Flotillin-1 contributes to invasion and metastasis in triple negative breast cancer (TNBC) and is modified post-translationally through palmitoylation. Palmitoylation, the process of conjugating ...palmitoyl-CoA to proteins, plays an essential role in protein stability and trafficking. Thus far, there has not been any investigation into the role of flotillin-1 palmitoylation in the context of metastasis in vivo. To address the role of flotillin-1 palmitoylation in metastasis, MDA-MB-231 cells expressing palmitoylation defective flotillin-1 constructs were used as models. Compared to flotillin-1 WT expressing tumors, flotillin-1 palmitoylation defective displayed abrogated tumor progression and lung metastasis in vivo in both spontaneous and experimental models. Further mechanistic investigation led to the identification of zDHHC5 as the main palmitoyl acyltransferase responsible for palmitoylating endogenous flotillin-1. Modulation of flotillin-1 palmitoylation status through mutagenesis, zDHHC5 silencing, and 2-bromopalmitate inhibition all resulted in the proteasomal degradation of flotillin-1 protein. To assess if flotillin-1 palmitoylation can be inhibited for potential clinical relevance, we designed a competitive peptide fused to a cell penetrating peptide sequence, which displayed efficacy in blocking flotillin-1 palmitoylation in vitro without altering palmitoylation of other zDHHC5 substrates, highlighting its specificity. Additionally, TNBC xenograft tumor models expressing a doxycycline inducible flotillin-1 palmitoylation inhibiting peptide displayed attenuated tumor growth and lung metastasis. Collectively, these results reveal a novel palmitoylation dependent mechanism which is essential for the stability of flotillin-1 protein. More specifically, disruption of flotillin-1 palmitoylation through mutagenesis or competitive peptide promoted flotillin-1 protein degradation, subsequently impeding its tumor promoting and metastasis-inducing effects in TNBC tumor models.
We evaluated the role of everolimus in the prevention of ductal carcinoma
(DCIS) to invasive ductal carcinoma (IDC) progression.
The effects of everolimus on breast cancer cell invasion, DCIS ...formation, and DCIS progression to IDC were investigated in a 3D cell culturing model, intraductal DCIS xenograft model, and spontaneous MMTV-Her2/neu mouse model. The effect of everolimus on matrix metalloproteinase 9 (MMP9) expression was determined with Western blotting and IHC in these models and in patients with DCIS before and after a window trial with rapamycin. Whether MMP9 mediates the inhibition of DCIS progression to IDC by everolimus was investigated with knockdown or overexpression of MMP9 in breast cancer cells.
Everolimus significantly inhibited the invasion of human breast cancer cells
. Daily intragastric treatment with everolimus for 7 days significantly reduced the number of invasive lesions from intraductal DCIS foci and inhibited DCIS progression to IDC in the MMTV-Her2/neu mouse mammary tumor model. Mechanistically, everolimus treatment decreased the expression of MMP9 in the
and
models, and in breast tissues from patients with DCIS treated with rapamycin for 1 week. Moreover, overexpression of MMP9 stimulated the invasion, whereas knockdown of MMP9 inhibited the invasion of breast cancer cell-formed spheroids
and DCIS
. Knockdown of MMP9 also nullified the invasion inhibition by everolimus
and
.
Targeting mTORC1 can inhibit DCIS progression to IDC via MMP9 and may be a potential strategy for DCIS or early-stage IDC therapy.
To overcome resistance to antihormonal and HER2-targeted agents mediated by cyclin D1-CDK4/6 complex, we proposed an oral combination of the HER2 inhibitor tucatinib, aromatase inhibitor letrozole, ...and CDK4/6 inhibitor palbociclib (TLP combination) for treatment of HR+/HER2+ metastatic breast cancer (MBC).
Phase Ib/II TLP trial (NCT03054363) enrolled patients with HR+/HER2+ MBC treated with ≥2 HER2-targeted agents. The phase Ib primary endpoint was safety of the regimen evaluated by NCI CTCAE version 4.3. The phase II primary endpoint was efficacy by median progression-free survival (mPFS).
Forty-two women ages 22 to 81 years were enrolled. Patients received a median of two lines of therapy in the metastatic setting, 71.4% had visceral disease, 35.7% had CNS disease. The most common treatment-emergent adverse events (AE) of grade ≥3 were neutropenia (64.3%), leukopenia (23.8%), diarrhea (19.0%), and fatigue (14.3%). Tucatinib increased AUC10-19 hours of palbociclib 1.7-fold, requiring palbociclib dose reduction from 125 to 75 mg daily. In 40 response-evaluable patients, mPFS was 8.4 months, with similar mPFS in non-CNS and CNS cohorts (10.0 months vs. 8.2 months; P = 0.9). Overall response rate was 44.5%, median duration of response was 13.9 months, and clinical benefit rate was 70.4%; 60% of patients were on treatment for ≥6 months, 25% for ≥1 year, and 10% for ≥2 years. In the CNS cohort, 26.6% of patients remained on study for ≥1 year.
TLP combination was safe and tolerable. AEs were expected and manageable with supportive therapy and dose reductions. TLP showed excellent efficacy for an all-oral chemotherapy-free regimen warranting further testing. See related commentary by Huppert and Rugo, p. 4993.
Purpose
Cancer stem cells (CSCs) are highly tumorigenic, spared by chemotherapy, sustain tumor growth, and are implicated in tumor recurrence after conventional therapies in triple negative breast ...cancer (TNBC). Lysine-specific histone demethylase 1A (KDM1A) is highly expressed in several human malignancies and CSCs including TNBC. However, the precise mechanistic role of KDM1A in CSC functions and therapeutic utility of KDM1A inhibitor for treating TNBC is poorly understood.
Methods
The effect of KDM1A inhibition on cell viability, apoptosis, and invasion were examined by Cell Titer Glo, Caspase 3/7 Glo, and matrigel invasion assays, respectively. Stemness and self-renewal of CSCs were examined using mammosphere formation and extreme limiting dilution assays. Mechanistic studies were conducted using RNA-sequencing, RT-qPCR, Western blotting and reporter gene assays. Mouse xenograft and patient derived xenograft models were used for preclinical evaluation of KDM1A inhibitor.
Results
TCGA data sets indicated that KDM1A is highly expressed in TNBC. CSCs express high levels of KDM1A and inhibition of KDM1A reduced the CSCs enrichment in TNBC cells. KDM1A inhibition reduced cell viability, mammosphere formation, self-renewal and promoted apoptosis of CSCs. Mechanistic studies suggested that IL6-JAK-STAT3 and EMT pathways were downregulated in KDM1A knockdown and KDM1A inhibitor treated cells. Importantly, doxycycline inducible knockout of KDM1A reduced tumor progression in orthotopic xenograft models and KDM1A inhibitor NCD38 treatment significantly reduced tumor growth in patient derived xenograft (PDX) models.
Conclusions
Our results establish that KDM1A inhibition mitigates CSCs functions via inhibition of STAT3 and EMT signaling, and KDM1A inhibitor NCD38 may represent a novel class of drug for treating TNBC.
Abstract Sacituzumab Govitecan (SG) is an antibody-drug conjugate that has demonstrated efficacy in patients with TROP-2 expressing epithelial cancers. In a xenograft model of intracranial breast ...cancer, SG inhibited tumor growth and increased mouse survival. We conducted a prospective window-of-opportunity trial (NCT03995706) at the University of Texas Health Science Center at San Antonio to examine the intra-tumoral concentrations and intracranial activity of SG in patients undergoing craniotomy for breast cancer with brain metastases (BCBM) or recurrent glioblastoma (rGBM). We enrolled 25 patients aged ≥18 years diagnosed with BCBM and rGBM to receive a single intravenous dose of SG at 10 mg/kg given one day before resection and continued on days 1 and 8 of 21-day cycles following recovery. The PFS was 8 months and 2 months for BCBM and rGBM cohorts, respectively. The OS was 35.2 months and 9.5 months, respectively. Grade≥3 AE included neutropenia (28%), hypokalemia (8%), seizure (8%), thromboembolic event (8%), urinary tract infection (8%) and muscle weakness of the lower limb (8%). In post-surgical tissue, the median total SN-38 was 249.8 ng/g for BCBM and 104.5 ng/g for rGBM, thus fulfilling the primary endpoint. Biomarker analysis suggests delivery of payload by direct release at target site and that hypoxic changes do not drive indirect release. Secondary endpoint of OS was 35.2 months for the BCBM cohort and 9.5 months for rGBM. Non-planned exploratory endpoint of ORR was 38% for BCBM and 29%, respectively. Exploratory endpoint of Trop-2 expression was observed in 100% of BCBM and 78% of rGBM tumors. In conclusion, SG was found to be well tolerated with adequate penetration into intracranial tumors and promising preliminary activity within the CNS. Trial Registration: Trial (NCT03995706) enrolled at Clinical Trials.gov as Neuro/Sacituzumab Govitecan/Breast Brain Metastasis/Glioblastoma/Ph 0: https://clinicaltrials.gov/study/NCT03995706?cond=NCT03995706 .
Despite improvements in overall survival, only a modest percentage of patients survives high-risk medulloblastoma. The devastating side effects of radiation and chemotherapy substantially reduce ...quality of life for surviving patients. Here, using genomic screens, we identified miR-584-5p as a potent therapeutic adjuvant that potentiates medulloblastoma to radiation and vincristine. MiR-584-5p inhibited medulloblastoma growth and prolonged survival of mice in pre-clinical tumor models. MiR-584-5p overexpression caused cell cycle arrest, DNA damage, and spindle defects in medulloblastoma cells. MiR-584-5p mediated its tumor suppressor and therapy-sensitizing effects by targeting HDAC1 and eIF4E3. MiR-584-5p overexpression or HDAC1/eIF4E3 silencing inhibited medulloblastoma stem cell self-renewal without affecting neural stem cell growth. In medulloblastoma patients, reduced expression of miR-584-5p correlated with increased levels of HDAC1/eIF4E3. These findings identify a previously undefined role for miR-584-5p/HDAC1/eIF4E3 in regulating DNA repair, microtubule dynamics, and stemness in medulloblastoma and set the stage for a new way to treat medulloblastoma using miR-584-5p.
Abstract Protein imprinting in hydrogels is a method to produce materials capable of selective recognition and capture of a target protein. Here we report on the imprinting of fluorescently-labeled ...maltose binding protein (MBP) in acrylamide (AAm)/N-isopropylacrylamide (NIPAm) hydrogels. The targeting efficiency and selectivity of protein recognition is usually characterized by the imprinting factor, which in the simplest case is the ratio of protein uptake in an imprinted film divided by the uptake by the corresponding non-imprinted film. Our objective in this work is to study the dynamics of protein binding and elution in imprinted and non-imprinted films to elucidate the processes that control protein recognition. Protein elution from imprinted and non-imprinted films suggests that imprinting results in sites with a distribution of binding energies, and that only a relatively small fraction of these sites exhibit strong binding.
BRCA1-associated basal-like breast cancer originates from luminal progenitor cells. Breast epithelial cells from cancer-free BRCA1 mutation carriers are defective in luminal differentiation. However, ...how BRCA1 deficiency leads to lineage-specific differentiation defect is not clear. BRCA1 is implicated in resolving R-loops, DNA-RNA hybrid structures associated with genome instability and transcriptional regulation. We recently showed that R-loops are preferentially accumulated in breast luminal epithelial cells of BRCA1 mutation carriers. Here, we interrogate the impact of a BRCA1 mutation-associated R-loop located in a putative transcriptional enhancer upstream of the ERα-encoding ESR1 gene. Genetic ablation confirms the relevance of this R-loop-containing region to enhancer-promoter interactions and transcriptional activation of the corresponding neighboring genes, including ESR1, CCDC170 and RMND1. BRCA1 knockdown in ERα+ luminal breast cancer cells increases intensity of this R-loop and reduces transcription of its neighboring genes. The deleterious effect of BRCA1 depletion on transcription is mitigated by ectopic expression of R-loop-removing RNase H1. Furthermore, RNase H1 overexpression in primary breast cells from BRCA1 mutation carriers results in a shift from luminal progenitor cells to mature luminal cells. Our findings suggest that BRCA1-dependent R-loop mitigation contributes to luminal cell-specific transcription and differentiation, which could in turn suppress BRCA1-associated tumorigenesis.
Obesity is associated with a worse breast cancer prognosis and elevated levels of inflammation, including greater cyclooxygenase-2 (COX-2) expression and activity in adipose-infiltrating macrophages. ...The product of this enzyme, the proinflammatory eicosanoid prostaglandin E2 (PGE2), stimulates adipose tissue aromatase expression and subsequent estrogen production, which could promote breast cancer progression. This study demonstrates that daily use of a nonsteroidal anti-inflammatory drug (NSAID), which inhibits COX-2 activity, is associated with reduced estrogen receptor α (ERα)-positive breast cancer recurrence in obese and overweight women. Retrospective review of data from ERα-positive patients with an average body mass index of >30 revealed that NSAID users had a 52% lower recurrence rate and a 28-month delay in time to recurrence. To examine the mechanisms that may be mediating this effect, we conducted in vitro studies that utilized sera from obese and normal-weight patients with breast cancer. Exposure to sera from obese patients stimulated greater macrophage COX-2 expression and PGE2 production. This was correlated with enhanced preadipocyte aromatase expression following incubation in conditioned media (CM) collected from the obese-patient, sera-exposed macrophages, an effect neutralized by COX-2 inhibition with celecoxib. In addition, CM from macrophage/preadipocyte cocultures exposed to sera from obese patients stimulated greater breast cancer cell ERα activity, proliferation, and migration compared with sera from normal-weight patients, and these differences were eliminated or reduced by the addition of an aromatase inhibitor during CM generation. Prospective studies designed to examine the clinical benefit of NSAID use in obese patients with breast cancer are warranted.
We conducted a first-in-human dose-escalation study with the oral FASN inhibitor TVB-2640 to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D), as monotherapy and with a ...taxane.
This completed open-label outpatient study was conducted at 11 sites in the United States and United Kingdom. Patients with previously-treated advanced metastatic solid tumors and adequate performance status and organ function were eligible. TVB-2640 was administered orally daily until PD. Dose escalation initially followed an accelerated titration design that switched to a standard 3 + 3 design after Grade 2 toxicity occurred. Disease-specific cohorts were enrolled at the MTD. Statistical analyses were primarily descriptive. Safety analyses were performed on patients who received at least 1 dose of study drug. (Clinicaltrials.gov identifier NCT02223247)
The study was conducted from 21 November 2013 to 07 February 2017. Overall, 136 patients received TVB-2640, 76 as monotherapy (weight-based doses of 60 mg/m2 to 240 mg/m2 and flat doses of 200 and 250 mg) and 60 in combination, (weight-based doses of 60 mg/m2 to 100 mg/m2 and flat dose of 200 mg) (55 paclitaxel, 5 docetaxel). DLTs with TVB-2640 were reversible skin and ocular effects. The MTD/RP2D was 100 mg/m2. The most common TEAEs (n,%) with TVB-2640 monotherapy were alopecia (46; 61%), PPE syndrome (35; 46%), fatigue (28; 37%), decreased appetite (20; 26%), and dry skin (17; 22%), and with TVB-2640+paclitaxel were fatigue (29 ; 53%), alopecia (25; 46%), PPE syndrome (25; 46%), nausea (22; 40%), and peripheral neuropathy (20; 36%). One fatal case of drug-related pneumonitis occurred with TVB-2640+paclitaxel; no other treatment-related deaths occurred. Target engagement (FASN inhibition) and inhibition of lipogenesis were demonstrated with TVB-2640. The disease control rate (DCR) with TVB-2640 monotherapy was 42%; no patient treated with monotherapy had a complete or partial response (CR or PR). In combination with paclitaxel, the PR rate was 11% and the DCR was 70%. Responses were seen across multiple tumor types, including in patients with KRASMUT NSCLC, ovarian, and breast cancer.
TVB-2640 demonstrated potent FASN inhibition and a predictable and manageable safety profile, primarily characterized by non-serious, reversible adverse events affecting skin and eyes. Further investigation of TVB-2640 in patients with solid tumors, particularly in KRASMUT lung, ovarian, and breast cancer, is warranted.
This trial was funded by 3-V Biosciences, Inc. (now known as Sagimet Biosciences Inc.).
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