Identification of STAG2-Mutant Bladder Cancers by Immunohistochemistry.

Bladder cancer is the fifth most common cancer in the United States. Most bladder cancers are early-stage lesions confined to the mucosa or submucosa and are therefore classified as non-muscle-invasive bladder cancer (NMIBC). A minority of tumors are diagnosed after they have invaded the underlying detrusor muscle and are classified as muscle-invasive bladder cancer (MIBC). Mutational inactivation of the STAG2 tumor suppressor gene is common in bladder cancer, and we and others have recently demonstrated that STAG2 mutation status can be used as an independent prognostic biomarker to predict whether NMIBC will recur and/or progress to MIBC. Here we describe an immunohistochemistry-based assay for identifying the STAG2 mutational status of bladder tumors.

STAG2 as a prognostic biomarker in low-grade non-muscle invasive bladder cancer.

OBJECTIVE: Improvements to bladder cancer risk stratification guidelines are needed to better tailor post-operative surveillance and adjuvant therapy to individual patients. We previously identified STAG2 as a commonly mutated tumor suppressor gene in bladder cancer and an independent predictor of progression in NMIBC. Here we test the value of combining STAG2 immunostaining with other risk stratification biomarkers in NMIBC, and as an individual biomarker in MIBC. MATERIALS AND METHODS: STAG2 immunohistochemistry was performed on a progressor-enriched cohort of tumors from 297 patients with NMIBC, and on tumors from 406 patients with MIBC from Aarhus University Hospital in Denmark. Survival analysis was performed using Kaplan-Meier survival analysis, the log rank test, and Cox proportional hazards models. RESULTS: STAG2-negative low-grade NMIBC tumors were 2.5 times less likely to progress to muscle invasion than STAG2-positive low-grade NMIBC tumors (Log-rank test, P = 0.008). In a composite group of patients with AUA intermediate and high-risk NMIBC tumors, STAG2-negative tumors were less likely to progress (Log-rank test, P = 0.02). In contrast to NMIBC, we show that STAG2 is not useful as a prognostic biomarker in MIBC. CONCLUSIONS: STAG2 immunostaining can be used to subdivide low-grade NMIBC tumors into two groups with substantially different risks of disease progression. Furthermore, STAG2 immunostaining may be useful to enhance NMIBC risk stratification guidelines, though larger cohorts are needed to solidify this conclusion in individual risk groups. STAG2 is not useful as a biomarker in MIBC. Further study of the use of STAG2 immunostaining as a biomarker for predicting the clinical behavior in NMIBC is warranted.

Reciprocal Regulation of DUSP9 and DUSP16 Expression by HIF1 Controls ERK and p38 MAP Kinase Activity and Mediates Chemotherapy-Induced Breast Cancer Stem Cell Enrichment.

Triple-negative breast cancer (TNBC) has a poor prognosis due to its aggressive characteristics and lack of targeted therapies. Cytotoxic chemotherapy may reduce tumor bulk, but leaves residual disease due to the persistence of chemotherapy-resistant breast cancer stem cells (BCSC), which are critical for tumor recurrence and metastasis. Here, we demonstrate that hypoxia-inducible factor (HIF)-1-dependent regulation of mitogen-activated protein kinase (MAPK) signaling pathways contributes to chemotherapy-induced BCSC enrichment. Chemotherapy increased DUSP9 expression and decreased DUSP16 expression in a HIF1-dependent manner, leading to inhibition of ERK and activation of p38 signaling pathways, respectively. Inhibition of ERK caused transcriptional induction of the pluripotency factor Nanog through decreased inactivating phosphorylation of FoxO3, while activation of p38 stabilized Nanog and Klf4 mRNA through increased inactivating phosphorylation of RNA-binding protein ZFP36L1, both of which promoted specification of the BCSC phenotype. Inhibition of HIF1 or p38 signaling blocked chemotherapy-induced pluripotency factor expression and BCSC enrichment. These surprising results delineate a mechanism by which a transcription factor switches cells from ERK to p38 signaling in response to chemotherapy and suggest that therapeutic targeting of HIF1 or the p38 pathway in combination with chemotherapy will block BCSC enrichment and improve outcome in TNBC.Significance: These findings provide a molecular mechanism that may account for the increased relapse rate of women with TNBC who are treated with cytotoxic chemotherapy and suggest that combining chemotherapy with an inhibitor of HIF1 or p38 activity may increase patient survival. Cancer Res; 78(15); 4191-202. ©2018 AACR.

Chemotherapy induces enrichment of CD47+/CD73+/PDL1+ immune evasive triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is treated with cytotoxic chemotherapy and is often characterized by early relapse and metastasis. To form a secondary (recurrent and/or metastatic) tumor, a breast cancer cell must evade the innate and adaptive immune systems. CD47 enables cancer cells to evade killing by macrophages, whereas CD73 and PDL1 mediate independent mechanisms of evasion of cytotoxic T lymphocytes. Here, we report that treatment of human or murine TNBC cells with carboplatin, doxorubicin, gemcitabine, or paclitaxel induces the coordinate transcriptional induction of CD47, CD73, and PDL1 mRNA and protein expression, leading to a marked increase in the percentage of CD47+CD73+PDL1+ breast cancer cells. Genetic or pharmacological inhibition of hypoxia-inducible factors (HIFs) blocked chemotherapy-induced enrichment of CD47+CD73+PDL1+ TNBC cells, which were also enriched in the absence of chemotherapy by incubation under hypoxic conditions, leading to T cell anergy or death. Treatment of mice with cytotoxic chemotherapy markedly increased the intratumoral ratio of regulatory/effector T cells, an effect that was abrogated by HIF inhibition. Our results delineate an HIF-dependent transcriptional mechanism contributing to TNBC progression and suggest that combining chemotherapy with an HIF inhibitor may prevent countertherapeutic induction of proteins that mediate evasion of innate and adaptive antitumor immunity.

Fractalkine induces angiogenic potential in CX3CR1-expressing monocytes.

We report the unique role of CX3CL1 (or fractalkine) on CD11b+ myelomonocytic cells expressing CX3CR1, the only known receptor for CX3CL1, in promoting blood perfusion recovery. In a mouse ischemic hind-limb model, CD11b+ CX3CR1+ cells migrated to ischemic femoral muscles through CX3CL1-mediated chemotaxis. CD11b+ CX3CR1+ macrophages isolated from ischemic tissues [tissue (T)-CD11b+ CX3CR1+ ] of muscle exert a proangiogenic effect through platelet factor-4 (CXCL4; PF-4) production. PF-4 does not promote angiogenesis by itself but, instead, increases VEGF-mediated angiogenesis. Despite proangiogenic effects of muscle-derived T-CD11b+ CX3CR1+ macrophages, their clinical implementation is limited because muscle excision is required for cell harvesting. Therefore, we focused on the more accessible bone marrow (BM)-CD11b+ CX3CR1+ monocytes, which migrate from BM into ischemic muscles via CX3CL1-mediated chemotaxis. PF-4 expression was not detected in BM-CD11b+ CX3CR1+ monocytes under normal conditions, but CX3CL1 (50 ng/ml) induced high PF-4 expression and enabled BM-CD11b+ CX3CR1+ monocytes to achieve a similar angiogenic potential to that of T-CD11b+ CX3CR1+ macrophages ex vivo. Furthermore, we were able to identify a subset of monocytes that express CD11b and CX3CR1 in human peripheral blood and confirmed the proangiogenic effect of CX3CL1 treatment. Thus, CX3CL1-treated CD11b+ CX3CR1+ monocytes may be of potential therapeutic use to significantly accelerate recovery of blood perfusion in ischemic diseases.

The prognostic significance of CD11b+CX3CR1+ monocytes in patients with newly diagnosed diffuse large B-cell lymphoma.

Despite their critical roles in angiogenesis and host immunosuppression within the tumor microenvironment, the prognostic significance of myeloid-lineage cells expressing CD11b and CX3CR1 in diffuse large B-cell lymphoma (DLBCL) has not been well studied. We prospectively enrolled newly-diagnosed DLBCL patients at two Korean institutions between May 2011 and Aug 2015. CD11b+CX3CR1+ cells were analyzed by flow cytometry using peripheral blood (PB) and bone marrow (BM) aspirate samples before treatments. Eighty-nine patients (52 males) were enrolled. The median age was 65 years (range, 19-88 years). Thirty-seven patients (42%) were classified as high-intermediate or high risk according to the National Comprehensive Cancer Network International Prognostic Index (NCCN-IPI). Patients were categorized into either high or low PB-/BM-CD11b+CX3CR1+ monocyte group according to the cutoffs identified by the receiver-operating-characteristics analysis (PB, 3.68%; BM, 3.45%). The high PB-CD11b+CX3CR1+ monocyte group was significantly associated with high-intermediate and high risk NCCN-IPI group (P = 0.004). With a median follow-up of 27.7 months (range, 1.7-60.4 months), the low PB-CD11b+CX3CR1+ monocyte group showed significantly better overall survival (OS) than the high PB-CD11b+CX3CR1+ monocyte group (3-year, 92.3% vs. 51.2%, respectively; P < 0.001). In contrast, no significant difference was observed between the high and low BM-CD11b+CX3CR1+ monocyte groups. Among patients with high-intermediate to high risk NCCN-IPI, the high PB-CD11b+CX3CR1+ monocyte group showed significantly worse OS than the low PB-CD11b+CX3CR1+ monocyte group (3-year, 29.3% vs. 80.2%, respectively; P = 0.008). Taken together, PB-CD11b+CX3CR1+ monocyte percentage correlates with the NCCN-IPI risk stratification, which enables identification of subgroups with extremely poor clinical outcomes.

Chemotherapy-Induced Ca2+ Release Stimulates Breast Cancer Stem Cell Enrichment.

Breast cancer stem cells (BCSCs) play a critical role in tumor recurrence and metastasis. Exposure of breast cancer cells to chemotherapy leads to an enrichment of BCSCs. Here, we find that chemotherapy induces the expression of glutathione S-transferase omega 1 (GSTO1), which is dependent on hypoxia-inducible factor 1 (HIF-1) and HIF-2. Knockdown of GSTO1 expression abrogates carboplatin-induced BCSC enrichment, decreases tumor initiation and metastatic capacity, and delays tumor recurrence after chemotherapy. GSTO1 interacts with the ryanodine receptor RYR1 and promotes calcium release from the endoplasmic reticulum. Increased cytosolic calcium levels activate PYK2 → SRC → STAT3 signaling, leading to increased expression of pluripotency factors and BCSC enrichment. HIF inhibition blocks chemotherapy-induced GSTO1 expression and BCSC enrichment. Combining HIF inhibitors with chemotherapy may improve clinical outcome in breast cancer.

PHGDH Expression Is Required for Mitochondrial Redox Homeostasis, Breast Cancer Stem Cell Maintenance, and Lung Metastasis.

Intratumoral hypoxia stimulates enrichment of breast cancer stem cells (BCSC), which are critical for metastasis and patient mortality. Here we report a metabolic adaptation that is required for hypoxia-induced BCSC enrichment and metastasis. Hypoxia-inducible factors coordinately regulate expression of genes encoding phosphoglycerate dehydrogenase (PHGDH) and five downstream enzymes in the serine synthesis pathway and mitochondrial one-carbon (folate) cycle. RNAi-mediated silencing of PHGDH expression in both estrogen receptor-positive and negative breast cancer cells led to decreased NADPH levels, disturbed mitochondrial redox homeostasis, and increased apoptosis, which abrogated BCSC enrichment under hypoxic conditions. PHGDH-deficient cells exhibited increased oxidant levels and apoptosis, as well as loss of BCSC enrichment, in response to treatment with carboplatin or doxorubicin. PHGDH-deficient cells were relatively weakly tumorigenic and tumors that did form were deficient in BCSCs, abolishing metastatic capacity. Our findings highlight a role for PHGDH in the formation of secondary (recurrent or metastatic) tumors, with potential implications for therapeutic targeting of advanced cancers. Cancer Res; 76(15); 4430-42. ©2016 AACR.