Faculty Bibliography

Inflammatory breast cancer (IBC) is a highly aggressive phenotype of breast cancer that is characterized by a high incidence early metastasis. We previously reported a significant association of human cytomegalovirus (HCMV) DNA in the carcinoma tissues of IBC patients but not in the adjacent normal tissues. HCMV-infected macrophages serve as "mobile vectors" for spreading and disseminating virus to different organs, and IBC cancer tissues are highly infiltrated by tumor-associated macrophages (TAMs) that enhance IBC progression and promote breast cancer stem cell (BCSC)-like properties. Therefore, there is a need to understand the role of HCMV-infected TAMs in IBC progression. The present study aimed to test the effect of the secretome (cytokines and secreted factors) of TAMs derived from HCMV⁺ monocytes isolated from IBC specimens on the proliferation, invasion, and BCSC abundance when tested on the IBC cell line SUM149. HCMV⁺ monocytes were isolated from IBC patients during modified radical mastectomy surgery and tested in vitro for polarization into TAMs using the secretome of SUM149 cells. MTT, clonogenic, invasion, real-time PCR arrays, PathScan Intracellular Signaling array, and cytokine arrays were used to characterize the secretome of HCMV⁺ TAMs for their effect on the progression of SUM149 cells. The results showed that the secretome of HCMV⁺ TAMs expressed high levels of IL-6, IL-8, and MCP-1 cytokines compared to HCMV^- TAMs. In addition, the secretome of HCMV⁺ TAMs induced the proliferation, invasion, colony formation, and expression of BCSC-related genes in SUM149 cells compared to mock untreated cells. In addition, the secretome of HCMV⁺ TAMs activated the phosphorylation of intracellular signaling molecules p-STAT3, p-AMPKα, p-PRAS40, and p-SAPK/JNK in SUM149 cells. In conclusion, this study shows that the secretome of HCMV⁺ TAMs enhances the proliferation, invasion, colony formation, and BCSC properties by activating the phosphorylation of p-STAT3, p-AMPKα, p-PRAS40, and p-SAPK/JNK intracellular signaling molecules in IBC cells.

Little is known regarding T cell translational regulation. We demonstrate that T follicular helper (TFH) cells use a previously unknown mechanism of selective messenger RNA (mRNA) translation for their differentiation, role in B cell maturation, and in autoimmune pathogenesis. We show that TFH cells have much higher levels of translation factor eIF4E than non-TFH CD4⁺ T cells, which is essential for translation of TFH cell fate-specification mRNAs. Genome-wide translation studies indicate that modest down-regulation of eIF4E activity by a small-molecule inhibitor or short hairpin RN impairs TFH cell development and function. In mice, down-regulation of eIF4E activity specifically reduces TFH cells among T helper subtypes, germinal centers, B cell recruitment, and antibody production. In experimental autoimmune encephalomyelitis, eIF4E activity down-regulation blocks TFH cell participation in disease pathogenesis while promoting rapid remission and spinal cord remyelination. TFH cell development and its role in autoimmune pathogenesis involve selective mRNA translation that is highly druggable.

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 (AB) for growth and survival in the brain parenchyma. Melanoma-secreted AB activates surrounding astrocytes to a pro-metastatic, anti-inflammatory phenotype and prevents phagocytosis of melanoma by microglia. Finally, we demonstrate that pharmacological inhibition of AB decreases brain metastatic burden.

PURPOSE/OBJECTIVE:Inflammatory breast cancer is a deadly and aggressive type of breast cancer. A key challenge relates to the need for a more detailed, formal, objective definition of IBC, the lack of which compromises clinical care, hampers the conduct of clinical trials, and hinders the search for IBC-specific biomarkers and treatments because of the heterogeneity of patients considered to have IBC. METHODS:Susan G. Komen, the Inflammatory Breast Cancer Research Foundation, and the Milburn Foundation convened patient advocates, clinicians, and researchers to review the state of IBC and to propose initiatives to advance the field. After literature review of the defining clinical, pathologic, and imaging characteristics of IBC, the experts developed a novel quantitative scoring system for diagnosis. RESULTS:The experts identified through consensus several "defining characteristics" of IBC, including factors related to timing of onset and specific symptoms. These reflect common pathophysiologic changes, sometimes detectable on biopsy in the form of dermal lymphovascular tumor emboli and often reflected in imaging findings. Based on the importance and extent of these characteristics, the experts developed a scoring scale that yields a continuous score from 0 to 48 and proposed cut-points for categorization that can be tested in subsequent validation studies. CONCLUSION/CONCLUSIONS:To move beyond subjective 'clinical diagnosis' of IBC, we propose a quantitative scoring system to define IBC, based on clinical, pathologic, and imaging features. This system is intended to predict outcome and biology, guide treatment decisions and inclusion in clinical trials, and increase diagnostic accuracy to aid basic research; future validation studies are necessary to evaluate its performance.

GGGGCC (G4C2) repeat expansion in the first intron of C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia. Repeat-containing RNA is translated into dipeptide repeat (DPR) proteins, some of which are neurotoxic. Using dynamic ribosome profiling, we identified three translation initiation sites in the intron upstream of (G4C2) repeats; these sites are detected irrespective of the presence or absence of the repeats. During translocation, ribosomes appear to be stalled on the repeats. An AUG in the preceding C9ORF72 exon initiates a uORF that inhibits downstream translation. Polysome isolation indicates that unspliced (G4C2) repeat-containing RNA is a substrate for DPR protein synthesis. (G4C2) repeat-containing RNA translation is 5' cap-independent but inhibited by the initiation factor DAP5, suggesting an interplay with uORF function. These results define novel translational mechanisms of expanded (G4C2) repeat-containing RNA in disease.

Inflammatory Breast Cancer (IBC) is a rare and aggressive type of breast cancer with a poor prognosis. Its management is challenging because of a lack of targeted therapies, increased metastatic potential, and high recurrence rates. Interest in using platinum agents such as carboplatin emerged from data suggesting frequent DNA repair defects in breast cancer. Because studies show that medicinal mushroom Ganoderma lucidum (GLE) sensitizes cancer cells to radiation and other drugs; herein, we aimed to investigate the therapeutic potential of GLE, alone or in combination with carboplatin in breast cancer models. Our studies were focused on the regulation of the DNA Damage Response (DDR) and on cancer cell stemness. Carboplatin and GLE were tested in vitro using the IBC cell line, SUM-149, breast cancer non-IBC cells, MDA-MB-231, and in vivo using IBC xenograft models. Our results show that the GLE/carboplatin combination decreased cell viability, induced cell death by two different mechanisms, and delayed the response to DNA damage. Furthermore, the combination suppressed mammosphere formation and the expression of cancer stemness proteins. In xenograft models, the combination showed significant tumor growth inhibitory effects without systemic toxicity. This study emphasizes the potential of this dual therapy for IBC patients.

Regulatory T cells (Treg cells) inhibit effector T cells and maintain immune system homeostasis. Treg cell maturation in peripheral sites requires inhibition of protein kinase mTORC1 and TGF-beta-1 (TGF-beta). While Treg cell maturation requires protein synthesis, mTORC1 inhibition downregulates it, leaving unanswered how Treg cells achieve essential mRNA translation for development and immune suppression activity. Using human CD4⁺ T cells differentiated in culture and genome-wide transcription and translation profiling, here we report that TGF-beta transcriptionally reprograms naive T cells to express Treg cell differentiation and immune suppression mRNAs, while mTORC1 inhibition impairs translation of T cell mRNAs but not those induced by TGF-beta. Rather than canonical mTORC1/eIF4E/eIF4G translation, Treg cell mRNAs utilize the eIF4G homolog DAP5 and initiation factor eIF3d in a non-canonical translation mechanism that requires cap-dependent binding by eIF3d directed by Treg cell mRNA 5' noncoding regions. Silencing DAP5 in isolated human naive CD4⁺ T cells impairs their differentiation into Treg cells. Treg cell differentiation is mediated by mTORC1 downregulation and TGF-beta transcriptional reprogramming that establishes a DAP5/eIF3d-selective mechanism of mRNA translation.