Triple-negative breast cancers (TNBCs), which lack specific targeted therapy options, evolve into highly chemo-resistant tumors that metastasize to multiple organs simultaneously. We have previously shown that TNBCs maintain an activated WNT10B-driven network that drives metastasis. Pharmacologic inhibition by ICG-001 decreases β-catenin-mediated proliferation of multiple TNBC cell lines and TNBC patient-derived xenograft (PDX)-derived cell lines. In vitro, ICG-001 was effective in combination with the conventional cytotoxic chemotherapeutics, cisplatin and doxorubicin, to decrease the proliferation of MDA-MB-231 cells. In contrast, in TNBC PDX-derived cells doxorubicin plus ICG-001 was synergistic, while pairing with cisplatin was not as effective. Mechanistically, cytotoxicity induced by doxorubicin, but not cisplatin, with ICG-001 was associated with increased cleavage of PARP-1 in the PDX cells only. In vivo, MDA-MB-231 and TNBC PDX orthotopic primary tumors initiated de novo simultaneous multi-organ metastases, including bone metastases. WNT monotherapy blocked multi-organ metastases as measured by luciferase imaging and histology. The loss of expression of the WNT10B/β-catenin direct targets HMGA2, EZH2, AXIN2, MYC, PCNA, CCND1, transcriptionally active β-catenin, SNAIL and vimentin both in vitro and in vivo in the primary tumors mechanistically explains loss of multi-organ metastases. WNT monotherapy induced VEGFA expression in both tumor model systems, whereas increased CD31 was observed only in the MDA-MB-231 tumors. Moreover, WNT-inhibition sensitized the anticancer response of the TNBC PDX model to doxorubicin, preventing simultaneous metastases to the liver and ovaries, as well as to bone. Our data demonstrate that WNT-inhibition sensitizes TNBC to anthracyclines and treats multi-organ metastases of TNBC.

The oxygen-responsive hypoxia inducible factor (HIF)-1 promotes several steps of the metastatic cascade. A hypoxic gene signature is enriched in triple-negative breast cancers (TNBCs) and is correlated with poor patient survival. Inhibiting the HIF transcription factors with small molecules is challenging; therefore, we sought to identify genes downstream of HIF-1 that could be targeted to block invasion and metastasis. Creatine kinase brain isoform (CKB) was identified as a highly differentially expressed gene in a screen of HIF-1 wild type and knockout mammary tumor cells derived from a transgenic model of metastatic breast cancer. CKB is a cytosolic enzyme that reversibly catalyzes the phosphorylation of creatine, generating phosphocreatine (PCr) in the forward reaction, and regenerating ATP in the reverse reaction. Creatine kinase activity is inhibited by the creatine analog cyclocreatine (cCr). Loss- and gain-of-function genetic approaches were used in combination with cCr therapy to define the contribution of CKB expression or creatine kinase activity to cell proliferation, migration, invasion, and metastasis in ER-negative breast cancers. CKB was necessary for cell invasion in vitro and strongly promoted tumor growth and lung metastasis in vivo. Similarly, cyclocreatine therapy repressed cell migration, cell invasion, the formation of invadopodia and lung metastasis. Moreover, in common TNBC cell line models, the addition of cCr to conventional cytotoxic chemotherapy agents was either additive or synergistic to repress tumor cell growth.

The Ccr4-Not complex functions as an effector of multiple signaling pathways that control gene transcription and mRNA turnover. Consequently, Ccr4-Not contributes to a diverse array of processes, which includes a significant role in cell metabolism. Yet a mechanistic understanding of how it contributes to metabolism is lacking. Herein, we provide evidence that Ccr4-Not activates nutrient signaling through the essential target of rapamycin complex 1 (TORC1) pathway. Ccr4-Not disruption reduces global TORC1 signaling, and it also upregulates expression of the cell wall integrity (CWI) pathway terminal kinase Mpk1. Although CWI signaling represses TORC1 signaling, we find that Ccr4-Not loss inhibits TORC1 independently of CWI activation. Instead, we demonstrate that Ccr4-Not promotes the function of the vacuole V-ATPase, which interacts with the Gtr1 GTPase-containing EGO complex to stimulate TORC1 in response to nutrient sufficiency. Bypassing the V-ATPase requirement in TORC1 activation using a constitutively active Gtr1 mutant fully restores TORC1 signaling in Ccr4-Not deficient cells. Transcriptome analysis and functional studies revealed that loss of the Ccr4 subunit activates the TORC1 repressed retrograde signaling pathway to upregulate mitochondrial activity. Blocking this mitochondrial upregulation in Ccr4-Not deficient cells further represses TORC1 signaling, and it causes synergistic deficiencies in mitochondrial-dependent metabolism. These data support a model whereby Ccr4-Not loss impairs V-ATPase dependent TORC1 activation that forces cells to enhance mitochondrial metabolism to sustain a minimal level of TORC1 signaling necessary for cell growth and proliferation. Therefore, Ccr4-Not plays an integral role in nutrient signaling and cell metabolism by promoting V-ATPase dependent TORC1 activation. Eukaryotic cells dynamically control nutrient signaling pathways to regulate metabolism, anabolic growth, and cell proliferation through molecular effectors that remain poorly understood. The Ccr4-Not complex is a multiprotein regulatory factor conserved from yeast to humans that integrates both mRNA degradation and ubiquitin ligase activities. While budding yeast Ccr4-Not represses mitochondrial metabolism to allow normal glycolytic metabolism, how it does so is not clear. Herein, we demonstrate that Ccr4-Not activates nutrient signaling through the conserved target of rapamycin complex 1 (TORC1) pathway by promoting the function of the vacuolar V-ATPase proton pump that acidifies the vacuole and activates TORC1. Ccr4-Not deficient cells exhibit decreased V-ATPase stability, leading to defects in both vacuole acidification and TORC1 signaling. This TORC1 defect causes an adaptive upregulation of mitochondrial metabolism to maintain a minimal level of TORC1 activity. This study defines a previously unknown role for Ccr4-Not as a key activator of a conserved eukaryotic nutrient signaling pathway highly relevant to human health and disease.

Background: Uveal melanoma (UVM) is a rare cancer that shows sex difference in incidence and survival, with little previous report for the underlying mechanism. Methods: This study used the SEER data (1974–2016) for an age-dependent analysis on sex difference in UVM, and further used the TCGA-UVM genomics dataset for analyzing the differential gene expression profiles in tumors from men and women. Results: Our results demonstrate a sex difference in older age (≥40 years) but not in younger patients, with men exhibiting a higher incidence rate than women. However, younger women have shown a continuous increasing trend since 1974. Examining the 11 major oncogenes and tumor suppressors in UVM revealed that EIF1AX showed a significant sex difference in mRNA accumulation and copy number variation, with female tumors expressing higher levels of EIF1AX and exhibiting more variations in copy numbers. EIF1AX mRNA levels were significantly inversely correlated with EIF1AX copy numbers in female tumors only, but not in male tumors. Differential gene expression analysis at the whole genomic level identified a set of 92 protein-coding and 16 RNA-coding genes which exhibited differential expression in men and women (fold of change cutoff at 1.7, adjusted p value < 0.05, FDR < 0.05). Network analysis showed significant difference in immune response and in disulfide bond formation, with EGR1/EGR2 and PDIA2 genes as regulators for immune response and disulfide bond formation, respectively. The melanocortin pathway which is linked to both melanin synthesis and obesity seems to be altered with unclear significance, as the sex difference in POMC, DCT/TYRP2, and MRAP2 was observed but with no clear direction. Conclusion: This study reveals possible mechanisms for the sex difference in tumorigenesis of UVM which has potentials for better understanding and prevention of UVM.

TSC renal cystic disease is poorly understood and has no approved treatment. In a new principal cell-targeted murine model of Tsc cystic disease, the renal cystic epithelium is mostly composed of type A intercalated cells with an intact Tsc2 gene confirmed by sequencing, although these cells exhibit a Tsc-mutant disease phenotype. We used a newly derived targeted murine model in lineage tracing and extracellular vesicle (EV) characterization experiments and a cell culture model in EV characterization and cellular induction experiments to understand TSC cystogenesis. Using lineage tracing experiments, we found principal cells undergo clonal expansion but contribute very few cells to the cyst. We determined that cystic kidneys contain more interstitial EVs than noncystic kidneys, excrete fewer EVs in urine, and contain EVs in cyst fluid. Moreover, the loss of Tsc2 gene in EV-producing cells greatly changes the effect of EVs on renal tubular epithelium, such that the epithelium develops increased secretory and proliferative pathway activity. We demonstate that the mTORC1 pathway activity is independent form the EV production, and that the EV effects for a single cell line can vary significantly. TSC cystogenesis involves significant contribution from genetically intact cells conscripted to the mutant phenotype by mutant cell derived EVs.

Androgen receptor splice variants (AR-SVs) contribute to the aggressive growth of castration-resistant prostate cancer (CRPC). AR-SVs, including AR-V7, are expressed in ~30% of CRPC, but minimally in treatment-naïve primary prostate cancer (PCa). Compared to Caucasian American (CA) men, African American (AA) men are more likely to be diagnosed with aggressive/potentially lethal PCa and have shorter disease-free survival. Expression of a truncated AR in an aggressively growing patient-derived xenograft developed with a primary PCa specimen from an AA patient led us to hypothesize that the expression of AR-SVs could be an indicator of aggressive growth both in PCa progression and at the CRPC stage in AA men. Tissue microarrays (TMAs) were created from formalin-fixed paraffin-embedded (FFPE) prostatectomy tumor blocks from 118 AA and 115 CA treatment-naïve PCa patients. TMAs were stained with AR-V7-speicifc antibody and with antibodies binding to the N-terminus domain (NTD) and ligand-binding domain (LBD) of the AR. Since over 20 AR-SVs have been identified, and most AR-SVs do not as yet have a specific antibody, we considered a 2.0-fold or greater difference in the NTD vs. LBD staining as indication of potential AR-SV expression. Two AA, but no CA, patient tumors stained positively for AR-V7. AR staining with NTD and LBD antibodies was robust in most patients, with 21% of patients staining at least 2-fold more for NTD than LBD, indicating that AR-SVs other than AR-V7 are expressed in primary treatment-naïve PCa. About 24% of the patients were AR-negative, and race differences in AR expression were not statistically significant. These results indicate that AR-SVs are not restricted to CRPC, but also are expressed in primary PCa at higher rate than previously reported. Future investigation of the relative expression of NTD vs. LBD AR-SVs could guide the use of newly developed treatments targeting the NTD earlier in the treatment paradigm.