Unresponsive Breast Cancer Research Articles

Abstract 696: Sensitizing breast cancers to immune checkpoint inhibitors through CD27 agonism and vaccination against tumor-associated antigen

Abstract Project objective: Despite the recent revolution in immune checkpoint inhibitors (ICIs), only modest improvement in overall survival and likely caused by not enough potent cellular immunity among BC patients. Our lab has been focus on inducing cellular immunity against HER2+ BC through vaccination against the tumor-associated antigen HER2. Approximately 20 years ago, we performed an experimental pilot study by administrating HER2 peptide and recombinant protein pulsed dendritic cells (DC vaccine) to six patients with refractory HER2+ advanced or metastatic (stage II (≥ 6 +LN), III, or stage IV) BC. We followed the patients on 2019 found that all of the six patients were still alive, 18 years after vaccination. Their blood sample were analyzed with cytometry by time-of-flight (CyTOF) and found there is a significantly increased presence of CD27 expressing memory T cells in response to HER2 peptide stimulation. Recent report on the SARS-CoV2 mRNA vaccine also suggested that CD27 expressing memory T cells plays a critical role in long-lasting cellular immunity against SARS-CoV2 infection. Therefore, we hypothesized that CD27 plays a critical role in cellular immunity against BC, and the stimulation of CD27 expressing T cells with mAb targeting CD27 significantly increase the cellular immunity triggered by vaccination against tumor-associated antigen. Results: We recapitulate the rise of CD27+ antigen specific T cells among the vaccinated patients using a transgenic mouse model expressing human CD27. When combined the adenoviral-vector based HER2 (Ad-HER2) vaccination with a single dose of human aCD27 antibody (Varlilumab), we found there is a robust increase in the HER2 specific T cells compared to vaccination alone, especially CD27+CD44+ memory CD4 T cells, even after 120 days post vaccination. Using an ICI-insensitive syngeneic HER2+ BC models, we found 50% of mice in the combination group of aCD27 antibody plus Ad-HER2 showed total tumor regression by the end of study. When combined with anti-PD1 antibody, the combination of AdHER2 and Varlilumab leads to total tumor regression in 90% of tumor bearing mice with syngeneic HER2+ BC, indicating that the vaccination against tumor associated antigen HER2 plus anti-CD27 antibody sensitized ICI-insensitive HER2+ BC toward ICI. Conclusions: Our data demonstrates that the administration of anti-CD27 antibody significantly increase the long term presence of CD27+ antigen specific memory T cells after vaccination against tumor associated antigen HER2. As consequence, combination of anti-CD27 with HER2 sensitized the immune unresponsive breast cancer toward anti-PD1 antibody. Our study suggests that the vaccination against tumor-associated antigen with mAb targeting CD27 leads to the robust cellular immunity, which is required for successful ICIs against breast cancer. Citation Format: Bin-Jin Hwang, Erika Crosby, Timothy Trotter, Li-Chung Tsao, Tao Wang, Xiao Yang, Herbert Kim Lyerly, Zachary Hartman. Sensitizing breast cancers to immune checkpoint inhibitors through CD27 agonism and vaccination against tumor-associated antigen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 696.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 620: Evidence for combination tamoxifen and betulinic acid to treat hormone responsive- and unresponsive breast cancer by attenuation of Pygopus expression

Abstract The presence of the estrogen and progesterone hormone receptors (ER and PR) in advanced breast cancer tumour nuclei is predictive for response to hormone disruptive therapy. However, the accuracy of ER and PR testing can be variable and although patients receive significant benefit from hormone therapy, they ultimately become refractory to treatment. The chromatin remodeling protein: human Pygopus2 (hPygo2), is important for oncogenic growth and cell cycle progression and may serve as a diagnostic or targetable biomarker for breast cancer. Here we demonstrate that ER-alpha (Erα)−Sp1 transcription factor (Sp1) complexes associate with chromatin at adjacent motifs within the hPygo2 promoter in both hormone receptor positive and negative breast cancer cells. Erα agonists induced, while antagonists inhibited hPygo2 mRNA and protein expression. Erα and Sp1 occupancy at the hPygo2 promoter required intact promoter binding sites and functional DNA binding domains. Estrogen (E2) increased, while Fulvestrant decreased, Erα binding to the hPygo2 promoter with no effect on Sp1. Furthermore, Sp1-directed siRNA reduced Sp1 occupancy at the hPygo2 promoter and decreased hPygo2 mRNA and protein expression in both Erα-expressing (+) and Erα-nonexpressing (-) cells. Betulinic acid, an Sp1 inhibitor, caused cell cycle arrest, reduced Sp1 binding to the hPygo2 promoter and reduced hPygo2 expression in Erα- cell lines. The activation of hPygo2 expression either cooperatively or independently by Erα and Sp1 suggests their potential diagnostic and therapeutic benefit for both hormone responsive and unresponsive breast cancer. Citation Format: Youlian Tzenov, Philip Andrews, Cathy Popadiuk, Kenneth R. Kao. Evidence for combination tamoxifen and betulinic acid to treat hormone responsive- and unresponsive breast cancer by attenuation of Pygopus expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 620. doi:10.1158/1538-7445.AM2014-620

Association of hypoxia inducible factor-1 alpha and carbonic anhydrase IX overexpression and survival in HER2/neu positive hormone-unresponsive breast cancer patients

e22078 Background: Hypoxia is common in solid tumors with overexpression of hypoxia inducible factor-1 alpha (HIF-1 alpha) and carbonic anhydrase IX (CA IX). In our study, we evaluated HIF-1alpha and CA IX expression in Her-2/neu positive hormone- unresponsive breast cancer patients. Methods: One hundred and eleven patients with operable breast cancer were identified who had paraffin-embedded tissue blocks available. Estrogen and progesterone receptors and Her-2/neu expression were evaluated by immunohistochemistry (IHC). FISH was used for IHC 2+ cases. We assigned two groups with 56 patients who have ER and PR receptor positive, Her-2/neu negative (group 1) and with 55 patients who have ER and PR negative, Her-2/neu positive (group 2). HIF 1 alpha and CA IX expressions were investigated and results were compared for both groups. Additionally, we investigated the association between HIF 1 alpha and CA IX expressions with stage, grade, lymph node metastasis, tumor dimension and menopause status. Results: Median age of the patients was 52 years (range, 34–77) in group 1 and 47 years (range, 27–83) in group 2. Nuclear HIF-1alpha expression was detected in 26 (46.4%) and 46 patients (83.6%) in group 1 and 2, respectively (p<0.0001). Membranous CA IX expression was detected in 25 (46.4%) and 37 (67.3%) patients in group 1 and 2, respectively (p<0.013). In group 1, median disease free survival (DFS) has not been reached. In group 2, median DFS was 46 months (95%CI: 42.98–49.02) (p= 0.0308). In group 1, median overall survival (OS) was 108 months (95%CI: 90.65–125.35) whereas in group 2, median OS was 75 months (95 % CI: 64.94- 85.06) (p= 0.0339). Conclusions: HIF-1 alpha and CA IX overexpressions were more frequently observed in ER and PR negative, Her-2/neu positive breast cancer. This was associated with shorter disease-free and overall survival. No significant financial relationships to disclose.

Benzo-[a]-pyrene increases invasion in MDA-MB-231 breast cancer cells via increased COX-II expression and prostaglandin E2 (PGE2) output

Benzo-[a]-pyrene (B[a]P), a carcinogenic component of cigarette smoke, has been shown to increase both COX-II expression and prostaglandin output in vascular smooth muscle and oral epithelial cells. In addition, invasive breast cancer cells have been reported to over express COX-II and PGE(2). Therefore, the objective of this study was to quantify the effect of increasing B[a]P concentrations on COX-II expression, PGE(2) output, and invasion using MDA-MB-231 cells, an invasive estrogen unresponsive breast cancer cell line. B[a]P significantly increased invasion in MDA-MB-231 cells at concentrations greater than 4 x 10(-8) M. Treatment of MDA-MB-231 cells with Vomitoxin (a selective COX-II inducer) enhanced invasion whereas co-treatment with NS398 (a selective COX-II inhibitor) attenuated B[a]P-induced invasion in MDA-MB-231 cells. Immunohistochemical staining and Western blots demonstrated a significant B[a]P treatment-induced increase in both the number of COX-II immunopositive MDA-MB-231 cells and COX-II protein levels. Moreover, B[a]P-treatment induced a profound (46 fold) increase in PGE(2) production by MDA-MB-231 cells. The aryl hydrocarbon receptor (AhR) antagonists resveratrol (RES) and alpha-naphthaflavone (alpha-NF) had no effect on their own, whereas B[a]P-induced invasion was significantly inhibited by co-treatment with RES and alpha-NF. Our data demonstrate that B[a]P-induced changes in invasion are mediated through augmented COX-II expression and PGE(2) production involving an AhR regulated pathway. Moreover, these results suggest a potential role for the AhR signalling pathway in breast cancer invasion.

IRS-1 expression and activation are not sufficient to activate downstream pathways and enable IGF-I growth response in estrogen receptor negative breast cancer cells.

IGF-responsive breast cancer cells activate insulin receptor substrate (IRS)-1 after IGF-I treatment. To determine if IRS-1 expression was sufficient to enable IGF-responsiveness, two IGF-I unresponsive breast cancer cell lines (MDA-MB-435A and MDA-MB-468) were transfected with IRS-1. While IGF-I caused tyrosine phosphorylation of IRS-1 in both transfected cell lines, increased MAP kinase activity was not seen. IGF-I treatment of 435A IRS-1 transfected cells resulted in minimal increased PI3 kinase activity associated with IRS-1, while IRS-2/PI3 kinase was greatly reduced. In MDA-MB-468 IRS-1 transfected cells, IGF-I caused increased IRS-1 associated PI3 kinase activity compared to parental cells, but at levels far below those observed in IGF-responsive MCF-7 cells. The transfected cells were also not responsive to IGF-I in monolayer growth. Thus, IRS-1 expression and activation alone are insufficient to mediate a proliferative response to IGF-I in breast cancer cells, and it is likely that maximal activation of downstream signaling pathways must also occur.

Pro-protein convertase gene expression in human breast cancer.

As a first step towards elucidating the role that pro-protein convertases play in the growth regulation of breast cancer, we studied the gene expression of 6 known human convertase members (PC1/PC3, PC2, furin/PACE, PACE4, PC5/PC6 and PC7/LPC) in human breast cancer tumors and cell lines. PC1, furin, PACE4 and PC7 mRNAs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification in all 7 human breast cancer cell lines and 30 breast tumor tissues tested. PC5 expression was detected in 2/30 tumor tissues. PC2 mRNA, however, was not detected. In situ hybridization localized furin mRNA to the tumor cells; adjacent fibrous stroma and blood vessel elements were negative for furin gene expression. Thirty breast tumors with varying quantities of estrogen and progesterone receptors were assayed for furin, PACE4 and PC1 mRNAs by quantitative RT-PCR, and 22 tumors were assayed for PC7 mRNA. An apparent association was observed only between PACE4 and estrogen receptors. No statistically significant correlation was found between the levels of steroid receptors and the expression of human furin, PCI and PC7 genes. Convertase mRNA levels appeared similar in both the estrogen-responsive and -unresponsive breast cancer cell lines. Also, proprotein convertase mRNAs were not detected in 9 histologically normal human breast tissues. These results suggest that elevated expression of some members of the pro-protein convertase gene family is a characteristic of human breast cancer, an event which may be important for human breast tumorigenesis.

Pro‐protein convertase gene expression in human breast cancer

As a first step towards elucidating the role that pro-protein convertases play in the growth regulation of breast cancer, we studied the gene expression of 6 known human convertase members (PC1/PC3, PC2, furin/PACE, PACE4, PC5/PC6 and PC7/LPC) in human breast cancer tumors and cell lines. PC1, furin, PACE4 and PC7 mRNAs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification in all 7 human breast cancer cell lines and 30 breast tumor tissues tested. PC5 expression was detected in 2/30 tumor tissues. PC2 mRNA, however, was not detected. In situ hybridization localized furin mRNA to the tumor cells; adjacent fibrous stroma and blood vessel elements were negative for furin gene expression. Thirty breast tumors with varying quantities of estrogen and progesterone receptors were assayed for furin, PACE4 and PC1 mRNAs by quantitative RT-PCR, and 22 tumors were assayed for PC7 mRNA. An apparent association was observed only between PACE4 and estrogen receptors. No statistically significant correlation was found between the levels of steroid receptors and the expression of human furin, PC1 and PC7 genes. Convertase mRNA levels appeared similar in both the estrogen-responsive and -unresponsive breast cancer cell lines. Also, pro-protein convertase mRNAs were not detected in 9 histologically normal human breast tissues. These results suggest that elevated expression of some members of the pro-protein convertase gene family is a characteristic of human breast cancer, an event which may be important for human breast tumorigenesis.Int. J. Cancer 71: 966-971, 1997. © 1997 Wiley-Liss Inc.

Hormonal regulation of estrogen receptor messenger ribonucleic acid in T47Dco and MCF-7 breast cancer cells.

Using a combination of hormone-binding assays, immunologic techniques, and mRNA hybridizations we have measured the estrogen receptor (ER) content and studied the hormonal regulation of ER mRNA in one estrogen responsive and one estrogen unresponsive breast cancer cell line, MCF-7 and T47Dco, respectively. Estradiol binding could be detected in cytosol from MCF-7 cells but not in T47Dco cells. However, when measured by an enzyme-linked immunosorbent assay, T47Dco cells were found to contain approximately 15 fmol ER/mg cytosolic protein or 10% of the ER content in MCF-7 cells. Immunologically reactive ER in T47Dco cells was indistinguishable in size (approximately equal to 68 KD) from the ER in MCF-7 cells, as shown by Western blotting using a monoclonal antihuman ER antibody. Quantification of ER mRNA in MCF-7 and T47Dco cells indicated that T47Dco cells contained approximately 50% of the ER mRNA levels found in MCF-7 cells. This basal level of ER mRNA in T47Dco cells was not decreased by estradiol treatment, as opposed to in MCF-7 cells where estradiol caused 40-60% decrease in the ER mRNA expression. Also, estradiol did not increase the progesterone receptor (PR) mRNA levels in T47Dco cells whereas in MCF-7 cells an approximately 5-fold increase of the PR mRNA levels occurred after estradiol treatment. However, incubation of the cells with the synthetic progestin R5020 decreased the ER mRNA levels to approximately the same degree in both cell lines. In conclusion, we have shown that estrogen down-regulates ER mRNA and up-regulates PR mRNA in MCF-7 cells. Neither of these estrogenic effects were seen in T47Dco cells. It appears that the steroid-resistance in T47Dco cells does not occur as a consequence of a complete absence of ER mRNA or protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen and oncogene mediated growth regulation of human breast cancer cells

The mechanism of growth control in estrogen-dependent and -independent human breast cancer is not completely understood. We have used both hormonally responsive and unresponsive breast cancer cells in culture to study the role of estrogens, oncogenes, and growth factors in their malignant transformation. MCF-7, an estrogen-receptor containing cell line, requires estradiol for tumor formation in vivo and is growth stimulated by estradiol and growth inhibited by antiestrogens in vitro. The growth regulation of MCF-7 cells by estrogens and antiestrogens may be linked to changes in several growth-related enzymes and polypeptide growth factors. Growth-acting polypeptides that are estradiolinducible include IGF-I, TGF-α, and PDGF. Induction of at least two growth-related enzymes, thymidine kinase and dihydrofolate reductase is by transcriptional regulation of their mRNAs. To understand the natural progression of human breast cancer, we have experimentally constructed a hormone-independent fully tumorigenic cell line from the non-tumorigenic MCF-7 cells by introduction of an activated oncogene, v-ras h , into these cells by DNA-mediated gene transfer. Acquisition of the activated ras gene confers hormone autonomy on the previously hormone-dependent tumorigenicity and results in upregulation in secretion of some of the growth factors in amounts compared to estradiol stimulation. The transfected cells also become refractory to growth regulation by estradiol and antiestrogens in culture, although estrogen responses persist. Hormone-independent breast cancer cells in culture show high constitutive growth factor secretion. Direct infusion of some of the authentic growth factors and medium conditioned by estrogen-independent cells into athymic ovariectomized mice suggests a direct involvement of some of the polypeptides in the in vivo progression of tumors by these cells. Thus, aberrant production of growth factors, triggered either by activated oncogenes and estrogen stimulation in hormone-dependent cells, or by increased constitutive production in hormone-independent cells may in an autocrine, paracrine, or endocrine manner be associated with neoplastic growth of breast cancer.