Identification of nuclear export inhibitor-based combination therapies in preclinical models of triple-negative breast cancer

An estimated 284,000 Americans will be diagnosed with breast cancer in 2021. Of these individuals, 15–20% have basal-like triple-negative breast cancer (TNBC), which is known to be highly metastatic. Chemotherapy is standard of care for TNBC patients, but chemoresistance is a common clinical problem. There is currently a lack of alternative, targeted treatment strategies for TNBC; this study sought to identify novel therapeutic combinations to treat basal-like TNBCs. For these studies, four human basal-like TNBC cell lines were utilized to determine the cytotoxicity profile of 1363 clinically-used drugs. Ten promising therapeutic candidates were identified, and synergism studies were performed in vitro. Two drug combinations that included KPT-330, an XPO1 inhibitor, were synergistic in all four cell lines. In vivo testing of four basal-like patient-derived xenografts (PDX) identified one combination, KPT-330 and GSK2126458 (a PI3K/mTOR inhibitor), that decreased tumor burden in mice significantly more than monotherapy with either single agent. Bulk and single-cell RNA-sequencing, immunohistochemistry, and analysis of published genomic datasets found that XPO1 was abundantly expressed in human basal-like TNBC cell lines, PDXs, and patient tumor samples. Within basal-like PDXs, XPO1 overexpression was associated with increased proliferation at the cellular level. Within patient datasets, XPO1 overexpression was correlated with greater rates of metastasis in patients with basal-like tumors. These studies identify a promising potential new combination therapy for patients with basal-like breast cancer.     

Specific Inhibition of the Nuclear Exporter Exportin-1 Attenuates Kidney Cancer Growth

Purpose

Despite the advent of FDA-approved therapeutics to a limited number of available targets (kinases and mTOR), PFS of kidney cancer (RCC) has been extended only one to two years due to the development of drug resistance. Here, we evaluate a novel therapeutic for RCC which targets the exportin-1 (XPO1) inhibitor.

Materials and Methods

RCC cells were treated with the orally available XPO1 inhibitor, KPT-330, and cell viability and Annexin V (apoptosis) assays, and cell cycle analyses were performed to evaluate the efficacy of KPT-330 in two RCC cell lines. Immunoblotting and immunofluorescence analysis were performed to validate mechanisms of XPO1 inhibition. The efficacy and on-target effects of KPT-330 were further analyzed in vivo in RCC xenograft mice, and KPT-330-resistant cells were established to evaluate potential mechanisms of KPT-330 resistance.

Results

KPT-330 attenuated RCC viability through growth inhibition and apoptosis induction both in vitro and in vivo, a process in which increased nuclear localization of p21 by XPO1 inhibition played a major role. In addition, KPT-330 resistant cells remained sensitive to the currently approved for RCC multi-kinase inhibitors (sunitinib, sorafenib) and mTOR inhibitors (everolimus, temsirolimus), suggesting that these targeted therapeutics would remain useful as second line therapeutics following KPT-330 treatment.

Conclusion

The orally-available XPO1 inhibitor, KPT-330, represents a novel target for RCC whose in vivo efficacy approaches that of sunitinib. In addition, cells resistant to KPT-330 retain their ability to respond to available RCC therapeutics suggesting a novel approach for treatment in KPT-330-naïve as well as -resistant RCC patients.     

Antitumor efficacy of XPO1 inhibitor Selinexor in KRAS-mutant lung adenocarcinoma patient-derived xenografts

Gain-of-function Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations occur in 25% of lung adenocarcinomas, and these tumors are challenging to treat. Some preclinical work, largely based on cell lines, suggested KRAS^mut lung cancers are especially dependent on the nuclear export protein exportin-1 (XPO1), while other work supports XPO1 being a broader cancer dependency. To investigate the sensitivity of KRAS^mut lung cancers to XPO1 inhibition in models that more closely match clinical tumors, we treated 10 independently established lung cancer patient-derived tumor xenografts (PDXs) with the clinical XPO1 inhibitor, Selinexor. Monotherapy with Selinexor reduced tumor growth in all KRAS^mut PDXs, which included 4 different codon mutations, and was more effective than the clinical MEK1/2 inhibitor, Trametinib. Selinexor was equally effective in KRAS^G12C and KRAS^G12D tumors, with TP53 mutations being a biomarker for a weaker drug response. By mining genome-wide dropout datasets, we identified XPO1 as a universal cancer cell dependency and confirmed this functionally in two KRAS^WT PDX models harboring kinase drivers. However, targeted kinase inhibitors were more effective than Selinexor in these models. Our findings support continued investigation of XPO1 inhibitors in KRAS^mut lung adenocarcinoma, regardless of the codon alteration.     

Novel Small Molecule XPO1/CRM1 Inhibitors Induce Nuclear Accumulation of TP53, Phosphorylated MAPK and Apoptosis in Human Melanoma Cells

XPO1/CRM1 is a key nuclear exporter protein that mediates translocation of numerous cellular regulatory proteins. We investigated whether XPO1 is a potential therapeutic target in melanoma using novel selective inhibitors of nuclear export (SINE). In vitro effects of SINE on cell growth and apoptosis were measured by MTS assay and flow cytometry [Annexin V/propidium iodide (PI)], respectively in human metastatic melanoma cell lines. Immunoblot analysis was used to measure nuclear localization of key cellular proteins. The in vivo activity of oral SINE was evaluated in NOD/SCID mice bearing A375 or CHL-1 human melanoma xenografts. SINE compounds induced cytostatic and pro-apoptotic effects in both BRAF wild type and mutant (V600E) cell lines at nanomolar concentrations. The cytostatic and pro-apoptotic effects of XPO1 inhibition were associated with nuclear accumulation of TP53, and CDKN1A induction in the A375 cell line with wild type TP53, while pMAPK accumulated in the nucleus regardless of TP53 status. The orally bioavailable KPT-276 and KPT-330 compounds significantly inhibited growth of A375 (p<0.0001) and CHL-1 (p = 0.0087) human melanoma cell lines in vivo at well tolerated doses. Inhibition of XPO1 using SINE represents a potential therapeutic approach for melanoma across cells with diverse molecular phenotypes by promoting growth inhibition and apoptosis.     

Nucleo-cytoplasmic transport as a therapeutic target of cancer

Shuttling of specific proteins out of the nucleus is essential for the regulation of the cell cycle and proliferation of both normal and malignant tissues. Dysregulation of this fundamental process may affect many other important cellular processes such as tumor growth, inflammatory response, cell cycle, and apoptosis. It is known that XPO1 (Exportin-1/Chromosome Region Maintenance 1/CRM1) is the main mediator of nuclear export in many cell types. Nuclear proteins exported to the cytoplasm by XPO1 include the drug targets topoisomerase IIα (topo IIα) and BCR-ABL and tumor suppressor proteins such as Rb, APC, p53, p21, and p27. XPO1 can mediate cell proliferation through several pathways: (i) the sub-cellular localization of NES-containing oncogenes and tumor suppressor proteins, (ii) the control of the mitotic apparatus and chromosome segregation, and (iii) the maintenance of nuclear and chromosomal structures. The XPO1 protein is elevated in ovarian carcinoma, glioma, osteosarcoma, pancreatic and cervical cancer. There is a growing body of research indicating that XPO1 may have an important role as a prognostic marker in solid tumors. Because of this, nuclear export inhibition through XPO1 is a potential target for therapeutic intervention in many cancers. The best understood XPO1 inhibitors are the small molecule nuclear export inhibitors (NEIs; Leptomycin B and derivatives, ratjadones, PKF050-638, valtrate, ACA, CBS9106, selinexor/KPT-330, and verdinexor/KPT-335). Selinexor and verdinexor are orally bioavailable, highly potent, small molecules that are classified as Selective Inhibitors of Nuclear Export (SINE). KPT-330 is the only NEI currently in Phase I/II human clinical trials in hematological and solid cancers. Of all the potential targets in nuclear cytoplasmic transport, the nuclear export receptor XPO1 remains the best understood and most advanced therapeutic target for the treatment of cancer.     

Selinexor Overcomes Hypoxia-Induced Drug Resistance in Multiple Myeloma

Increased levels of the nuclear export protein, exportin 1 (XPO1), were demonstrated in multiple myeloma (MM) patients. Targeting XPO1 with selinexor (the selective inhibitor of nuclear export; SINE compound KPT-330) demonstrates broad antitumor activity also in patient cells resistant to bortezomib; hence, it is a promising target in MM patients. Hypoxia is known to mediate tumor progression and drug resistance (including bortezomib resistance) in MM cells. In this study, we tested the effects of selinexor alone or in combination with bortezomib in normoxia and hypoxia on MM cell survival and apoptosis in vitro and in vivo. In vitro, selinexor alone decreased survival and increased apoptosis, resensitizing MM cells to bortezomib. In vivo, we examined the effects of selinexor alone on tumor initiation and tumor progression, as well as selinexor in combination with bortezomib, on tumor growth in a bortezomib-resistant MM xenograft mouse model. Selinexor, used as a single agent, delayed tumor initiation and tumor progression, prolonging mice survival. In bortezomib-resistant xenografts, selinexor overcame drug resistance, significantly decreasing tumor burden and extending mice survival when combined with bortezomib.     

Genetic and Epigenetic Regulation of TOX3 Expression in Breast Cancer

Genome wide association studies (GWAS) have identified low penetrance and high frequency single nucleotide polymorphisms (SNPs) that contribute to genetic susceptibility of breast cancer. The SNPs at 16q12, close to the TOX3 and CASC16 genes, represent one of the susceptibility loci identified by GWAS, showing strong evidence for breast cancer association across various populations. To examine molecular mechanisms of TOX3 regulation in breast cancer, we investigated both genetic and epigenetic factors using cell lines and datasets derived from primary breast tumors available through The Cancer Genome Atlas (TCGA). TOX3 expression is highly up-regulated in luminal subtype tumors compared to normal breast tissues or basal-like tumors. Expression quantitative trait loci (eQTL) analyses revealed significant associations of rs3803662 and rs4784227 genotypes with TOX3 expression in breast tumors. Bisulfite sequencing of four CpG islands in the TOX3 promoter showed a clear difference between luminal and basal-like cancer cell lines. 5-Aza-2’-deoxycytidine treatment of a basal-like cancer cell line increased expression of TOX3. TCGA dataset verified significantly lower levels of methylation of the promoter in luminal breast tumors with an inverse correlation between methylation and expression of TOX3. Methylation QTL (mQTL) analyses showed a weak or no correlation of rs3803662 or rs4784227 with TOX3 promoter methylation in breast tumors, indicating an independent relationship between the genetic and epigenetic events. These data suggest a complex system of TOX3 regulation in breast tumors, driven by germline variants and somatic epigenetic modifications in a subtype specific manner.     

Annexin A1 Preferentially Predicts Poor Prognosis of Basal-Like Breast Cancer Patients by Activating mTOR-S6 Signaling

IntroductionAnnexin A1 (ANXA1) is an anti-inflammatory protein reported to play a role in cell proliferation and apoptosis, and to be deregulated in breast cancer. The exact role of annexin A1 in the biology of breast cancer remains unclear. We hypothesized that the annexin A1 plays an oncogenic role in basal subtype of breast cancer by modulating key growth pathway(s).MethodsBy mining the Cancer Genome Atlas (TCGA)-Breast Cancer dataset and manipulating annexin A1 levels in breast cancer cell lines, we studied the role of annexin A1 in breast cancer and underlying signaling pathways.ResultsOur in-silico analysis of TCGA-breast cancer dataset demonstrated that annexin A1 mRNA expression is higher in basal subtype compared to luminal and HER2 subtypes. Within the basal subtype, patients show significantly poorer overall survival associated with higher expression of annexin A1. In both TCGA patient samples and cell lines, annexin A1 levels were significantly higher in basal-like breast cancer than luminal and Her2/neu-positive breast cancer. Stable annexin A1 knockdown in TNBC cell lines suppressed the mTOR-S6 pathway likely through activation of AMPK but had no impact on the MAPK, c-Met, and EGFR pathways. In a cell migration assay, annexin A1-depleted TNBC cells showed delayed migration as compared to wild-type cells, which could be responsible for poor patient prognosis in basal like breast cancers that are known to express higher annexin A1.ConclusionsOur data suggest that annexin A1 is prognostic only in patients with basal like breast cancer. This appears to be in part due to the role of annexin A1 in activating mTOR-pS6 pathway.     

Metabotropic Glutamate Receptor-1 Contributes to Progression in Triple Negative Breast Cancer

TNBC is an aggressive breast cancer subtype that does not express hormone receptors (estrogen and progesterone receptors, ER and PR) or amplified human epidermal growth factor receptor type 2 (HER2), and there currently exist no targeted therapies effective against it. Consequently, finding new molecular targets in triple negative breast cancer (TNBC) is critical to improving patient outcomes. Previously, we have detected the expression of metabotropic glutamate receptor-1 (gene: GRM1; protein: mGluR1) in TNBC and observed that targeting glutamatergic signaling inhibits TNBC growth both in vitro and in vivo. In this study, we explored how mGluR1 contributes to TNBC progression, using the isogenic MCF10 progression series, which models breast carcinogenesis from nontransformed epithelium to malignant basal-like breast cancer. We observed that mGluR1 is expressed in human breast cancer and that in MCF10A cells, which model nontransformed mammary epithelium, but not in MCF10AT1 cells, which model atypical ductal hyperplasia, mGluR1 overexpression results in increased proliferation, anchorage-independent growth, and invasiveness. In contrast, mGluR1 knockdown results in a decrease in these activities in malignant MCF10CA1d cells. Similarly, pharmacologic inhibition of glutamatergic signaling in MCF10CA1d cells results in a decrease in proliferation and anchorage-independent growth. Finally, transduction of MCF10AT1 cells, which express c-Ha-ras, using a lentiviral construct expressing GRM1 results in transformation to carcinoma in 90% of resultant xenografts. We conclude that mGluR1 cooperates with other factors in hyperplastic mammary epithelium to contribute to TNBC progression and therefore propose that glutamatergic signaling represents a promising new molecular target for TNBC therapy.     

RB1 Status in Triple Negative Breast Cancer Cells Dictates Response to Radiation Treatment and Selective Therapeutic Drugs

Triple negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which only chemotherapy and radiation therapy are currently available. The retinoblastoma (RB1) tumor suppressor is frequently lost in human TNBC. Knockdown of RB1 in luminal BC cells was shown to affect response to endocrine, radiation and several antineoplastic drugs. However, the effect of RB1 status on radiation and chemo-sensitivity in TNBC cells and whether RB1 status affects response to divergent or specific treatment are unknown. Using multiple basal-like and claudin-low cell lines, we hereby demonstrate that RB-negative TNBC cell lines are highly sensitive to gamma-irradiation, and moderately more sensitive to doxorubicin and methotrexate compared to RB-positive TNBC cell lines. In contrast, RB1 status did not affect sensitivity of TNBC cells to multiple other drugs including cisplatin (CDDP), 5-fluorouracil, idarubicin, epirubicin, PRIMA-1^met, fludarabine and PD-0332991, some of which are used to treat TNBC patients. Moreover, a non-biased screen of ∼3400 compounds, including FDA-approved drugs, revealed similar sensitivity of RB-proficient and -deficient TNBC cells. Finally, ESA⁺/CD24^−/low/CD44⁺ cancer stem cells from RB-negative TNBC lines were consistently more sensitive to gamma-irradiation than RB-positive lines, whereas the effect of chemotherapy on the cancer stem cell fraction varied irrespective of RB1 expression. Our results suggest that patients carrying RB-deficient TNBCs would benefit from gamma-irradiation as well as doxorubicin and methotrexate therapy, but not necessarily from many other anti-neoplastic drugs.     

Targeting XPO1 enhances innate immune response and inhibits KSHV lytic replication during primary infection by nuclear stabilization of the p62 autophagy adaptor protein

Nucleocytoplasmic transport of signaling modulators is essential for regulating cellular responses to extracellular stimulation and stress, as well as pathogen infection. Exportin 1 (XPO1), also known as chromosomal maintenance 1 (CRM1), mediates nuclear export of proteins, rRNAs, snRNAs, and some mRNAs. In this study, we have identified an essential role of XPO1 in regulating Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic replication during primary infection of primary human umbilical vein endothelial cells. Treatment with an XPO1 inhibitor KPT-8602 and short hairpin RNA (shRNA)-mediated knockdown of XPO1 reduced KSHV lytic replication but had no effect on KSHV entry and trafficking. XPO1 inhibition induced retention of autophagy adaptor protein p62 (SQSTM1) in the nucleus, which enhanced activation of TBK1 and IRF3. As a result, nuclear accumulation of p62 increased expression of innate immune-related genes including IRF7, ISG15, IFIT1, IFIT2, and IFIT3, leading to a reduction of KSHV lytic replication. These results illustrate a novel mechanism by which XPO1 mediates innate immune response and KSHV replication, and identify XPO1 as a potential therapeutic target and KPT-8602 as a promising therapeutic agent for KSHV infection.Subject terms: Mechanisms of disease, Infection     

Angiogenesis and Antiangiogenesis in Triple-Negative Breast cancer

Several data support a central role for angiogenesis in breast cancer growth and metastasis. Observational studies have demonstrated that microvascular density (MVD) is a prognostic factor in invasive breast cancer, whereas others reached the opposite conclusion. Vascular endothelial growth factor is the most important angiogenic factor with proven significance in breast cancer, as it has been assessed in both experimental and clinical studies. Triple-negative breast cancer (TNBC) is a type of breast cancer which lacks estrogen, progesterone, and HER-2/neu receptors. MVD in both basal-like and TNBC is significantly higher than in non–basal-like and non-TNBC. In breast cancer and other malignancies, the development of agents that inhibit tumor angiogenesis has been an active area of investigation. In TNBC, clinical trials combining targeted agents and chemotherapy have failed to show substantial survival improvement. There is evidence that patients with TNBC may have a greater probability of obtaining some kind of clinical efficacy benefit from bevacizumab-based therapy.     

LSD1 Overexpression Is Associated with Poor Prognosis in Basal-Like Breast Cancer,and Sensitivity to PARP Inhibition

LSD1, a lysine-specific histone demethylase, is overexpressed in several types of cancers and linked to poor outcomes. In breast cancer, the significance of LSD1 overexpression is not clear. We have performed an in silico analysis to assess the relationship of LSD1 expression to clinical outcome. We demonstrate that LSD1 overexpression is a poor prognostic factor in breast cancer, especially in basal-like breast cancer, a subtype of breast cancer with aggressive clinical features. This link is also observed in samples of triple negative breast cancer. Interestingly, we note that overexpression of LSD1 correlates with down-regulation of BRCA1 in triple negative breast cancer. This phenomenon is also observed in in vitro models of basal-like breast cancer, and is associated with an increased sensitivity to PARP inhibitors. We propose therefore that high expression levels of the demethylase LSD1 is a potential prognostic factor of poor outcome in basal-like breast cancer, and that PARP inhibition may be a therapeutic strategy of interest in this poor prognostic subtype with overexpression of LSD1.     

Midostaurin preferentially attenuates proliferation of triple-negative breast cancer cell lines through inhibition of Aurora kinase family

Background

Breast cancer is classified into three subtypes by the expression of biomarker receptors such as hormone receptors and human epidermal growth factor receptor 2. Triple-negative breast cancer (TNBC) expresses none of these receptors and has an aggressive phenotype with a poor prognosis, which is insensitive to the drugs that target the hormone receptors and human epidermal growth factor receptor 2. It is, thus, required to develop an effective therapeutic reagent to treat TNBC.

Results

The study using a panel of 19 breast cancer cell lines revealed that midostaurin, a multi-target protein kinase inhibitor, suppresses preferentially the growth of TNBC cells comparing with non-TNBC cells. Clustering analysis of the drug activity data for the panel of cancer cell lines predicted that midostaurin shares the target with Aurora kinase inhibitors. Following studies indicated that midostaurin attenuates the phosphorylation reaction mediated by Aurora kinase in the cells and directly inhibits this protein kinase in vitro, and that this reagent induces apoptosis accompanying accumulation of 4N and 8N DNA cells in TNBC cells.

Conclusion

Midostaurin suppresses the proliferation of TNBC cells among the breast cancer cell lines presumably through the inhibition of the Aurora kinase family. The precise study of midostaurin on cell growth will contribute to the development of the drug for the treatment of TNBC.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0150-2) contains supplementary material, which is available to authorized users.     

XPO1 inhibition with selinexor synergizes with proteasome inhibition in neuroblastoma by targeting nuclear export of IkB

Across many cancer types in adults, upregulation of the nuclear-to-cytoplasmic transport protein Exportin-1 (XPO1) correlates with poor outcome and responsiveness to selinexor, an FDA-approved XPO1 inhibitor. Similar data are emerging in childhood cancers, for which selinexor is being evaluated in early phase clinical studies. Using proteomic profiling of primary tumor material from patients with high-risk neuroblastoma, as well as gene expression profiling from independent cohorts, we have demonstrated that XPO1 overexpression correlates with poor patient prognosis. Neuroblastoma cell lines are also sensitive to selinexor in the low nanomolar range. Based on these findings and knowledge that bortezomib, a proteasome inhibitor, blocks degradation of XPO1 cargo proteins, we hypothesized that combination treatment with selinexor and bortezomib would synergistically inhibit neuroblastoma cellular proliferation. We observed that selinexor promoted nuclear retention of IkB and that bortezomib augmented the ability of selinexor to induce cell-cycle arrest and cell death by apoptosis. This synergy was abrogated through siRNA knockdown of IkB. The synergistic effect of combining selinexor and bortezomib in vitro provides rationale for further investigation of this combination treatment for patients with high-risk neuroblastoma.     

CHFR regulates chemoresistance in triple-negative breast cancer through destabilizing ZEB1

Failures to treat triple-negative breast cancer (TNBC) are mainly due to chemoresistance or radioresistance. We and others previously discovered that zinc finger E-box-binding homeobox 1 (ZEB1) is a massive driver causing these resistance. However, how to dynamically modulate the intrinsic expression of ZEB1 during cell cycle progression is elusive. Here integrated affinity purification combined with mass spectrometry and TCGA analysis identify a cell cycle-related E3 ubiquitin ligase, checkpoint with forkhead and ring finger domains (CHFR), as a key negative regulator of ZEB1 in TNBC. Functional studies reveal that CHFR associates with and decreases ZEB1 expression in a ubiquitinating-dependent manner and that CHFR represses fatty acid synthase (FASN) expression through ZEB1, leading to significant cell death of TNBC under chemotherapy. Intriguingly, a small-molecule inhibitor of HDAC under clinical trial, Trichostatin A (TSA), increases the expression of CHFR independent of histone acetylation, thereby destabilizes ZEB1 and sensitizes the resistant TNBC cells to conventional chemotherapy. In patients with basal-like breast cancers, CHFR levels significantly correlates with survival. These findings suggest the therapeutic potential for targeting CHFR-ZEB1 signaling in resistant malignant breast cancers.Subject terms: Cancer therapy, Cell death, Post-translational modifications