibitor PP242. Platinum resistance is shown to be associated with activating phosphorylation of AKT and CHK1, inactivating phosphorylation of 4E-BP1, the negative regulator of eIF4E, which promotes increased cap-dependent mRNA translation and increased levels of CHK1 and BRCA1 proteins. Animals with platinum-resistant OVCAR-3 tumors treated with carboplatin plus mTORC1/2 inhibition had significantly longer median survival and strikingly reduced metastasis compared with animals treated with carboplatin plus everolimus, which inhibits only mTORC1. Reduced tumor growth, metastasis, and increased survival by mTORC1/2 inhibition with carboplatin treatment was associated with reduced AKT-activating phosphorylation and increased 4E-BP1 hypophosphorylation (activation). We conclude that mTORC1/2 inhibition is superior to mTORC1 inhibition in reversing platinum resistance in tumors and strongly impairs AKT activation, DNA repair responses, and translation, promoting improved survival in the background of platinum resistance. Mol Cancer Ther; 15(7); 1557–67. ©2016 AACR.

Supplementary Figure S1: Growth inhibitory 50% (GI50) determination of single agent everolimus, PP242 and carboplatin using OVCAR-3 cells; Supplementary Figure S2: mTORC1 and mTORC1/2 inhibition sensitizes platinum resistant SKOV3 cells to carboplatin-mediated growth inhibition; Supplementary Figure S3: qRT-PCR analysis of select mRNAs in mTORC1 and mTORC1/2 inhibited cells with and without carboplatin treatment.

Brain metastasis is a significant cause of morbidity and mortality in multiple cancer types and represents an unmet clinical need. The mechanisms that mediate metastatic cancer growth in the brain parenchyma are largely unknown. Melanoma, which has the highest rate of brain metastasis among common cancer types, is an ideal model to study how cancer cells adapt to the brain parenchyma. Our unbiased proteomics analysis of melanoma short-term cultures revealed that proteins implicated in neurodegenerative pathologies are differentially expressed in melanoma cells explanted from brain metastases compared with those derived from extracranial metastases. We showed that melanoma cells require amyloid beta (Aβ) for growth and survival in the brain parenchyma. Melanoma-secreted Aβ activates surrounding astrocytes to a prometastatic, anti-inflammatory phenotype and prevents phagocytosis of melanoma by microglia. Finally, we demonstrate that pharmacologic inhibition of Aβ decreases brain metastatic burden. Significance: Our results reveal a novel mechanistic connection between brain metastasis and Alzheimer's disease, two previously unrelated pathologies; establish Aβ as a promising therapeutic target for brain metastasis; and demonstrate suppression of neuroinflammation as a critical feature of metastatic adaptation to the brain parenchyma. This article is highlighted in the In This Issue feature, p. 1171

Supplementary Materials and Methods for additional procedures

Inflammatory breast cancer (IBC) is a highly aggressive form of breast cancer that displays profound cancer stem cell (CSC) and mesenchymal features that promote rapid metastasis. Another hallmark of IBC is high infiltration of M2 tumor-associated (immune-suppressing) macrophages. The molecular mechanism that drives these IBC phenotypes is not well understood. Using patient breast tumor specimens, breast cancer cell lines, and a patient-derived xenograft model of IBC, we demonstrate that IBC strongly expresses IL8 and growth-regulated oncogene (GRO) chemokines that activate STAT3, which promotes development of high levels of CSC-like cells and a mesenchymal phenotype. We also show that IBC expresses high levels of many monocyte recruitment and macrophage polarization factors that attract and differentiate monocytes into tumor-promoting, immune-suppressing M2-like macrophages. The M2 macrophages in turn were found to secrete high levels of IL8 and GRO chemokines, thereby creating a feed-forward chemokine loop that further drives an IBC epithelial-to-mesenchymal transition. Our study uncovers an intricate IBC-initiated autocrine–paracrine signaling network between IBC cells and monocytes that facilitates development of this highly aggressive form of breast cancer.Significance:This study uncovers a signaling network in which IBC cells commandeer macrophages to become tumor-promoting, and they in turn drive IBC cells to be more cancer stem-like, mesenchymal, and aggressive.

Figure S5. VEGF-D 5'UTR mRNA exhibits two structural forms that allow the binding of different proteins

Figure S2. Expression of endogenous VEGF-D in mice tissues

Figure S7. Tumor growth after NSAID treatment

The vascular endothelial growth factor VEGF-D promotes metastasis by inducing lymphangiogenesis and dilatation of the lymphatic vasculature, facilitating tumor cell extravasion. Here we report a novel level of control for VEGF-D expression at the level of protein translation. In human tumor cells, VEGF-D colocalized with eIF4GI and 4E-BP1, which can program increased initiation at IRES motifs on mRNA by the translational initiation complex. In murine tumors, the steady-state level of VEGF-D protein was increased despite the overexpression and dephosphorylation of 4E-BP1, which downregulates protein synthesis, suggesting the presence of an internal ribosome entry site (IRES) in the 5′ UTR of VEGF-D mRNA. We found that nucleolin, a nucleolar protein involved in ribosomal maturation, bound directly to the 5′UTR of VEGF-D mRNA, thereby improving its translation following heat shock stress via IRES activation. Nucleolin blockade by RNAi-mediated silencing or pharmacologic inhibition reduced VEGF-D translation along with a subsequent constriction of lymphatic vessels in tumors. Our results identify nucleolin as a key regulator of VEGF-D expression, deepening understanding of lymphangiogenesis control during tumor formation. Cancer Res; 76(15); 4394–405. ©2016 AACR.

Figure S8. Schematic representation of VEGF-D synthesis in tumor cells

Supplementary Data from Identification of Markers of Taxane Sensitivity Using Proteomic and Genomic Analyses of Breast Tumors from Patients Receiving Neoadjuvant Paclitaxel and Radiation

Supplementary figure legend

FS1: Four panels (A-D) demonstrating that SUM190 cells are enriched in cancer stem-like cells, shown by immunoblot analysis, cell flow cytometry and by increased cell invasion. FS2: Three panels (A-C) showing that co-culture of IBC cells with monocytes-macrophages increases IBC cell mesenchymal characteristics, shown by immunoblot analysis and invasion assays. FS3: Three immunoblot panels showing that addition of IL8 and GRO cytokines to the media of IBC cells promotes phosphorylation of STAT3 and cancer stem cell-like and mesenchymal characteristics. FS4: Four panels showing that IBC cells secrete attracting and type II polarization factors for monocytes and macrophages. Shown by function assays, cytokine sandwich blot assays, flow cytometry and immunohistochemistry.

TS1 describes the characteristics of the cell lines used in this study, including hormone receptor status and type of breast cancer. TS2 lists the antibodies used and the method by which immunohistochemistry stained breast cancer specimens were scored. TS3 describes the levels of IL8, Jak2, P-Ja2, STAT3, P-STAT3 and monocyte macrophages (CD68, CD163) in normal breast tissue and IBC specimens. TS4 lists the expression levels of 80 cytokines, chemokines and growth factors in the conditioned media from SUM149 cells. Data were obtained from at least three independent studies. TS5 lists the sequences of siRNAs that were used for gene silencing in this study. TS6 lists the primer sequences and the genes targeted for qPCR studies carried out.

Figure S6. Polysome profiling of 4T1 cells

Figure S4. Lymphangiogenesis and tumor gowth are not affected by 4T1 or 67NR lentiviral transduction

Purpose: To identify molecular markers of pathologic response to neoadjuvant paclitaxel/radiation treatment, protein and gene expression profiling were done on pretreatment biopsies.Experimental Design: Patients with high-risk, operable breast cancer were treated with three cycles of paclitaxel followed by concurrent paclitaxel/radiation. Tumor tissue from pretreatment biopsies was obtained from 19 of the 38 patients enrolled in the study. Protein and gene expression profiling were done on serial sections of the biopsies from patients that achieved a pathologic complete response (pCR) and compared to those with residual disease, non-pCR (NR).Results: Proteomic and validation immunohistochemical analyses revealed that α-defensins (DEFA) were overexpressed in tumors from patients with a pCR. Gene expression analysis revealed that MAP2, a microtubule-associated protein, had significantly higher levels of expression in patients achieving a pCR. Elevation of MAP2 in breast cancer cell lines led to increased paclitaxel sensitivity. Furthermore, expression of genes that are associated with the basal-like, triple-negative phenotype were enriched in tumors from patients with a pCR. Analysis of a larger panel of tumors from patients receiving presurgical taxane-based treatment showed that DEFA and MAP2 expression as well as histologic features of inflammation were all statistically associated with response to therapy at the time of surgery.Conclusion: We show the utility of molecular profiling of pretreatment biopsies to discover markers of response. Our results suggest the potential use of immune signaling molecules such as DEFA as well as MAP2, a microtubule-associated protein, as tumor markers that associate with response to neoadjuvant taxane–based therapy. Clin Cancer Res; 16(2); 681–90

Figure S3. Podoplanin staining of lymphatic vessels

p23 is a heat shock protein 90 (Hsp90) cochaperone located in both the cytoplasm and nucleus that stabilizes unliganded steroid receptors, controls the catalytic activity of certain kinases, regulates protein-DNA dynamics, and is upregulated in several cancers. We had previously shown that p23-overexpressing MCF-7 cells (MCF-7+p23) exhibit increased invasion without affecting the estrogen-dependent proliferative response, which suggests that p23 differentially regulates genes controlling processes linked to breast tumor metastasis. To gain a comprehensive view of the effects of p23 on estrogen receptor (ER)-dependent and -independent gene expression, we profiled mRNA expression from control versus MCF-7+p23 cells in the absence and presence of estrogen. A number of p23-sensitive target genes involved in metastasis and drug resistance were identified. Most striking is that many of these genes are also misregulated in invasive breast cancers, including PMP22, ABCC3, AGR2, Sox3, TM4SF1, and p8 (NUPR1). Upregulation of the ATP-dependent transporter ABCC3 by p23 conferred resistance to the chemotherapeutic agents etoposide and doxorubicin in MCF-7+p23 cells. MCF-7+p23 cells also displayed higher levels of activated Akt and an expanded phosphoproteome relative to control cells, suggesting that elevated p23 also enhances cytoplasmic signaling pathways. For breast cancer patients, tumor stage together with high cytoplasmic p23 expression more accurately predicted disease recurrence and mortality than did stage alone. High nuclear p23 was found to be associated with high cytoplasmic p23, therefore both may promote tumor progression and poor prognosis by increasing metastatic potential and drug resistance in breast cancer patients. Cancer Res; 70(21); 8446–56. ©2010 AACR.

Supplementary Figure 2 - PDF file 19K, S2. Plating efficiency of MB-435 cells under hypoxia on plastic or vitronectin with beta3 silencing

Supplementary Figures 1-8, Tables 1-7 from High Levels of Hsp90 Cochaperone p23 Promote Tumor Progression and Poor Prognosis in Breast Cancer by Increasing Lymph Node Metastases and Drug Resistance

Supplementary Figure Legends - PDF file 59K, Figure legends for Sup. Figures 1 and 2

Supplementary Figure 1 - PDF file 20K, S1. Plating efficiency of MB-435 and MB-231 cells on different ECM matrices

Spurred by the lack of progress in improving outcomes in patients with inflammatory breast cancer (IBC), a deadly and aggressive type of breast cancer, Susan G. Komen, the Inflammatory Breast Cancer Research Foundation, and the Milburn Foundation convened patient advocates, breast cancer researchers, and clinicians to review the state of IBC care and research globally, and propose specific initiatives to move the field forward. The outcome of the discussions was abundantly clear: the field needs a formal definition of IBC. Without a clear definition, both patient care and research suffer. Diagnosis remains subjective and treatment variable. Clinical trials become inherently underpowered by inclusion of subjects with locally advanced, but not inflammatory, breast cancer. Similarly, the search for a molecular sine qua non or unifying pathway aberration is hindered by inclusion of bio-specimens from subjects with a different disease. The goal is to move beyond the subjective ‘clinical diagnosis’ to a set of specific diagnostic criteria and scoring system that will advance IBC research and facilitate the discoveries that will improve care of IBC patients. Here we propose a definition of IBC, based on a review of clinical, and pathologic features. Table 1. Proposed Scoring System for inflammatory breast cancer diagnosisPriority FactorCharacteristicScore3213Timing of signs/symptoms≤ 3 months3-6 months> 6 months3Skin changesPeau d’orangeSkin edema/thickening (≥ 1/3 of the breast)Focal skin edema/thickening (< 1/3 of the breast)3Swelling/engorgement of the breastClinically apparent enlargement of the breast or new asymmetry in breast sizeBreast edema identified on imaging but not clinically detectable2Erythema or other skin discoloration: pink, red, darkened, bruising/purplish or serpiginous in characterNearly complete involvement of the breastPartial involvement of the breastMinimal involvement or ambiguous color change2Nipple abnormalitiesNew nipple inversionNew nipple flattening or other asymmetryCrusting of the nipple/areola without other nipple changes2Lymphatic emboliDermal lymphatic emboli present without evidence of direct involvement of the dermis or epidermisNon-dermal lymphatic embolic present in breast parenchyma or stroma1Breast imagingDiffuse involvement of breast parenchyma, with or without dominant massEnlargement of non-axillary nodes (internal mammary, supraclavicular, subpectoral etc.) With increased awareness of IBC, patients are coming to medical attention earlier when clinical characteristics may not be as profound as historically depicted. Not all criterion are required for a diagnosis. To determine the total score, multiply the priority factor by the score for each criteria, then add the subtotals for each criteria. A priority factor is used to weigh criterion more heavily that are key to a consistent diagnosis of IBC to better define breast cancers that are ambiguous. While we cannot currently recommend a molecular-genetic profile that unambiguously identifies IBC, we will also provide a panel of prioritized biomarkers that are strongly associated with IBC and deserve further study in cohorts meeting our diagnostic criteria. We acknowledge that our proposed scoring system requires validation and refinement. If accurate, we expect patients with IBC based on these criteria to have a different clinical course and outcome compared to patients with “non-inflammatory” locally advanced breast cancer (LABC). Ultimately, we envision using the final disease classification and scoring system to develop a staging system specific to IBC. The discovery and validation of a distinct molecular or genetic profile that identifies IBC is of utmost importance to advancing research in this disease and cannot be accomplished without a clear and validated definition of IBC. Citation Format: Ginny Mason, Beth A Overmoyer, Wendy A. Woodward, Sunil Badve, Robert J. Schneider, Reshma Jagsi, Julie E. Lang, Mary Alpaugh, Krissa Smith, Kathy Miller. Inflammatory breast cancer (IBC) defined: Proposed common diagnostic criteria and scoring - Moving beyond the subjective ‘clinical diagnosis’ of IBC to advance research [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 P6-15-03.

Inflammatory breast cancer (IBC) is a highly aggressive form of breast cancer that displays profound cancer stem cell (CSC) and mesenchymal features that promote rapid metastasis. Another hallmark of IBC is high infiltration of M2 tumor-associated (immune-suppressing) macrophages. The molecular mechanism that drives these IBC phenotypes is not well understood. Using patient breast tumor specimens, breast cancer cell lines, and a patient-derived xenograft model of IBC, we demonstrate that IBC strongly expresses IL8 and growth-regulated oncogene (GRO) chemokines that activate STAT3, which promotes development of high levels of CSC-like cells and a mesenchymal phenotype. We also show that IBC expresses high levels of many monocyte recruitment and macrophage polarization factors that attract and differentiate monocytes into tumor-promoting, immune-suppressing M2-like macrophages. The M2 macrophages in turn were found to secrete high levels of IL8 and GRO chemokines, thereby creating a feed-forward chemokine loop that further drives an IBC epithelial-to-mesenchymal transition. Our study uncovers an intricate IBC-initiated autocrine–paracrine signaling network between IBC cells and monocytes that facilitates development of this highly aggressive form of breast cancer. Significance: This study uncovers a signaling network in which IBC cells commandeer macrophages to become tumor-promoting, and they in turn drive IBC cells to be more cancer stem-like, mesenchymal, and aggressive.

Brain metastasis is a significant cause of morbidity and mortality in multiple cancer types and represents an unmet clinical need. The mechanisms that mediate metastatic cancer growth in the brain parenchyma are largely unknown. Melanoma, which has the highest rate of brain metastasis among common cancer types, is an ideal model to study how cancer cells adapt to the brain parenchyma. Our unbiased proteomics analysis of melanoma short-term cultures revealed that proteins implicated in neurodegenerative pathologies are differentially expressed in melanoma cells explanted from brain metastases compared to those derived from extracranial metastases. We showed that melanoma cells require amyloid beta (Aβ) for growth and survival in the brain parenchyma. Melanoma-secreted Aβ activates surrounding astrocytes to a prometastatic, anti-inflammatory phenotype and prevents phagocytosis of melanoma by microglia. Finally, we demonstrate that pharmacological inhibition of Aβ decreases brain metastatic burden. Our results reveal a novel mechanistic connection between brain metastasis and Alzheimer’s disease - two previously unrelated pathologies, establish Aβ as a promising therapeutic target for brain metastasis, and demonstrate suppression of neuroinflammation as a critical feature of metastatic adaptation to the brain parenchyma. Citation Format: Kevin Kleffman, Grace Levinson, Indigo V. Rose, Lili Blumenberg, Sorin A. Shadaloey, Avantika Dhabaria, Eitan Wong, Francisco Galán-Echevarría, Alcida Karz, Diana Argibay, Richard Von-Itter, Alfredo Floristán, Gillian Baptiste, Nicole Eskow, James Tranos, Jenny Chen, Eleazar C. Vega Saenz de Miera, Melissa Call, Robert Rogers, George Jour, Youssef Zaim Wadghiri, Iman Osman, Yue Ming Li, Paul Mathews, Ronald Demattos, Beatrix Ueberheide, Kelly Ruggles, Shane A. Liddelow, Robert J. Schneider, Eva Hernando. Melanoma-secreted amyloid beta suppresses neuroinflammation and promotes brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB052.

This abstract was withdrawn by the authors.

The vascular endothelial growth factor VEGF-D promotes metastasis by inducing lymphangiogenesis and dilatation of the lymphatic vasculature, facilitating tumor cell extravasion. Here we report a novel level of control for VEGF-D expression at the level of protein translation. In human tumor cells, VEGF-D colocalized with eIF4GI and 4E-BP1, which can program increased initiation at IRES motifs on mRNA by the translational initiation complex. In murine tumors, the steady-state level of VEGF-D protein was increased despite the overexpression and dephosphorylation of 4E-BP1, which downregulates protein synthesis, suggesting the presence of an internal ribosome entry site (IRES) in the 5′ UTR of VEGF-D mRNA. We found that nucleolin, a nucleolar protein involved in ribosomal maturation, bound directly to the 5′UTR of VEGF-D mRNA, thereby improving its translation following heat shock stress via IRES activation. Nucleolin blockade by RNAi-mediated silencing or pharmacologic inhibition reduced VEGF-D translation along with a subsequent constriction of lymphatic vessels in tumors. Our results identify nucleolin as a key regulator of VEGF-D expression, deepening understanding of lymphangiogenesis control during tumor formation. Cancer Res; 76(15); 4394–405. ©2016 AACR.

There is considerable interest in the clinical development of inhibitors of mTOR complexes mTORC1 and 2. Because mTORC1 and its downstream mRNA translation effectors may protect against genotoxic DNA damage, we investigated the inhibition of mTORC1 and mTORC1/2 in the ability to reverse platinum resistance in tissue culture and in animal tumor models of serous ovarian cancer. Cell survival, tumor growth, PI3K–AKT–mTOR pathway signaling, DNA damage and repair response (DDR) gene expression, and translational control were all investigated. We show that platinum-resistant OVCAR-3 ovarian cancer cells are resensitized to low levels of carboplatin in culture by mTOR inhibition, demonstrating reduced survival after treatment with either mTORC1 inhibitor everolimus or mTORC1/2 inhibitor PP242. Platinum resistance is shown to be associated with activating phosphorylation of AKT and CHK1, inactivating phosphorylation of 4E-BP1, the negative regulator of eIF4E, which promotes increased cap-dependent mRNA translation and increased levels of CHK1 and BRCA1 proteins. Animals with platinum-resistant OVCAR-3 tumors treated with carboplatin plus mTORC1/2 inhibition had significantly longer median survival and strikingly reduced metastasis compared with animals treated with carboplatin plus everolimus, which inhibits only mTORC1. Reduced tumor growth, metastasis, and increased survival by mTORC1/2 inhibition with carboplatin treatment was associated with reduced AKT-activating phosphorylation and increased 4E-BP1 hypophosphorylation (activation). We conclude that mTORC1/2 inhibition is superior to mTORC1 inhibition in reversing platinum resistance in tumors and strongly impairs AKT activation, DNA repair responses, and translation, promoting improved survival in the background of platinum resistance. Mol Cancer Ther; 15(7); 1557–67. ©2016 AACR.

Regulatory T cells (Tregs) maintain immune system homeostasis in the organism. In the tumor microenvironment, however, Tregs can hinder the antitumor immune response. Treg maturation in peripheral sites (pTregs) requires inhibition of protein kinase mTORC1, which paradoxically blocks mRNA translation. mTORC1 phosphorylates (inactivates) 4E-BP1, a negative regulator of the translation initiation factor eIF4E. When mTORC1 is active, 4E-BP1 is hyperphosphorylated and releases eIF4E, which can then bind the mRNA cap structure and promote eIF4F complex assembly and ribosome recruitment. Inhibition of mTORC1 results in hypo-phosphorylation (activation) of 4E-BP1, which then binds and sequesters eIF4E, preventing its interaction with the cap and ribosome association to the mRNA. Using human CD4+ T cells differentiated in vitro, we show that mTORC1 inhibition strongly impairs protein synthesis, yet with exposure to TGFbeta is essential to mediate iTreg differentiation and immune suppression activity. We found that TGFbeta reprograms the T-cell transcriptome, while mTORC1 inhibition reprograms the translatome (genome-wide translation signature), mediating Treg differentiation. Genome-wide transcription and translation profiling identified TGFbeta-upregulated mRNAs resistant to mTORC1 inhibition, in part due to their 5'-untranslated regions, that induce Treg differentiation and immune-suppression activity. These include canonical pTreg fate-determining mRNAs FOXP3, CTLA-4, CD101 and CD103, among others. Inhibition of mTORC1 alone, or TGFbeta alone, induces iTregs with poor immune-suppression activity. Treg differentiation is therefore mediated by an mTORC1-independent privileged mRNA translation mechanism that converts activated CD4+ T cells to Tregs. These findings are quite relevant in the tumor microenvironment, where mTORC1-inhibiting conditions such as hypoxia and deprivation of nutrients and energy are present. Investigation of molecular targets acting in this mTORC1/eIF4E-independent translation of Treg mRNAs is ongoing. These targets could be drugged to selectively inhibit Treg differentiation in the tumor microenvironment, thus allowing effector cells to mount the response against cancer cells. Citation Format: Viviana Volta, Amanda Ernlund, Abhilash Gadi, Amanda Valeta-Magara, Columba de la Parra, Preeyam Patel, Robert J. Schneider. mTOR inhibition promotes differentiation of human regulatory T cells via privileged mRNA translation [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A26.

Acute myeloid leukemia (AML) is a clonal disorder of hematopoiesis originating in hematopoietic/stem progenitor cells characterized by impaired differentiation and the accumulation of immature blasts in the bone marrow. While genetic, transcriptional, and epigenetic alterations have been comprehensively characterized in human AML, few studies have assessed post-transcriptional and translational regulatory mechanisms in leukemic stem cells (LSCs). We previously identified a novel LSC marker, CD99, that enriches for LSCs among leukemic blasts. We now show that CD99high LSCs express low levels of transcripts encoding ribosomal proteins and exhibit reduced translation compared to non-LSC blasts as assessed by OP-Puro incorporation assays and sucrose gradient polysome profiling. This pattern recapitulates reductions in translation in normal hematopoietic stem cells (HSCs) compared to committed progenitors, suggesting common mechanisms regulating translation in normal and malignant hematopoietic cells. To examine global post-transcriptional and translational regulation in HSCs, we performed polysome profiling, RNA-sequencing and whole-proteomic analysis of HSC-enriched cells (Lin-Sca-1+c-Kit+; LSK) and committed myeloid progenitors (MPs; Lin-Sca-1-c-Kit+). While MP cells exhibit higher levels of global translation than LSK cells, LSK cells exhibit higher translational efficiencies (TEs) of transcripts required for HSC maintenance and self-renewal, and MP cells show increased TEs of genes associated with differentiation. Although HSC-enriched LSK cells exhibit lower levels of global translation than MP cells, they show activation of mTOR signaling and high TEs of mTOR dependent transcripts. In contrast, even though MP cells exhibit higher levels of translation than HSCs, Western blot analysis confirms that they lack significant mTOR protein expression or signaling. Indeed, mTOR expression in MP cells is rescued by proteasome inhibitors, and its expression is restored in the absence of the E3 ubiquitin ligase, c-Cbl, demonstrating its regulation by proteasomal degradation. Confirming that mTOR signaling is not required for MP function, mTOR inhibition with rapamycin in vivo or Torin1 ex vivo did not affect MP translation or differentiation. To determine if similar mTOR-independent mechanisms of translational activation are also present in malignant hematopoiesis, we analyzed CD99high LSCs and CD99low non-LSC AML blasts and observed higher mTOR signaling and lower global translation in LSCs, as well as near-absence of mTOR protein expression in non-LSCs, recapitulating the differences observed in LSK versus MP cells. Collectively, these findings establish the presence of developmental stage-specific mechanisms of translational regulation mediated by mTOR in both normal and malignant hematopoiesis. This abstract is also being presented as Poster B30. Citation Format: Christina C. Spevak, Harold K. Elias, Lavanya Kannan, Gaelle Martin, Shanmugapriya Selvaraj, William S. Eng, Amanda Ernlund, Vinagolu K. Rajasekhar, Carolien M. Woolthuis, Robert J. Schneider, Christopher Y. Park. Developmentally regulated mTOR degradation in normal and malignant hematopoiesis [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr PR06.

While translation initiation of most mammalian mRNAs is mediated by a 5' cap structure that binds eIF4E, inactivation/silencing of eIF4E, or quantitative sequestration of eIF4E by 4E-BPs through mTORC1 inhibition, does not impair translation of many capped mRNAs. This suggests an alternate mechanism may exist for cap-dependent but eIF4E/mTORC1-independent translation. DAP5 (NAT1, eIF4G2, p97) is an eIF4G family member that lacks the N-terminal binding domains for eIF4E and PABP. DAP5 was shown to mediate translation initiation through internal ribosome entry sites (IRES elements). We recently showed that DAP5 utilizes the cap binding activity of eIF3d to facilitate cap-dependent translation of a large number of capped mRNAs, making it a previous unknown major mechanism of cap-dependent but eIF4E/mTORC1-independent mRNA translation. Genome-wide transcriptomic and translatomic analyses indicate that DAP5 is required for translation of many transcription factor and cell receptor capped mRNAs and their mRNA targets involved in cell survival, motility, DNA repair, oncogenesis, translation initiation, and cancer cell metastasis. Up to 20% of cellular mRNAs utilize DAP5-eIF3d for cap-dependent mRNA translation. Mass spectrometry and in vivo cross-linking studies found that eIF3d is a direct binding partner of DAP5 but does not bind strongly to eIF4GI or II family members that bind eIF4E. In vitro translation and ribosome complex studies demonstrate that DAP5 and eIF3d are both essential for eIF4E and mTORC1-independent capped-mRNA translation. Given the prevalence of oncogenic and survival mRNAs directed in translation by DAP5-eIF3d, we analyzed the NCI human tissue genome cancer atlas (TCGA) and found that high expression of DAP5 mRNA is strongly associated with estrogen receptor-negative (ER-) breast cancer metastasis and poor survival. We therefore investigated the role of DAP5 in preclinical mouse models of highly metastatic ER- breast cancer. Reduction of DAP5 expression by ~70% almost eliminated metastasis to the lung and significantly increased survival with no impact on primary tumor growth. These and other studies indicate that the DAP5-eIF3d complex is a significant alternate mechanism for cap-dependent, eIF4E/mTORC1-independent mRNA translation despite lacking eIF4E and PABP binding, that DAP5 directs translation of many capped mRNAs that are involved in cell stress and survival pathways, is essential for promoting breast cancer metastasis, and that most mRNAs translated by DAP5 do not utilize an IRES. Citation Format: Columba de la Parra, Amanda Ernlund, Amandine Alard, Kelly Ruggles, Beatrix Ueberheide, Olga Katsara, Robert J. Schneider. An mTOR/eIF4E-independent translation mechanism promotes breast cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA13.

We previously reported that dietary daidzein, a soy isoflavone, increased breast cancer progression in a nude mouse model of breast cancer metastasis. Further studies indicated that equol, a metabolite of the soy isoflavone daidzein is responsible for the cancer promoting effects of soy isoflavones in breast cancer cells in vitro. We reported that equol increases the expression of the eukaryotic protein synthesis initiation factor eIF4G1, and the transcription factor c-Myc. Increased eIF4G in response to equol resulted in the non-canonical protein synthesis of pro-cancer molecules such as Cyclin D, Bcl-XL, p120, matrix metalloproteinases, etc., as confirmed by ribosome profiling and eIF4G1 knockdown studies. This study tested the hypothesis that equol promoted breast cancer progression via eIF4G1 upregulation. Immunocompromised nude mice were used to establish mammary fatpad tumors from human metastatic breast cancer cells expressing eIF4G1 knockdowns via a Tetracycline-inducible promoter. Oral gavage of 50 mg/kg BW Doxycycline 3X a week for 4 weeks was enough to knockdown eIF4G1 expression by ~80%. Therefore, one week following tumor establishment, the mice were treated with vehicle (90% corn oil, 10% ethanol), equol, Doxycycline or, equol and Doxycycline. Data show that as expected, equol treatment increased tumor growth relative to vehicle treatment. Moreover, eIF4G1 knockdown abolished the effect of equol on tumor growth. Interestingly, the effect of eIF4G1 knockdown on mammary tumor growth was not evident until 55 days of doxycycline treatment. Therefore, we tested the cell and tumor lysates for potential expression of the eIF4G1 isoform Dap5 when eIF4G1 is knocked out. Dap5 protein was expressed in eIF4G1 knockdown cells and was increased by equol treatment, indicating that Dap5 may compensate for eIF4G1 knockdown by inducing alternative cap-mediated protein synthesis. In conclusion, the cancer promoting effect of equol is abolished in breast tumors where eIF4G1 expression was stably knocked down, thus, validating our hypothesis that equol increases breast cancer progression via upregulation of eIF4G1 dependent protein synthesis initiation. Citation Format: Ailed M. Cruz-Collazo, Columba De la Parra, Robert Schneider, Suranganie Dharmawardhane. Dietary soy isoflavone equol promotes breast cancer progression via regulation of protein synthesis initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4390.

Regulatory T-cells (Tregs) maintain immune system homeostasis in the organism. In the tumor micro-environment, however, Tregs can hinder antitumor immune response. Treg maturation in peripheral sites (pTregs) requires inhibition of protein kinase mTORC1, which paradoxically blocks mRNA translation. mTORC1 phosphorylates (inactivates) 4E-BP1, a negative regulator of the translation initiation factor eIF4E. When mTORC1 is active, 4E-BP1 is hyperphosphorylated and releases eIF4E, which can then bind the mRNA cap structure and promote eIF4F complex assembly and ribosome recruitment. Inhibition of mTORC1 results in hypo-phosphorylation (activation) of 4E-BP1, which then binds and sequesters eIF4E, preventing its interaction with the cap and ribosome association to the mRNA. Using human CD4+ T-cells differentiated in vitro, we show that mTORC1 inhibition strongly impairs protein synthesis, yet with exposure to TGFbeta, is essential to mediate iTreg differentiation and immune suppression activity. We found that TGFbeta reprograms the T-cell transcriptome, while mTORC1 inhibition reprograms the translatome (genome-wide translation signature), mediating Treg differentiation. Genome-wide transcription and translation profiling identified TGFbeta-upregulated mRNAs resistant to mTORC1 inhibition, in part due to their 5’-untranslated regions, that induces Treg differentiation and immune suppression activity. These include canonical pTreg fate-determining mRNAs FOXP3, CTLA-4, CD101 and CD103, among others. Inhibition of mTORC1 alone or TGFbeta alone induces iTregs with poor immune suppression activity. Treg differentiation is therefore mediated by an mTORC1-independent privileged mRNA translation mechanism that converts activated CD4+ T-cells to Tregs. These findings are quite relevant in the tumor microenvironment, where mTORC1-inhibiting conditions such as hypoxia and deprivation of nutrients and energy are present. Investigation of molecular targets acting in this mTORC1/eIF4E-independent translation of Treg mRNAs is ongoing. These targets could be drugged to selectively inhibit Treg differentiation in the tumor microenvironment, thus allowing effector cells to mount the response against cancer cells. Citation Format: Viviana Volta, Amanda Ernlund, Columba De la Parra, Abilash Gadi, Amanda Valeta-Magara, Robert J. Schneider. Exploiting mTORC1-independent protein synthesis in Tregs to boost antitumor immune response [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A222.

The tumor microenvironment is a crucial player in the ability of cancer cells to acquire the ability to survive under the hypoxic environment and promote migration and invasion. Translational regulation is an essential part of cancer development and progression. Protein synthesis consumes considerable cellular metabolic energy and is therefore highly regulated, in turn controlling tumor cell proliferation and survival in extreme tumor–host conditions. Protein synthesis is typically downregulated by hypoxia, impairing cell proliferation and migration. Here, we show that breast cancer cells expressing integrin αvβ3, when engaging the extracellular matrix (ECM) protein vitronectin, strongly upregulate both mTOR activity and cap-dependent mRNA translation, which overrides their inhibition by hypoxia and facilitates tumor cell invasion. Interaction of vitronectin with integrin αvβ3 results in the continued activation of the kinase mTOR despite hypoxia through a mechanism that is dependent on integrin-linked kinase but is independent of focal adhesion kinase. Continuous activation of mTOR despite hypoxia involves release of translation initiation factor eIF4E from its repressor protein 4E-BP1, which is required for vitronectin-mediated tumor cell invasion. As integrin αvβ3 is associated with breast cancer cell invasion and metastasis to bone, we propose that the interaction with specific ECM proteins can influence cancer cell invasion, in part, by hyperactivation of mTOR, thereby promoting and sustaining protein synthesis under hypoxic conditions. Cancer Res; 73(14); 4571–8. ©2013 AACR.

BACKGROUND: RAD001 is an oral mTOR inhibitor that has exhibited activity in breast cancer. Triple negative breast cancer cells are unable to repair double stranded DNA breaks and hence have sensitivity to platinum agents that cause interstrand cross-links. Rapamycin acts synergistically with platinum agents to induce apoptosis and inhibit proliferation in at least two different breast cancer cell lines (including ER/PR negative cell lines). We propose that combination RAD001 and carboplatin may have activity in triple-negative breast cancer. MATERIALS AND METHODS: The primary objective of the study is to determine clinical benefit (complete remission (CR) + partial remission (PR) + stable disease (SD more than 6 months)) and the toxicity of this combination in triple negative metastatic breast cancer who have had 0–3 prior chemotherapy regimens for metastatic disease. Twenty-five subjects were to be entered in this Phase II study. This design has greater than 80% power to test the null hypothesis that the clinical benefit rate is less than or equal to 10% versus the alternative hypothesis that clinical benefit rate is greater than or equal to 30%. Prior carboplatin is allowed. Women with treated brain metastasis are eligible. Secondary objectives are to determine progression free survival and relationship between pretreatment sensitivity (biopsy at baseline) and clinical response (biopsy post 2 cycles) using IHC staining for abundance of key proteins in the Akt-mTOR pathway and their activity using surrogate phosphorylation site-specific antibodies. According to the original study plan, carboplatin AUC 6, was to be given intravenously every three weeks. Five mg of RAD001 was to be given daily with a 3 patient run-in and then 10 mg daily if there were no dose-limiting toxicities. Due to a surprising amount of thrombocytopenia with this combination the dose of carboplatin was first amended to AUC 5 and most recently to AUC 4 with 5 mg of RAD001 (and no plan to escalate to 10 mg). RESULTS: 23 patients of a planned 25 have been recruited thus far. Median age is 59. Of the 20 patients assessable for response at this time, there have been 1 CR, 5 PRs, 8 SDs and 6 PDs. One SD was achieved in a patient progressing on single agent Carboplatin at study entry. Median duration of CR+ SD +PR thus far is 13 weeks (range: 6–74 weeks). 5 of 22 patients assessable for toxicity had grade 3/4 thrombocytopenia and 4 patients had grade 3 neutropenia (no febrile neutropenia). 13 out of eighteen patients have had treatment held and/or dose reductions secondary to hematological toxicity, however, since amendment for starting dose of Carboplatin to AUC 4 the regimen has been very well tolerated with only 1 out of eleven patients with grade 3 neutropenia and grade 3 thrombocytopenia. 1 patient suffered from grade 3 dehydration. The estimated clinical benefit rate is 45% (95% confidence interval: 23%, 67%). Median time to progression or death is 85 days from start of treatment. CONCLUSIONS: Our study has met the primary end point of demonstrating clinical benefit in triple negative metastatic breast cancer. Dose limiting thrombocytopenia was an unexpected side effect requiring protocol amendment. We continue to accrue study subjects at the amended dosing. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-20-05.

Background: RAD001 is an oral mTOR inhibitor that has exhibited activity in breast cancer. Triple negative breast cancer cells are unable to repair double stranded DNA breaks and hence have sensitivity to platinum agents that cause interstrand cross-links. Rapamycin acts synergistically with platinum agents to induce apoptosis and inhibit proliferation in at least two different breast cancer cell lines (including ER/PR negative cell lines). We propose that combination RAD001 and carboplatin may have activity in triple-negative breast cancer. Methods: The primary objective of the study is to determine clinical benefit (complete remission (CR) + partial remission (PR) + stable disease (SD)) and the toxicity of this combination in women with triple negative metastatic breast cancer who have had 0–3 prior chemotherapy regimens for metastatic disease. Secondary objectives are to determine progression free survival as well as investigating the relationship between pretreatment sensitivity (biopsy at baseline) and clinical response (biopsy post 2 cycles) using IHC staining for abundance of key proteins in the Akt-mTOR pathway and their activity using surrogate phosphorylation site-specific antibodies (Akt and phospho-serine 473, phospho-threonine and phospho-threonine 308 Akt; mTOR and phospho-serine 2448 mTOR; ribosome protein S6 kinase (S6K) and phospho-threonine 378 S6K; 4E-BP1 and phospho-serine 65 4E-BP1). Prior carboplatin is allowed. Women with treated brain metastasis are eligible. According to the original study plan, carboplatin AUC 6, was to be given intravenously every three weeks. Five mg of RAD001 was to be given daily with a 3 patient run-in and then 10 mg daily if there were no dose-limiting toxicities. Due to a unexpected amount of thrombocytopenia with this combination the dose of carboplatin was first amended to AUC 5 and most recently to AUC 4 with 5 mg of RAD001 (and no plan to escalate to 10 mg). Results: Fourteen patients of a planned 25 have been recruited thus far. Median age is 58.5. Median number of prior regimens is 2 (0-3). Of the 7 patients assessable for response at this time, there have been 2 PR's and 5 patients with SD. One SD was achieved in a patient progressing on single agent carboplatin at study entry. Median duration of SD + PR is 28.5 weeks (5 patients have ongoing response ranging from 8–46.5 weeks). Five of 8 patients assessable for toxicity had grade 3 or 4 thrombocytopenia and 2 patients had grade 3 neutropenia. No cases of febrile neutropenia were observed. Four patients have required blood transfusion and one patient has required platelet transfusion. All patients have had treatment held and/or dose reductions secondary to hematological toxicity, however, since amended carboplatin dose the regimen has been very well tolerated with only one out of six patients) with grade 3 neutropenia and grade 3 thrombocytopenia. There have been no non-hematological grade 3 or 4 toxicities. Conclusions: Clinical benefit was observed in all 7 assessable patients. Dose limiting thrombocytopenia was an unexpected side effect requiring protocol amendment. We continue to accrue study subjects at the amended dosing. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-17-07.

p23 is a heat shock protein 90 (Hsp90) cochaperone located in both the cytoplasm and nucleus that stabilizes unliganded steroid receptors, controls the catalytic activity of certain kinases, regulates protein-DNA dynamics, and is upregulated in several cancers. We had previously shown that p23-overexpressing MCF-7 cells (MCF-7+p23) exhibit increased invasion without affecting the estrogen-dependent proliferative response, which suggests that p23 differentially regulates genes controlling processes linked to breast tumor metastasis. To gain a comprehensive view of the effects of p23 on estrogen receptor (ER)-dependent and -independent gene expression, we profiled mRNA expression from control versus MCF-7+p23 cells in the absence and presence of estrogen. A number of p23-sensitive target genes involved in metastasis and drug resistance were identified. Most striking is that many of these genes are also misregulated in invasive breast cancers, including PMP22, ABCC3, AGR2, Sox3, TM4SF1, and p8 (NUPR1). Upregulation of the ATP-dependent transporter ABCC3 by p23 conferred resistance to the chemotherapeutic agents etoposide and doxorubicin in MCF-7+p23 cells. MCF-7+p23 cells also displayed higher levels of activated Akt and an expanded phosphoproteome relative to control cells, suggesting that elevated p23 also enhances cytoplasmic signaling pathways. For breast cancer patients, tumor stage together with high cytoplasmic p23 expression more accurately predicted disease recurrence and mortality than did stage alone. High nuclear p23 was found to be associated with high cytoplasmic p23, therefore both may promote tumor progression and poor prognosis by increasing metastatic potential and drug resistance in breast cancer patients. Cancer Res; 70(21); 8446–56. ©2010 AACR.

Breast cancer is the most commonly diagnosed malignancy among women in the U.S. and the second leading cause of cancer-related deaths in women. The estrogen receptor-positive (ER+) breast cancer subtype comprises the majority (70-80%) of breast cancer cases and the majority of late recurrences. Anti-estrogen therapy with tamoxifen remains a cornerstone of adjuvant therapy for ER+ breast cancers in the premenopausal setting, but resistance is inevitable for a third of patients, leading to metastatic disease, which is responsible for the majority of breast cancer deaths. Our work demonstrates that tamoxifen resistance in ER+ breast cancer involves selective mRNA translation mediated by hyperactivation of mTOR and MNK1, two kinases that play major roles in mRNA translation. The hyperactivation of mTOR and MNK1 converges on eukaryotic translation initiation factor 4E (eIF4E) to increase its availability and mRNA selectivity. Using tamoxifen-sensitive and -resistant ER+ cells, we conducted genome-wide transcriptomic and translatomic analyses +/- silencing of eIF4E. We identified key genes responsible for tamoxifen resistance, whose mRNAs are selectively translationally upregulated with increased levels of eIF4E. RUNX2 is a key mRNA that is selectively translationally upregulated, which we show provides resistance by opposing estrogenic signaling and possibly phenotypically reprogramming the cell. Silencing of RUNX2 strongly resensitizes the cells to tamoxifen. We also show that RUNX2 impairs cellular proliferation and cell survival of previously drug-resistant cells in the presence of tamoxifen. Silencing of key translationally upregulated mRNAs, such as RUNX2, can partially restore endocrine therapy sensitivity. Using a tamoxifen-resistant ER+ PDX model, and patient-derived sensitive and resistant tumor tissues (no recurrence or recurrence within 5 years), we validated the hyperactivation of mTOR and MNK1, the overexpression of eIF4E, and selective mRNA translation in tamoxifen resistance. We hypothesize that selection for increased expression of RUNX2 and other selectively translated mRNAs promotes a more “basal-like,” estrogen-independent phenotype. We aim to determine how tamoxifen resistance translationally reprograms breast cancer cells to a different breast cancer phenotype (e.g., cancer stem cell-like, basal-like, luminal with disrupted ER signaling). Citation Format: Sofia Bakogianni, Phillip Geter, Amanda Ernlund, Rezina Arju, Abhilash Gadi, Robert J. Schneider. Translational reprogramming of breast cancer and its role in tamoxifen resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5886.

This abstract was not presented at the symposium.

High-grade breast cancers are better adapted to hypoxia and more resistant to chemotherapy and radiotherapy. Constitutive activation of the transcription factor nuclear factor-κB (NF-κB) increases in breast tumors and in breast cancer cell lines, where it promotes chemoradiation resistance, in part by activation of antiapoptotic genes. The role for up-regulation of NF-κB in breast cancer progression is less clear. Here, we first show that whereas the constitutive activity of NF-κB is incrementally elevated from immortalized breast epithelial to frank transformed invasive ductal breast cancer cell lines (∼3-fold, ±0.1-fold, P < 0.05), inflammatory cytokine-inducible activity is further increased (up to 9-fold, ±0.9-fold, P < 0.05). We then show that inhibition of NF-κB activity selectively sensitizes transformed but not immortalized cells to killing by ionizing radiation or low levels of tumor necrosis factor (TNF) by up to 10-fold (±1-fold, P < 0.05) but has little effect on hypoxia-mediated cell death. Prolonged cultivation of immortalized and partially transformed cells in TNF selected for cells displaying stable constitutive and strongly inducible overexpression of NF-κB even in the absence of TNF. Stable acquisition of increased NF-κB activity conferred resistance to ionizing radiation or inflammatory cytokines, which was dependent on elevated NF-κB activity, but had no effect on transformation potential measured by in vitro and in vivo parameters. Thus, TNF and possibly other inflammatory cytokines in the tumor-stroma matrix likely select for breast cancer cells that stably overexpress NF-κB, leading to greater cancer cell survival. Greater cell survival despite increased genomic injury may permit increased acquisition of malignant genetic alterations as well as resistance to chemoradiation therapy. (Mol Cancer Res 2008;6(1):78–88)

Metastasis is the cause of 90% of cancer-related deaths. Selective mRNA translation has been found to be crucial for breast cancer development, progression and metastasis; however, there is a poor biological and mechanistic understanding of the role of translation in these processes. Our work and others have pioneered an understanding of translational regulation in breast cancer. We have previously shown that overexpression of initiation factor eIF4GI in advanced breast cancer cells selectively increases the translation of mRNAs encoding survival, cell cycle inhibition, and DNA damage and repair proteins, among other DNA damage-protective mRNAs. eIF4G consists of three family members: eIF4GI, eIF4GII, and the poorly studied eIF4G homolog, DAP5, the subject of this study. DAP5 lacks the N-terminal domain for eIF4E and PABP binding. DAP5 is therefore suspected to promote eIF4E-independent or IRES-driven translation of mRNAs involved in cell stress and oncogenesis, but its role in metastasis hasn’t been investigated. We analyzed the NCI human tissue genome cancer atlas (TCGA) and found that higher DAP5 mRNA expression is strongly associated with estrogen receptor negative breast cancer metastasis with lower overall survival. We then engineered a highly metastatic breast cancer cell line MDA-MB-231TR to express dox-inducible shRNA to DAP5 or a non-silencing (NS) control. Tumors were developed in clinically relevant orthotopic mouse models, with and without DAP5 silencing by addition of dox to the drinking water. Remarkably, reduction of DAP5 expression by ~70% had no impact on primary tumor growth but fully eliminated metastasis to the lung. Genome-wide transcriptome and translatome analysis of DAP5 and in vivo ultraviolet-crosslinking and high-throughput sequencing (HITS-CLIP) identified specific mRNAs that interact directly with DAP5. A significant fraction of DAP5 mRNA targets are involved in cell death and survival, cell proliferation, cell mobility, DNA repair and translation initiation and do not have IRESs, suggesting a novel role for DAP5 in a non-classical translation initiation process upon cellular stress and metastasis. Our results suggest that the translation initiation factor DAP5 likely plays a critical role in breast cancer metastasis by a unique mechanism for metastasis-specific translation initiation and provides new concepts for therapeutic strategies involving translational regulation. Citation Format: Columba de la Parra, Amandine Alard, Amanda Ernlund, Robert J. Schneider. Translation initiation factor DAP5 plays an essential role in translational control of breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4481. doi:10.1158/1538-7445.AM2017-4481

INTRODUCTION: Ovarian cancer is the most lethal gynecologic malignancy. Initial treatment with platinum and taxane drugs are effective, however inevitably these patients will recur and become resistant to cytotoxic therapies. Thus, new alternatives for durable treatments need to be identified. One approach is to explore the heterogeneity of epithelial ovarian cancer and identify the subpopulation of cells that are resistant to treatment. It has been hypothesized, that cancer stem cells (CSCs) can survive chemotherapy and have enhanced tumor-initiating capabilities. The AKT-PI3K-mTOR pathway is well studied and has been shown to be critical for tumor cell survival. This pathway closely regulates both mTOR complexes (mTORC1 and mTORC2). mTORC1 regulates protein synthesis and autophagy whereas mTORC2 regulates cell motility. mTORC1 and 2 ultimately affect cell proliferation, survival and angiogenesis, all inherently important in tumorgenesis. OBJECTIVES: Rapalogs are specific and potent inhibitors of mTORC1 but have no effect of mTORC2. We investigate the efficacy of dual mTORC1/2 inhibitors and its effect on the CSC population. METHODS: Carboplatin resistant ovarian cancer cell line OVCAR3 were cultured and treated with dimethyl sulfoxide (DMSO), carboplatin, or INK128, a potent dual inhibitor of mTORC1/2. Using cell surface markers CD133 and CD44, CSCs were quantified by flow cytometry. OVCAR3 spheroids were cultured as a surrogate for CSCs. Colony formation-survival assays were performed with spheroids treated with the above mentioned conditions. Analysis was performed using ArrayScan technology. Doxycyline inducible silencing cell lines derived from OVCAR3 were generated; Non-silenced (Nsi), Raptor (Sh-mTORC1) and Rictor (Sh-mTORC2). Colony formation assays using spheroids from silenced lineages were performed to evaluate growth and survival. Western blot analysis was performed to confirm silencing of targets, and to evaluate protein expression in CSCs. RESULTS: 4.87% of the untreated OVCAR3 population were CSCs. INK128 treatment of the cells confered a 2 fold increase of CD44+/CD133+ cells, presumably CSCs, P CONCLUSIONS: The PI3K/AKT/mTOR pathway appears to have a role in the biogenesis of platinum resistant ovarian cancer cells. The inhibition of mTORC1/2 delays growth of OVCAR3 cells, and may have a role in activating in stem like CSCs. However, it does not appear to selectively inhibit CD133+/CD44+ cells. These findings suggest that targeting CSCs may lead to advances in ovarian cancer treatment. Citation Format: Jing-Yi Chern MD, Melissa Frey MD, Fernanda Musa MD, Amandine Alard PhD, Stephanie V. Blank, MD, Robert Schneider, PhD. EFFICACY OF MTORC1/2 INHIBITION ON OVARIAN CANCER STEM CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-051.

PURPOSE: To investigate the effects of PF-477736, a small molecular checkpoint kinase 1 (Chk1) inhibitor with and without carboplatin on cell apoptosis and proliferation in a p53-mutated platinum resistant ovarian cancer cell line. METHODS: OVCAR3 cells were treated with PF-477736 alone and in combination with carboplatin. Western blot analyses were performed to assess the protein expression of Chk1, phosphorylated Chk1, H2AX, and phosphorylated H2AX. Cell proliferation was evaluated with colony formation assays 10 days following treatment and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assays over the course of 5 days following treatment. Cell apoptosis was measured with flow cytometry after staining with FITC Annexin V and propidium iodide. Paired t-tests were performed for comparative analyses. RESULTS: PF-477736 decreased the expression of phosphorylated Chk1 on serine 249 within 24 hours following treatment as seen on Western blot assay. Total Chk1 expression was unchanged after treatment with PF-477736. The combination treatment of PF-477736 and carboplatin effectively decreased OVCAR3 cell proliferation. Colony formation assays revealed fewer colonies after dual treatment with PF-477736 and carboplatin compared to PF-477736 alone (average colony count 1.5 vs 13.6, p = 0.03) and compared to carboplatin alone (average colony count 1.5 vs 9.7, p < 0.001). MTT assays demonstrated a synergistic effect with PF-477736 and carboplatin: dual treated cells had lower mean dye absorbance values than PF-477736 alone (mean absorbance fold change 1.7 vs 3.2, p = 0.009) and carboplatin alone (mean absorbance fold change 1.7 vs 5.6, p < 0.001). OVCAR3 cells after treatment with PF-477736 and carboplatin had higher rates of apoptosis. On flow cytometry, a higher proportion of cells stained with the apoptotic markers Annexin V and propidium iodide after treatment with PF-477736 and carboplatin compared to PF-477736 alone (percentage of apoptotic cells 34.9% vs 13.0%, p = 0.004) and carboplatin alone (percentage of apoptotic cells 34.9% vs 16.0%, p = 0.008). In addition, western blot analysis of the cell lysates following dual treatment of PF-477736 and carboplatin showed increased expression of phosphorylated H2AX, a marker of double-stranded DNA damage while there was minimal expression with PF-477736 alone and carboplatin alone. CONCLUSIONS: Carboplatin and PF-477736, a small molecule Chk1 inhibitor, synergistically decreased cell proliferation and increased cell apoptosis in OVCAR3 cells, an in vitro model of platinum resistant ovarian cancer. PF-477736 may be a potential new therapeutic strategy for the resensitization of platinum resistant ovarian cancer. Citation Format: Jessica Lee MD, Stephanie V. Blank MD, Robert J. Schneider MD. THE SYNERGISTIC EFFECTS OF CARBOPLATIN AND PF–477736, A SMALL MOLECULE CHECKPOINT KINASE 1 INHIBITOR ON A PLATINUM RESISTANT OVARIAN CANCER CELL LINE [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-095.

Currently, ER+ breast cancer is treated with adjuvant antiestrogen therapies (i.e. tamoxifen and raloxifene). Despite maintaining expression of the estrogen receptor, many women treated with adjuvant therapy develop resistance several years after curative care. In fact, resistance to tamoxifen therapy is responsible for a vast majority of deaths resulting from metastatic disease. Our lab and others have shown that upregulation of the abundance and/or activity of translation initiation factors eIF4E and eIF4G1 occurs widely in advanced breast cancer, selectively upregulating translation of certain mRNAs required for survival, proliferation, and metastasis. Therefore, we hypothesized that tamoxifen resistance in ER+ breast cancer involves selective mRNA translation mediated by hyperactivation of the mTORC1 translational control pathway via regulating the availability and activity of eIF4E. Tamoxifen resistant (R) and sensitive (S) ER+ cell lines have been developed by several labs by long-term cultivation in tamoxifen and are used in my work. We define here, using the aforementioned cell lines, a molecular hub consisting of the two translational control pathways, PI3K/Akt/mTOR and MAPK/ERK/MNK which converge on translation initiation factor 4E (eIF4E) increasing its availability and activity. We show that blocking eIF4E-mediated translation was able to resensitize drug resistant cells to tamoxifen; conversely, overexpression of eIF4E, without concurrent mTORC1 hyperactivity, was not sufficient to generate resistance. Furthermore, knockdown of two genes identified from our genome-wide translation state analysis, TEX14 and RUNX2, significantly impacted drug resistance and serve as a major regulators in disease progression. Our findings demonstrate that eIF4E is a key player regulating tamoxifen resistance in ER+ disease. Citation Format: Phillip A. Geter, Robert J. Schneider. Translational control pathways form a molecular hub that regulates tamoxifen resistance in ER+ breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B33.

Introduction: Previously we showed that the incidence of multiple human cytomegalovirus (HCMV) genotypes in the carcinoma tissues of inflammatory breast cancer (IBC) patients plays essential role in the disease progression. Primary HCMV infection to monocytes induces differentiation and biological turnover of monocytes to macrophages. In addition infected macrophages serves as “mobile vectors” for virus spreading and dissemination to different organs mainly by transendothelial migration. In addition we screened for the infiltration of CD14+ and CD68+ monocytes/macrophages markers in the carcinoma tissues of IBC versus non-IBC patients we showed that of CD14+ cells highly infiltrate tumor microenvironment (TME) of IBC patients compared to non-IBC. Aims: In the present study we aim to 1) Assess the level of expression of MAC387 protein by monocytes/macrophages infiltrating TME of IBC versus non-IBC patients; 2) Test the correlation between the density of infiltrated MAC387+ cells and the incidence of different HCMV genotypes in carcinoma tissues of IBC versus non-IBC tissues. Since MAC387 found to be more common in cancers characterized by high metastatic properties we will also 3) determine whether the expression of MCA387 correlate with lymph-node metastasis and lymphovascular invasion in IBC versus non-IBC breast cancer patients. Materials and Methods: A total of 135 breast cancer patients (91 non-IBC and 44 IBC) were enrolled to the present study during the period of January 2012 to September 2015 from Ain Shams university Hospitals. Detection of MAC387 marker was assessed by immunohistochemistry and HCMV genotypes were detected using multiplex PCR methodology. Results: MAC387+ positive cells were more prevalent in IBC tissues than non-IBC tissues (p = 0.4). Incidence of higher number of MAC387+ cells were positively correlate with higher number of metastatic lymph nodes in both IBC and non-IBC patients r = 0.807 and 0.779 respectively. Moreover, Incidence of higher number of MAC387+ cells found to be positively correlate with lymphovascular invasion in IBC patients r = 0622. Detection of multiple HCMV genotypes was statistically higher (p = 0.04) in IBC tissues in comparison with non-IBC tissues. Moreover, triple negative non-IBC and IBC tissues showed higher incidence of multiple HCMV genotypes in comparison with hormonal positive non-IBC and IBC tissues. of the monocytes/macrophages MAC387+ positive cells were more prevalent in IBC tissues showed multiple HCMV genotypes in comparison with IBC tissues showed single HCMV genotype (p = 0.46). Conclusion: MAC387+ positive cells were more prevalent in IBC tissues and correlate with presence of multiple HCMV genotypes and high invasive properties of the disease. Citation Format: Hossam Taha Mohamed, Ramy Gadalla, Sherif Abdel Aziz Ibrahim, M. Akram Nouh, Mohamed El-Shinawi, Robert J. Schneider, Mona Mostafa Mohamed. High incidence of MAC387 positive cells in the carcinoma tissues of inflammatory breast cancer patients correlate with the detection of multiple human Cytomegalovirus genotypes and invasive properties of the disease. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3302.