Triple negative breast cancer (TNBC) is a deadly form of breast cancer due to the development of resistance to chemotherapy affecting over 30% of patients. New therapeutics and companion biomarkers ...are urgently needed. Recognizing the elevated expression of glucose transporter 1 (GLUT1, encoded by SLC2A1) and associated metabolic dependencies in TNBC, we investigated the vulnerability of TNBC cell lines and patient-derived samples to GLUT1 inhibition. We report that genetic or pharmacological inhibition of GLUT1 with BAY-876 impairs the growth of a subset of TNBC cells displaying high glycolytic and lower oxidative phosphorylation (OXPHOS) rates. Pathway enrichment analysis of gene expression data suggests that the functionality of the E2F pathway may reflect to some extent OXPHOS activity. Furthermore, the protein levels of retinoblastoma tumor suppressor (RB1) strongly correlate with the degree of sensitivity to GLUT1 inhibition in TNBC, where RB1-negative cells are insensitive to GLUT1 inhibition. Collectively, our results highlight a strong and targetable RB1-GLUT1 metabolic axis in TNBC and warrant clinical evaluation of GLUT1 inhibition in TNBC patients stratified according to RB1 protein expression levels.
Patient-derived tumor organoids (PDOs) are a highly promising preclinical model that recapitulates the histology, gene expression, and drug response of the donor patient tumor. Currently, PDO culture ...relies on basement-membrane extract (BME), which suffers from batch-to-batch variability, the presence of xenogeneic compounds and residual growth factors, and poor control of mechanical properties. Additionally, for the development of new organoid lines from patient-derived xenografts, contamination of murine host cells poses a problem. We propose a nanofibrillar hydrogel (EKGel) for the initiation and growth of breast cancer PDOs. PDOs grown in EKGel have histopathologic features, gene expression, and drug response that are similar to those of their parental tumors and PDOs in BME. In addition, EKGel offers reduced batch-to-batch variability, a range of mechanical properties, and suppressed contamination from murine cells. These results show that EKGel is an improved alternative to BME matrices for the initiation, growth, and maintenance of breast cancer PDOs.
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Finding a material that emulates the in vivo microenvironment to allow complex cell phenotypes in vitro and without biasing cell fate in vivo remains a significant challenge. ...Hyaluronan is over-expressed in breast cancer and in several other tumours. Mammary epithelial cells invade tissues through both active degradation of the matrix and ameboid-like mechanisms. Thus, we synthesized a defined, biomimetic hydrogel composed of hyaluronan (HA) and matrix metalloproteinase-cleavable (MMPx) crosslinker and used oxime crosslinking to ensure tunability of the resulting HA-MMPx hydrogel. This strategy allowed us to identify the optimal matrix to study the growth and polarization of healthy and diseased mammary epithelial cancer cells first in vitro and then in vivo. We then extended the platform to study nine different cancer types in vitro. We demonstrate that primary, patient-derived breast cancer cells from biopsies established organoids within HA-MMPx and, relative to Matrigel®, had different growth rates and responses to drugs, underscoring the importance of the extracellular environment to cell fate. We established patient-derived xenografts (PDX) using HA-MMPx in SCID mice and showed superior reproducibility compared to Matrigel®. Fascinatingly, PDX grown in HA-MMPx did not induce resident murine macrophage polarization whereas those grown in Matrigel® showed an increase in the proportion of alternatively activated cells, indicating that Matrigel® itself skewed macrophage polarization. Importantly, HA-MMPx did not bias the immune cell response in vivo and supported a diverse range of organoid phenotypes in vitro.
Statins, a family of FDA-approved cholesterol-lowering drugs that inhibit the rate-limiting enzyme of the mevalonate metabolic pathway, have demonstrated anticancer activity. Evidence shows that ...dipyridamole potentiates statin-induced cancer cell death by blocking a restorative feedback loop triggered by statin treatment. Leveraging this knowledge, we develop an integrative pharmacogenomics pipeline to identify compounds similar to dipyridamole at the level of drug structure, cell sensitivity and molecular perturbation. To overcome the complex polypharmacology of dipyridamole, we focus our pharmacogenomics pipeline on mevalonate pathway genes, which we name mevalonate drug-network fusion (MVA-DNF). We validate top-ranked compounds, nelfinavir and honokiol, and identify that low expression of the canonical epithelial cell marker, E-cadherin, is associated with statin-compound synergy. Analysis of remaining prioritized hits led to the validation of additional compounds, clotrimazole and vemurafenib. Thus, our computational pharmacogenomic approach identifies actionable compounds with pathway-specific activities.
Development of novel multimodality radiotherapy treatments in metastatic breast cancer, especially in the most aggressive triple negative (TNBC) subtype, is of significant clinical interest. Here we ...show that a novel inhibitor of Polo-Like Kinase 4 (PLK4), CFI-400945, in combination with radiation, exhibits a synergistic anti-cancer effect in TNBC cell lines and patient-derived organoids in vitro and leads to a significant increase in survival to tumor endpoint in xenograft models in vivo, compared to control or single-agent treatment. Further preclinical and proof-of-concept clinical studies are warranted to characterize molecular mechanisms of action of this combination and its potential applications in clinical practice.
•PLK4 inhibitor CFI-400945, combined with radiation, shows synergistic antiproliferative activity in immortalized breast cancer cell lines.•CFI-400945 in combination with radiation shows synergistic antiproliferative activity in breast cancer patient-derived organoids.•In MDA-MB-231 xenograft mice, CFI-400945 sensitizes to radiation and significantly improves survival to the tumour endpoint.
Telomerase defers the onset of telomere damage-induced signaling and cellular senescence by adding DNA onto chromosome ends. The ability of telomerase to elongate single-stranded telomeric DNA ...depends on the reverse transcriptase domain of TERT, and also relies on protein:DNA contacts outside the active site. We purified the N-terminus of human TERT (hTEN) from Escherichia coli, and found that it binds DNA with a preference for telomeric sequence of a certain length and register. hTEN interacted with the C-terminus of hTERT in trans to reconstitute enzymatic activity in vitro. Mutational analysis of hTEN revealed that amino acids Y18 and Q169 were required for telomerase activity in vitro, but not for the interaction with telomere DNA or the C-terminus. These mutants did not reconstitute telomerase activity in cells, maintain telomere length, or extend cellular lifespan. In addition, we found that T116/T117/S118, while dispensable in vitro, were required for cellular immortalization. Thus, the interactions of hTEN with telomere DNA and the C-terminus of hTERT are functionally separable from the role of hTEN in telomere elongation activity in vitro and in vivo, suggesting other roles for the protein and nucleic acid interactions of hTEN within, and possibly outside, the telomerase catalytic core.
Eukaryotic telomerase contains a telomerase reverse transcriptase (TERT) and an RNA template component that are essential for telomerase catalytic activity and several other telomerase-associated ...factors of which only a few appear to be integral enzyme components 1–3. The first essential telomerase protein identified was S. cerevisiae Est1p, whose deletion leads to ever-shorter telomeres despite the persistence of telomerase activity 4–6. Extensive genetic and biochemical data show that Est1p, via its interaction with the telomerase RNA and telomere end DNA binding complex Cdc13p/Stn1p/Ten1p, promotes the ability of telomerase to elongate telomeres in vivo 7–22. The characterization of Est1p homologs outside of yeast has not been documented. We report the characterization of two putative human homologs of Est1p, hEST1A and hEST1B. Both proteins specifically associated with telomerase activity in human cell extracts and bound hTERT in rabbit reticulocyte lysates independently of the telomerase RNA. Overproduction of hEST1A cooperated with hTERT to lengthen telomeres, an effect that was specific to cells containing telomerase activity. Like Est1p, hEST1A (but not hEST1B) exhibited a single-stranded telomere DNA binding activity. These results suggest that the telomerase-associated factor Est1p is evolutionarily conserved in humans.
Oxidative stress plays an important role in cancer development and treatment. Recent data implicate the tumor suppressor BRCA1 in regulating oxidative stress, but the molecular mechanism and the ...impact in BRCA1-associated tumorigenesis remain unclear. Here, we show that BRCA1 regulates Nrf2-dependent antioxidant signaling by physically interacting with Nrf2 and promoting its stability and activation. BRCA1-deficient mouse primary mammary epithelial cells show low expression of Nrf2-regulated antioxidant enzymes and accumulate reactive oxygen species (ROS) that impair survival in vivo. Increased Nrf2 activation rescues survival and ROS levels in BRCA1-null cells. Interestingly, 53BP1 inactivation, which has been shown to alleviate several defects associated with BRCA1 loss, rescues survival of BRCA1-null cells without restoring ROS levels. We demonstrate that estrogen treatment partially restores Nrf2 levels in the absence of BRCA1. Our data suggest that Nrf2-regulated antioxidant response plays a crucial role in controlling survival downstream of BRCA1 loss. The ability of estrogen to induce Nrf2 posits an involvement of an estrogen-Nrf2 connection in BRCA1 tumor suppression. Lastly, BRCA1-mutated tumors retain a defective antioxidant response that increases the sensitivity to oxidative stress. In conclusion, the role of BRCA1 in regulating Nrf2 activity suggests important implications for both the etiology and treatment of BRCA1-related cancers.
Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the worst prognosis and few effective therapies. Here we identified MS023, an inhibitor of type I protein ...arginine methyltransferases (PRMTs), which has antitumor growth activity in TNBC. Pathway analysis of TNBC cell lines indicates that the activation of interferon responses before and after MS023 treatment is a functional biomarker and determinant of response, and these observations extend to a panel of human-derived organoids. Inhibition of type I PRMT triggers an interferon response through the antiviral defense pathway with the induction of double-stranded RNA, which is derived, at least in part, from inverted repeat Alu elements. Together, our results represent a shift in understanding the antitumor mechanism of type I PRMT inhibitors and provide a rationale and biomarker approach for the clinical development of type I PRMT inhibitors.
Breast cancer is the most common female cancer, affecting approximately one in eight women during their life- time. Besides environmental triggers and hormones, inherited mutations in the breast ...cancer 1 （BRCA1） or BRCA2 genes markedly increase the risk for the development of breast cancer. Here, using two different mouse models, we show that genetic inactivation of the key osteoclast differentiation factor RANK in the mammary epithelium markedly delayed onset, reduced incidence, and attenuated progression of Brcal;p53 mutation-driven mammary cancer. Long-term pharmacological inhibition of the RANK ligand RANKL in mice abolished the occurrence of Brcal mutation-driven pre-neoplastic lesions. Mechanistically, genetic inactivation of Rank or RANKL/RANK blockade impaired proliferation and expansion of both murine Brcal;p53 mutant mammary stem cells and mammary progenitors from human BRCA1 mutation carriers. In addition, genome variations within the RANK locus were significantly associated with risk of developing breast cancer in women with BRCA1 mutations. Thus, RANKL/ RANK control progenitor cell expansion and tumorigenesis in inherited breast cancer. These results present a viable stratesy for the uossible prevention of breast cancer in BRCA1 mutant i~atients.
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