Inflammation is part of our body’s response to tissue injury and pathogens. It helps to recruit various immune cells to the site of inflammation and activates the production of mediators to mobilize ...systemic protective processes. However, chronic inflammation can increase the risk of diseases like cancer. Apart from cytokines and chemokines, lipid mediators, particularly sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), contribute to inflammation and cancer. S1P is an important player in inflammation-associated colon cancer progression. On the other hand, C1P has been recognized to be involved in cancer cell growth, migration, survival, and inflammation. However, whether C1P is involved in inflammation-associated cancer is not yet established. In contrast, few studies have also suggested that S1P and C1P are involved in anti-inflammatory pathways regulated in certain cell types. Ceramide is the substrate for ceramide kinase (CERK) to yield C1P, and sphingosine is phosphorylated to S1P by sphingosine kinases (SphKs). Biological functions of sphingolipid metabolites have been studied extensively. Ceramide is associated with cell growth inhibition and enhancement of apoptosis while S1P and C1P are associated with enhancement of cell growth and survival. Altogether, S1P and C1P are important regulators of ceramide level and cell fate. This review focuses on S1P and C1P involvement in inflammation and cancer with emphasis on recent progress in the field.
About 40,000 American women die from metastatic breast cancer each year despite advancements in treatment. Approximately, 15% of breast cancers are triple-negative for estrogen receptor, progesterone ...receptor, and HER2. Triple-negative cancer is characterized by more aggressive, harder to treat with conventional approaches and having a greater possibility of recurrence. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid signaling mediator has emerged as a key regulatory molecule in breast cancer progression. Therefore, we investigated whether cytosolic sphingosine kinase type 1 (SphK1) and nuclear sphingosine kinase type 2 (SphK2), the enzymes that make S1P are critical for growth and PI3K/AKT, ERK-MAP kinase mediated survival signaling of lung metastatic variant LM2-4 breast cancer cells, generated from the parental triple-negative MDA-MB-231 human breast cancer cell line. Similar with previous report, SphKs/S1P signaling is critical for the growth and survival of estrogen receptor positive MCF-7 human breast cancer cells, was used as our study control. MDA-MB-231 did not show a significant effect of SphKs/S1P signaling on AKT, ERK, and p38 pathways. In contrast, LM2-4 cells that gained lung metastatic phenotype from primary MDA-MB-231 cells show a significant effect of SphKs/S1P signaling requirement on cell growth, survival, and cell motility. PF-543, a selective potent inhibitor of SphK1, attenuated epidermal growth factor (EGF)-mediated cell growth and survival signaling through inhibition of AKT, ERK, and p38 MAP kinase pathways mainly in LM2-4 cells but not in parental MDA-MB-231 human breast cancer cells. Moreover, K-145, a selective inhibitor of SphK2, markedly attenuated EGF-mediated cell growth and survival of LM2-4 cells. We believe this study highlights the importance of SphKs/S1P signaling in metastatic triple-negative breast cancers and targeted therapies.
•Metastasis is the major cause of death of the advanced breast cancer patients.•Although many triple-negative breast cancers respond well to chemotherapy initially, they become aggressive, recur and metastasize once they develop resistance. There are few treatment options when this happens.•Our study highlights the importance of SphKs/S1P signaling in metastatic triple-negative metastatic breast cancers and targeted therapies.
Sphingosine kinase 2 (SphK2) is known to phosphorylate the nuclear sphingolipid metabolite to generate sphingosine‐1‐phosphate (S1P). Nuclear S1P is involved in epigenetic regulation of gene ...expression; however, the underlying mechanisms are not well understood. In this work, we have identified the role of nuclear S1P and SphK2 in regulating hypoxia‐responsive master transcription factors hypoxia‐inducible factor (HIF)‐1α/2α, and their functions in breast cancer, with a focus on triple‐negative breast cancer (TNBC). We have shown SphK2 is associated with HIF‐1α in protein complexes, and is enriched at the promoters of HIF target genes, including vascular endothelial growth factor (VEGF), where it enhances local histone H3 acetylation and transcription. S1P specifically binds to the PAS domains of HIF‐1α. SphK2, and HIF‐1α expression levels are elevated in metastatic estrogen receptor‐positive (ER+) and TNBC clinical tissue specimens compared to healthy breast tissue samples. To determine if S1P formation in the nucleus by SphK2 is a key regulator of HIF functions, we found using a preclinical TNBC xenograft mouse model, and an existing selective SphK2 inhibitor K‐145, that nuclear S1P, histone acetylation, HIF‐1α expression, and TNBC tumor growth were all reduced in vivo. Our results suggest that S1P and SphK2 in the nucleus are linked to the regulation of HIF‐1α/2α functions associated with breast cancer progression, and may provide potential therapeutic targets.
Sphingosine-1-phosphate (S1P), a bioactive sphingolipid mediator, has been implicated in regulation of many processes important for breast cancer progression. Previously, we observed that S1P is ...exported out of human breast cancer cells by ATP-binding cassette (ABC) transporter ABCC1, but not by ABCB1, both known multidrug resistance proteins that efflux chemotherapeutic agents. However, the pathologic consequences of these events to breast cancer progression and metastasis have not been elucidated. Here, it is demonstrated that high expression of ABCC1, but not ABCB1, is associated with poor prognosis in breast cancer patients. Overexpression of ABCC1, but not ABCB1, in human MCF7 and murine 4T1 breast cancer cells enhanced S1P secretion, proliferation, and migration of breast cancer cells. Implantation of breast cancer cells overexpressing ABCC1, but not ABCB1, into the mammary fat pad markedly enhanced tumor growth, angiogenesis, and lymphangiogenesis with a concomitant increase in lymph node and lung metastases as well as shorter survival of mice. Interestingly, S1P exported via ABCC1 from breast cancer cells upregulated transcription of sphingosine kinase 1 (SPHK1), thus promoting more S1P formation. Finally, patients with breast cancers that express both activated SPHK1 and ABCC1 have significantly shorter disease-free survival. These findings suggest that export of S1P via ABCC1 functions in a malicious feed-forward manner to amplify the S1P axis involved in breast cancer progression and metastasis, which has important implications for prognosis of breast cancer patients and for potential therapeutic targets.
Multidrug resistant transporter ABCC1 and activation of SPHK1 in breast cancer worsen patient's survival by export of S1P to the tumor microenvironment to enhance key processes involved in cancer progression.
.
In MCF-7 breast tumor cells, ionizing radiation promoted autophagy that was cytoprotective; pharmacological or genetic interference with autophagy induced by radiation resulted in growth suppression ...and/or cell killing (primarily by apoptosis). The hormonally active form of vitamin D, 1,25D
3
, also promoted autophagy in irradiated MCF-7 cells, sensitized the cells to radiation and suppressed the proliferative recovery that occurs after radiation alone. 1,25D
3
enhanced radiosensitivity and promoted autophagy in MCF-7 cells that overexpress Her-2/neu as well as in p53 mutant Hs578t breast tumor cells. In contrast, 1,25D
3
failed to alter radiosensitivity or promote autophagy in the BT474 breast tumor cell line with low-level expression of the vitamin D receptor. Enhancement of MCF-7 cell sensitivity to radiation by 1,25D
3
was not attenuated by a genetic block to autophagy due largely to the promotion of apoptosis via the collateral suppression of protective autophagy. However, MCF-7 cells were protected from the combination of 1,25D
3
with radiation using a concentration of chloroquine that produced minimal sensitization to radiation alone. The current studies are consistent with the premise that while autophagy mediates a cytoprotective function in irradiated breast tumor cells, promotion of autophagy can also confer radiosensitivity by vitamin D (1,25D
3
). As both cytoprotective and cytotoxic autophagy can apparently be expressed in the same experimental system in response to radiation, this type of model could be utilized to distinguish biochemical, molecular and/or functional differences in these dual functions of autophagy.
The vast majority of mortality in breast cancer results from distant metastasis. Brain metastases occur in as many as 30% of patients with advanced breast cancer, and the 1-year survival rate of ...these patients is around 20%. Pre-clinical animal models that reliably reflect the biology of breast cancer brain metastasis are needed to develop and test new treatments for this deadly condition. The patient-derived xenograft (PDX) model maintains many features of a donor tumor, such as intra-tumor heterogeneity, and permits the testing of individualized treatments. However, the establishment of orthotopic PDXs of brain metastasis is procedurally difficult. We have developed a method for generating such PDXs with high tumor engraftment and growth rates. Here, we describe this method and identify variables that affect its outcomes. We also compare the brain-orthotopic PDXs with ectopic PDXs grown in mammary pads of mice, and show that the responsiveness of PDXs to chemotherapeutic reagents can be dramatically affected by the site that they are in.
Due to the heterogeneous nature of breast cancer, metastasis organotropism has been poorly understood. This study assessed the specific cancer-related gene expression changes occurring with ...metastatic breast cancer recurrence to distant organs compared with non-metastatic breast cancer. We found that several secreted mediators encoding genes notably, LCN2 and S100A8 overexpressed at the distant metastatic site spine (LCN2, 5-fold; S100A8, 6-fold) and bone (LCN2, 5-fold; S100A8, 3-fold) vs. primary tumors in the syngeneic implantation/tumor-resection metastasis mouse model. In contrast, the ESM-1 encoding gene is overexpressed in the primary tumors and markedly downregulated at distant metastatic sites. Further digging into TCAGA-BRCA, SCAN-B, and METABRIC cohorts data analysis revealed that LCN2, S100A8, and ESM-1 mediators encoding individual gene expression scores were strongly associated with disease-specific survival (DSS) in the METABRIC cohort (hazard ratio (HR) > 1, p < 0.0004). The gene expression scores predicted worse clinically aggressive tumors, such as high Nottingham histological grade and advanced cancer staging. Higher gene expression score of ESM-1 gene was strongly associated with worse overall survival (OS) in the triple-negative breast cancer (TNBC) and hormonal receptor (HR)-positive/HER2-negative subtype in METABRIC cohort, HER2+ subtype in TCGA-BRCA and SCAN-B breast cancer cohorts. Our data suggested that mediators encoding genes with prognostic and predictive values may be clinically useful for breast cancer spine, bone, and lung metastasis, particularly in more aggressive subtypes such as TNBC and HER2+ breast cancer.
Abstract
Purpose: Breast cancer most often recurs and metastasizes to the distal organs that had their primary tumors surgically excised. Due to heterogeneous nature of the breast cancer, metastasis ...organotropism has poorly understood. In this study, we assessed the specific cancer-related gene expression changes occurring with metastatic breast cancer recurrence to distant organs comparing with non-metastatic breast cancer. Experimental design: We develop a clinically relevant metastatic syngeneic mouse model using two cell lines 4T1-luc2 that can only metastasize to lung and 4T1.2-luc2 metastasized to bone and lung. After the primary mammary tumor surgically excised from mice, we monitor mice using IVIS imaging for metastatic tumor lesion development in the distant organs followed by MRI scan to detect spinal bone metastases. Animals were sacrificed to collect spines, bones, and lung lesions for molecular analysis. We also validate the gene expression data using the RNA-seq cohort data from the Roswell Park Cancer Institute patient samples with advanced breast cancer bone metastases and matched non-metastatic breast cancer.
Results: Our cell lines, RNA-seq data analysis suggested that there are at least 50 genes which are statistically highly expressed in 4T1.2-luc2 compared to 4T1-luc2 cells. Out of which only a few genes are well established for cancer metastasis biology, include ANGPTL77, SERPINE2, TSPAN11, ESM1, LCN2 which are expressed more than 8 fold in 4T1.2-luc2 compared to 4T1-luc2 cells. To our surprise, we found that most of these genes are differentially expressed when metastasized to distant organ. Our data revealed that LCN2 (7 fold) and CD133 (above 20 fold) are overexpressed in the spine and bone compared to the primary or lung met lesions formed by 4T1.2-luc2. Conversely, LCN2 and CD133 genes are downregulated in breast cancer lung metastasis tissues, whereas CD151, EPHA2, and TWIST1 genes are highly overexpressed in lung metastatic lesion compared to primary, bone or spine. Further, the RNA-seq data of patient samples explained that LCN2, CD133 expressions are significantly higher (8 out of 10 patients) in advanced breast cancer bone metastases compared with matching non-metastatic breast cancer. Conclusion: Our data suggested that LCN2, CD133 would be a prognostic marker for breast cancer spinal bone metastasis, whereas CD151, TWIST1, EPHA2 would be a prognostic marker for lung metastasis and organ-specific relapse.
Citation Format: Aparna Maiti, Nitai C. Hait. LCN2, CD133 expressions in breast cancer spinal bone metastasis and CD151, TWIST1, EPHA2 in lung metastasis: Prognostic value and organ-specific relapse abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 39.
Abstract
Triple-negative breast cancers (TNBCs) have increased expression of hypoxia-inducible factor 1α (HIF1α) and HIF-direct genes that associated with decreased overall survival in patients. ...Previously we have shown that nuclear sphingosine-1-phosphate (S1P), a lipophilic pro-cancer signal mediator, a product of the catalytic reaction by sphingosine kinase 2 (SphK2), directly binds to and inhibits histone deacetylases-1 and -2 (HDAC1 and 2), thus, altered epigenetic landscape in breast cancer cells. SphK2/S1P signaling is essential for metastatic TNBC cell growth and survival. We hypothesize that SphK2/S1P epigenetically regulates HIFα functions in TNBC cells; TNBC subset with elevated SphK2 and HIF1α would be a marker for SphK2-mediated HIFα targeted therapy. METABRIC cohort gene expression analysis revealed that the mRNA expression of HIF1α mRNA and downstream angiogenesis gene, vascular endothelial growth factor B (VEGFB) are associated with overall poor survival for TNBC patients compared to non-TNBC patients. Here, we demonstrate that a subset of TNBC patient tissue samples nuclei has elevated HIF1α and SphK2 protein expressions. SphK2/S1P markedly enhances hypoxia-induced HIFα proximal promoter histone acetylation. SphK2/S1P is found in the nuclear protein complexes with HIFα. Moreover, S1P is docked well at the PASB domain of HIFα and facilitate HIFα-HIF1β heterodimer formation in hypoxia. SphK2/S1P markedly augmented HIF1α direct target gene HIF2α and VEGFA promoter occupancy in hypoxia. Down-regulation of SphK2 with siRNA markedly attenuated transcription of HIFα-direct target genes associated with cancer stem cells, angiogenesis, and metastasis. In agreement with an oncogenic role of SphK2, selective inhibition of SphK2 markedly reduced nuclear histone acetylation, HIF1α expression, and in vivo human TNBC tumor growth in mice. Our data strongly suggested that TNBC subset with elevated SphK2 and HIF1α would be a good candidate for SphK2 targeted therapy. Supported by Health Research, Inc. (HRI) Grant 71-4084-01 (NCH).
Citation Format: Nitai C. Hait, Aparna Maiti. Nuclear S1P binds PASB domain of HIFα to facilitate HIFα-HIF1β heterodimer formation in hypoxic TNBC cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3337.
Abstract
The hypoxia-inducible transcription factors (HIF)-1α and -2α play a critical role in cellular response to hypoxia in solid tumors. Elevated HIF-α expression correlates with poor patient ...survival in a large number of cancers including breast cancers. HIF-α activates expression of genes promoting angiogenesis, metastasis, increased tumor growth and resistance to treatments. Understanding transcriptional regulation HIF-α under physiologically relevant hypoxic conditions would be helpful to reduce breast cancer growth. Our recent studies identified that intracellular bioactive sphingosine-1-phosphate (S1P) generated from nuclear sphingosine kinase 2 (SphK2) acts as an endogenous modulator of histone deacetylases (HDACs) epigenetically regulates gene transcription in breast cancer cells. We wonder whether nuclear S1P epigenetically regulates acetylated histones, positive transcription marks in hypoxia and regulate transcription of HIF-α and target genes in breast cancer cells. We have used estrogen receptor positive human breast cancer MCF-7 cells, triple-negative human breast cancer MDA-231 cells, and lung metastatic LM-2-4 cells derived from MDA-231 cells cells for our studies. We found that hypoxic condition enhances nuclear bulk histone acetylation in breast cancer cells and enhances HIF-α synthesis. Down regulation of SphK2 with siRNA or using selective inhibitor of SphK2 (K145 compound) reduces nuclear S1P as well as bulk histone acetylation and consequently reduces nuclear HIF-α synthesis in breast cancer cells. Downregulation of cytosolic sphingosine kinase 1 (SphK1) with siRNA reduces cytosolic S1P does not alter nuclear bulk histone acetylation neither significantly alter nuclear HIF-α synthesis in breast cancer cells. We also found that targeting SphK2 with K145 compound dramatically reduces in vitro tumorosphere formation in breast cancer cells. Our data suggested that targeting the SphK2/S1P signaling could represent an attractive strategy for therapeutic intervention in hypoxic breast cancers. Supported by Health Research, Inc. (HRI) Grant 71-4084 (NCH), National Cancer Institute Grant R01CA160688 (KT).
Citation Format: Nitai C. Hait, Aparna Maiti. SphK2/S1P axis regulates hypoxia-mediated HIF-α synthesis in breast cancer cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1388. doi:10.1158/1538-7445.AM2017-1388
PDF
