Cardiac glycoside neriifolin exerts anti-cancer activity in prostate cancer cells by attenuating DNA damage repair through endoplasmic reticulum stress.

Prostate cancer (PCa) is one of the most common cancers in men. Patients with recurrent disease initially respond to androgen-deprivation therapy, but the tumor eventually progresses into castration-resistant PCa. Thus, new therapeutic approaches for PCa resistance to current treatments are urgently needed. Here, we report that cardiac glycoside neriifolin suppresses the malignancy of cancer cells via increasing DNA damage and apoptosis through activation of endoplasmic reticulum stress (ERS) in prostate cancers. We found that cardiac glycoside neriifolin markedly inhibited the cell growth and induced apoptosis in prostate cancer cells. Transcriptome sequence analysis revealed that neriifolin significantly induced DNA damage and double strand breaks (DSBs), validated with attenuation expression of genes in DSBs repair and increasing phosphorylated histone H2AX (γ-H2AX) foci formation, a quantitative marker of DSBs. Moreover, we found that neriifolin also activated ERS, evidenced by upregulation and activation of ERS related proteins, including eukaryotic initiation factor 2α (eIF2α), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and C/EBP homologous protein (CHOP) as well as downregulation of CCAATenhancerbinding protein alpha (C/EBP-α), a transcriptional factor that forms heterodimers with CHOP. In addition, neriifolin treatment dramatically inhibited the by tumor growth, which were reversed by CHOP loss or overexpression of C/EBP-α in nude mice. Mechanistically, neriifolin suppressed the tumor growth by increasing DNA damage and apoptosis through CHOP-C/EBP-α signaling axis of ERS in prostate cancers. Taken together, these results suggest that cardiac glycoside neriifolin may be a potential tumor-specific chemotherapeutic agent in prostate cancer treatment.

The feasibility of using an autologous GM-CSF-secreting breast cancer vaccine to induce immunity in patients with stage II-III and metastatic breast cancers.

PURPOSE: The antigenic targets of immunity and the role of vaccination in breast cancer are unknown. We performed a phase I study of an autologous GM-CSF-secreting breast cancer vaccine in patients with metastatic and stage II-III breast cancer. METHODS: Tumor cells from patients with metastatic (n = 15) and stage II-III (n = 7) disease were transduced with a replication-defective adenoviral vector encoding GM-CSF, and then irradiated. Twelve and seven patients with metastatic and stage II-III disease, respectively, received weekly vaccination for three weeks, followed by every other week until disease progression or vaccine supply was exhausted (metastatic) or until six total vaccine doses were administered (stage II-III). RESULTS: Among those patients with metastatic disease who received vaccinations, eight had progressive disease at two months, three had stable disease for 4-13 months, and one has had no evidence of disease for 13 years. Of the patients with stage II-III disease, five died of metastatic disease between 1.16 and 8.49 years after the start of vaccinations (median 6.24 years) and two are alive as of September 2021. Toxicities included injection site reactions, fatigue, fever, upper respiratory symptoms, joint pain, nausea, and edema. Four of five evaluable patients with metastatic disease developed a skin reaction with immune cell infiltration after the fifth injection of unmodified, irradiated tumor cells. CONCLUSION: We conclude that tumor cells can be harvested from patients with metastatic or stage II-III breast cancer to prepare autologous GM-CSF-secreting vaccines that induce coordinated immune responses with limited toxicity. TRIAL REGISTRATION AND DATE OF REGISTRATION: clinicaltrials.gov, NCT00317603 (April 25, 2006) and NCT00880464 (April 13, 2009).

Activation of the δ opioid receptor relieves cerebral ischemic injury in rats via EGFR transactivation.

Delta opioids are thought to relieve ischemic injury and have tissue-protective properties. However, the detailed mechanisms of delta opioids have not been well identified. Receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR), have been shown to mediate downstream signals of δ opioid receptor (δOR) activation through the metalloproteinase (MMP)-dependent EGF-like growth factor (HB-EGF) excretion pathway, which is called transactivation. In this study, to investigate the role of EGFR in δOR-induced anti-ischemic effects in the brain, we applied the middle cerebral artery occlusion (MCAO) model followed by reperfusion to mimic ischemic stroke injury in rats. Pre-treatment with the δOR agonist [D-ala2, D-leu5] enkephalin (DADLE) improved the neurologic deficits and the decreased infarct volume caused by cerebral ischemia/reperfusion injury, which were blocked by the EGFR inhibitor AG1478 and the MMP inhibitor GM6001, respectively. Further results indicated that DADLE activated EGFR, Akt and ERK1/2 and upregulated EGFR expression in the hippocampus in a time-dependent manner, which were inhibited by AG1478 and GM6001. The enzyme-linked immunosorbent assay (ELISA) results showed that δOR activation led to an increase in HB-EGF release, but HB-EGF in tissue was downregulated at the mRNA and protein levels. Moreover, this protective action caused by δOR agonists may involve attenuated hippocampal cellular apoptosis. Overall, these results demonstrate that MMP-mediated transactivation of EGFR is essential for δOR agonist-induced MCAO/reperfusion injury relief. These findings provide a potential molecular mechanism for the neuroprotective property of δOR and may add new insight into mitigating or preventing injury.

The neoepitope landscape of breast cancer: implications for immunotherapy.

BACKGROUND: Cancer immunotherapy with immune checkpoint blockade (CKB) is now standard of care for multiple cancers. The clinical response to CKB is associated with T cell immunity targeting cancer-induced mutations that generate novel HLA-binding epitopes (neoepitopes). METHODS: Here, we developed a rapid bioinformatics pipeline and filtering strategy, EpitopeHunter, to identify and prioritize clinically relevant neoepitopes from the landscape of somatic mutations. We used the pipeline to determine the frequency of neoepitopes from the TCGA dataset of invasive breast cancers. We predicted HLA class I-binding neoepitopes for 870 breast cancer samples and filtered the neoepitopes based on tumor transcript abundance. RESULTS: We found that the total mutational burden (TMB) was highest for triple-negative breast cancer, TNBC, (median = 63 mutations, range: 2-765); followed by HER-2(+) (median = 39 mutations, range: 1-1206); and lowest for ER/PR(+)HER-2(-) (median = 32 mutations, range: 1-2860). 40% of the nonsynonymous mutations led to the generation of predicted neoepitopes. The neoepitope load (NEL) is highly correlated with the mutational burden (R2 = 0.86). CONCLUSIONS: Only half (51%) of the predicted neoepitopes are expressed at the RNA level (FPKM≥2), indicating the importance of assessing whether neoepitopes are transcribed. However, of all patients, 93% have at least one expressed predicted neoepitope, indicating that most breast cancer patients have the potential for neo-epitope targeted immunotherapy.

The association of genomic lesions and PD-1/PD-L1 expression in resected triple-negative breast cancers.

BACKGROUND: Elevated PD-L1 expression on tumor cells, a context associated with an adaptive immune response, has been linked to the total burden of copy number variants (CNVs) in aneuploid tumors, to microsatellite instability (MSI), and to specific genomic driver lesions, including loss of PTEN, MYC amplification, and activating mutations in driver oncogenes such as KRAS and PIK3CA. Triple-negative breast cancers (TNBCs) typically have high levels of CNVs and diverse driver lesions in their genomes. Thus, there is significant interest in exploiting genomic data to develop predictive immunotherapy biomarkers for patients with TNBC. METHODS: Whole tissue samples from 55 resected TNBCs were screened by immunohistochemistry (IHC) for PD-1 and PD-L1 by using validated antibodies and established scoring methods for staining of tumor and non-tumor cells. In parallel, we interrogated biopsies from each resection with DNA content flow cytometry and sorted the nuclei of diploid, tetraploid, and aneuploid cell populations. CNVs were mapped with CNV oligonucleotide arrays by using purified (>95%) tumor populations. We generated whole exome data for 12 sorted tumor samples to increase the resolution within loci of interest and to incorporate somatic mutations into our genomic signatures. RESULTS AND CONCLUSIONS: PD-L1 staining was detected on tumor cells in 29 out of 54 (54%) evaluable cases and was associated with increased overall survival (P = 0.0024). High levels of PD-1 and PD-L1 (IHC ≥4) were present in 11 out of 54 (20%) and 20 out of 54 (37%) cases with staining of PD-L1 primarily on tumor cells for 17 out of 20 (85%) cases. The latter included tumors with both high (>50) and low (<20) numbers of CNVs. Notably, homozygous deletion of PTEN (n = 6) or activating mutation in PIK3CA (n = 1) was not associated with increased expression of either immune checkpoint activator in TNBC. In contrast, two treatment-naïve cases with EGFR driver amplicons had high PD-L1 tumor staining. High mutational load and predicted neoepitopes were observed in MSI+ and high CNV burden TNBCs but were not associated with high PD-L1 expression on tumor cells. Our results challenge current models of genomic-based immunotherapy signatures yet suggest that discrete genomic lesions may complement existing biomarkers to advance immune checkpoint therapies for patients with TNBC.

JAK2 and PD-L1 Amplification Enhance the Dynamic Expression of PD-L1 in Triple-negative Breast Cancer.

BACKGROUND: Activation of the JAK/STAT pathway is common in triple-negative breast cancer (TNBC) and affects the expression of genes controlling immune signaling. A subset of TNBC cases will have somatic amplification of chromosome 9p24.1, encoding PD-L1, PD-L2, and JAK2, which has been associated with decreased survival. MATERIALS AND METHODS: Eleven TNBC cell lines were evaluated using array comparative genomic hybridization. A copy number gain was defined as an array comparative genomic hybridization log2 ratio of ≥ 1. Cell surface expression of programmed cell death ligand 1 (PD-L1) was detected using flow cytometry and compared with the median fluorescence intensity of isotype control immunoglobulin. To selectively inhibit JAK2, lentiviral vectors encoding 2 different short hairpin RNA (shRNA) were generated. JAK2, STAT1, STAT3, phosphorylated (p) STAT1, and pSTAT3 expression were measured by immunoblot. Statistical significance was defined as P < .05. RESULTS: The cell line HCC70 had 9p24.1 copy number amplification that was associated with both increased JAK2 and pSTAT3; however, knockdown of JAK2 inhibited cell growth independently of 9p24.1 copy number status. In TNBC cell lines with 9p24.1 gain or amplification, PD-L1 expression rapidly and strikingly increased 5- to 38-fold with interferon-γ (P < .05), and inducible PD-L1 expression was completely blocked by JAK2 knockdown and the JAK1/2 inhibitor ruxolitinib. In tumor tissue, expression of interferon-γ-related genes correlated with 9p24.1 copy number status. CONCLUSION: These data suggest that the JAK2/STAT1 pathway in TNBC might regulate the dynamic expression of PD-L1 that is induced in the setting of an inflammatory response. Inhibition of JAK2 might provide a synergistic therapy when combined with other immunotherapies in the subset of TNBC with 9p24.1 amplification.

Development and validation of a novel clinical fluorescence in situ hybridization assay to detect JAK2 and PD-L1 amplification: a fluorescence in situ hybridization assay for JAK2 and PD-L1 amplification.

The amplification of chromosome 9p24.1 encoding PD-L1, PD-L2, and JAK2 has been reported in multiple types of cancer and is associated with poor outcome, upregulation of PD-L1, and activation of the JAK/STAT pathway. We have developed a novel fluorescence in situ hybridization assay which combines 3 probes mapping to 9p24.1 with a commercial chromosome 9 centromere (CEN9) probe for detection of the JAK2/9p24.1 amplification. JAK2 fluorescence in situ hybridization was compared with array-based comparative genomic hybridization in 34 samples of triple negative breast cancer tumor. By array-based comparative genomic hybridization, 15 had 9p24.1 copy-number gain (log2ratio>0.3) and 19 were classified as non-gain (log2ratio≤0.3). Copy-number gain was defined as JAK2/CEN9 ratio ≥1.1 or average JAK2 signals≥3.0. Twelve of 15 samples with copy-number gain by array-based comparative genomic hybridization were also detected by fluorescence in situ hybridization. Eighteen of 19 samples classified as copy-number non-gain by array-based comparative genomic hybridization were concordant by array-based comparative genomic hybridization. The sensitivity and specificity of the fluorescence in situ hybridization assay was 80% and 95%, respectively (P=0.02). The sample with the highest level of amplification by array-based comparative genomic hybridization (log2ratio=3.6) also scored highest by fluorescence in situ hybridization (ratio=8.2). There was a correlation between the expression of JAK2 and amplification status (Mean 633 vs 393, P=0.02), and there was a trend of association with PD-L1 RNA expression (Mean 46 vs 22, P=0.11). No significant association was observed between PD-L1 immunohistochemistry expression and copy-number gain status. In summary, the novel array-based comparative genomic hybridization assay for detection of chromosome 9p24.1 strongly correlates with the detection of copy-number gain by array-based comparative genomic hybridization. In triple negative breast cancer, this biomarker may identify a relevant subset of patients for targeted molecular therapies.

Strong adverse effect of epidermal growth factor receptor 2 overexpression on prognosis of patients with invasive lobular breast cancer: a comparative study with invasive ductal breast cancer in Chinese population.

The data on the outcome of breast invasive lobular carcinoma (ILC) are conflicting. In addition, the prognostic effect of molecular subtypes on ILC remains unclear. In this study, the clinicopathological and prognostic data between 269 ILC and 816 invasive ductal carcinoma (IDC) cases in a Chinese population were extensively compared, with a median follow-up time of 7.8 years. Compared with the IDC group, ILC tumors had more lymph node invasion, hormonal receptor positivity, and human epidermal growth factor receptor 2 (HER2) negativity. ILC patients showed overall survival (OS) and recurrence/metastasis-free survival (RFS) rates similar to those of IDC patients but exhibited worse disease-free survival (DFS) rate because of the higher rate of contralateral breast cancer (BC). Further analysis showed that OS, RFS, and DFS were similar between ILC and IDC patients in the subgroups of luminal A and triple-negative BC with HER2 negativity but were worse in ILC patients than those in IDC patients in the subgroups of luminal B and HER2 overexpression with positive HER2 expression. Multivariate analysis indicated HER2 positivity as an independent risk factor for OS, RFS, and DFS of ILC patients, which increased the risk in the ILC group than that in IDC group. The interaction of HER2 and ILC was also defined as an independent risk factor for OS, RFS, and DFS of the entire population. In conclusion, overexpression of HER2 exhibited stronger negative effect on the prognosis of ILC patients than that in IDC patients, suggesting that treatment targeting HER2 is crucial for this BC subgroup.

[A retrospective analysis of clinic-pathological characteristics and prognostic factor for 137 cases of breast cancer brain metastasis].

OBJECTIVE: To investigate the clinicopathological characteristics and prognosis in breast cancer with brain metastasis (BCBM). METHODS: The clinical data of 137 BCBM from June 2002 to June 2008 was reviewed and analyzed. Their molecular subtypes were categorized based on detection of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) expression. The focal area included 35 cases of triple-negative breast cancer (TNBC), 38 cases of HR (ER and PR) (-)/HER-2(+), 40 cases of HR(+)/HER-2(-), 24 cases of HR(+)/HER-2(+). The clinical characteristics and the outcome in patients with influence were analyzed. RESULTS: In 137 BCBM, the median overal survival after brain metastasis was 6.5 month. The median survivals of TNBC, HR(-)/HER-2(+), HR(+)/HER-2(-) and HR(+)/HER-2(+) were 5.0, 5.5, 10.0 and 9.5 months, respectively. The median survivals after brain metastasis of the breast cancer patients who received the combination therapy of whole brain radiation therapy (WBRT) and neurosurgery and/or stereotactic radiosurgery, received WBRT but not combination therapy and didn't receive WBRT were 15.0, 9.5 and 4.0 months, respectively. In univariate survival analysis, substyle, number of brain metastasis, brain metastasis as initial recurrence or not, brain-only metastases or not, the combination therapy status after brain metastasis were obviously correlated with the prognosis (χ(2) = 6.891 to 29.414, P < 0.05). Substyle (RR = 1.234, 95%CI: 1.057 to 1.440) and the combination therapy status after brain metastasis (RR = 1.838, 95%CI: 1.389 to 2.431) were independent prognostic factor in multivariable analysis (P < 0.05). CONCLUSIONS: TNBC confers a high risk of death after brain metastases. Systemic treatment via combined modalities are helpful for breast cancer patients, even after the detection of brain metastases.