Measuring functional HER2-driven signaling status ex vivo to predict response to HER2 therapy: Results from a mouse breast tumor xenograft study.

e23203 Background: Clinical trials indicate a weak correlation between HER2 expression levels and HER2 targeted therapy benefit. Other biological factors, such as HER2 signaling activity, may be important to measure when identifying patients for treatment with HER2 therapies. To measure the HER2 signaling activity of a patient’s live tumor cells, a new assay using an impedance biosensor, the CELx HER2 Signaling Function (CELx HSF) Test, was developed. In this study, we evaluated whether functional HER2 signaling status was more predictive of response to a dual-HER2 kinase inhibitor than HER2 receptor or gene amplification status, using breast tumor xenograft in vivo models. Methods: Two breast cancer cell lines were studied: HCC1954, a HER2+ cell line with normal HER2 signaling according to the CELx HSF Test, and BT483, a HER2- cell line with abnormally high HER2 signaling according to the CELx HSF Test. Thus, the HER2 receptor status of each cell line was opposite its HER2 signaling status. For each cell line, twenty 4-5 week old female NSG mice were injected with two million cells. Mice were randomly assigned to either a control group that received Captisol or a treatment group that received lapatinib at a dose of 20mg/kg daily for 16 days. Results: There was no significant difference in tumor volume between the control and lapatinib-treated groups in the mice injected with the HCC1954 (HER2+, normal HER2 signaling) cells. Average tumor sizes reached 1028.7 ± 166.9 mm3 for the control group and 893.8 ± 111.5 mm3 for the lapatinib-treated group (P = 0.285) by the end of the study. With the BT483 (HER2-, abnormal HER2 signaling) cells, lapatinib treatment significantly slowed down the increase in tumor size. Average tumor sizes reached 328.3 ± 54.9 mm3 for the control group and 192.4 ± 19.4 mm3 for the lapatinib-treated group (P = 0.0049). Conclusions: The results demonstrate that functional HER2-driven signaling status in live tumor cells is more correlative to response to lapatinib in mouse xenograft tumors than HER2 expression level. These findings provide strong evidence that HER2- breast cancer patients with abnormal HER2 signaling may respond to anti-HER2 therapies.

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Investigating New Therapeutic Strategies Targeting Hyperinsulinemia's Mitogenic Effects in a Female Mouse Breast Cancer Model

Endocrinology ◽

10.1210/en.2012-2263 ◽

2013 ◽

Vol 154 (5) ◽

pp. 1701-1710 ◽

Cited By ~ 21

Author(s):

Ran Rostoker ◽

Keren Bitton-Worms ◽

Avishay Caspi ◽

Zila Shen-Orr ◽

Derek LeRoith

Keyword(s):

Breast Cancer ◽

Tumor Growth ◽

Breast Tumor ◽

Experimental Studies ◽

Tumor Development ◽

Treated Group ◽

Tumor Growth Rate ◽

Breast Cancer Patients ◽

Mitogenic Effect

Abstract Epidemiological and experimental studies have identified hyperinsulinemia as an important risk factor for breast cancer induction and for the poor prognosis in breast cancer patients with obesity and type 2 diabetes. Recently it was demonstrated that both the insulin receptor (IR) and the IGF-IR mediate hyperinsulinemia's mitogenic effect in several breast cancer models. Although IGF-IR has been intensively investigated, and anti-IGF-IR therapies are now in advanced clinical trials, the role of the IR in mediating hyperinsulinemia's mitogenic effect remains to be clarified. Here we aimed to explore the potential of IR inhibition compared to dual IR/IGF-IR blockade on breast tumor growth. To initiate breast tumors, we inoculated the mammary carcinoma Mvt-1 cell line into the inguinal mammary fat pad of the hyperinsulinemic MKR female mice, and to study the role of IR, we treated the mice bearing tumors with the recently reported high-affinity IR antagonist-S961, in addition to the well-documented IGF-IR inhibitor picropodophyllin (PPP). Although reducing IR activation, with resultant severe hyperglycemia and hyperinsulinemia, S961-treated mice had significantly larger tumors compared to the vehicle-treated group. This effect maybe secondary to the severe hyperinsulinemia mediated via the IGF-1 receptor. In contrast, PPP by partially inhibiting both IR and IGF-IR activity reduced tumor growth rate with only mild metabolic consequences. We conclude that targeting (even partially) both IR and IGF-IRs impairs hyperinsulinemia's effects in breast tumor development while simultaneously sparing the metabolic abnormalities observed when targeting IR alone with virtual complete inhibition.

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Ductal tree ablation by local delivery of ethanol prevents tumor formation in an aggressive mouse model of breast cancer

Breast Cancer Research ◽

10.1186/s13058-019-1217-x ◽

2019 ◽

Vol 21 (1) ◽

Cited By ~ 1

Author(s):

Elizabeth Kenyon ◽

Jennifer J. Westerhuis ◽

Maximilian Volk ◽

Jeremy Hix ◽

Shatadru Chakravarty ◽

...

Keyword(s):

Breast Cancer ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Prophylactic Mastectomy ◽

Treated Group ◽

Tumor Formation ◽

Mammary Epithelial ◽

Tumor Incidence ◽

Control Group ◽

Untreated Control Group

Abstract Background Prophylactic mastectomy is the most effective intervention to prevent breast cancer. However, this major surgery has life-changing consequences at the physical, emotional, psychological, and social levels. Therefore, only high-risk individuals consider this aggressive procedure, which completely removes the mammary epithelial cells from which breast cancer arises along with surrounding tissue. Here, we seek to develop a minimally invasive procedure as an alternative to prophylactic mastectomy by intraductal (ID) delivery of a cell-killing solution that locally ablates the mammary epithelial cells before they become malignant. Methods After ID injection of a 70% ethanol-containing solution in FVB/NJ female animals, ex vivo dual stained whole-mount tissue analysis and in vivo X-ray microcomputed tomography imaging were used to visualize ductal tree filling, and histological and multiplex immunohistochemical assays were used to characterize ablative effects and quantitate the number of intact epithelial cells and stroma. After ID injection of 70% ethanol or other solutions in cancer-prone FVB-Tg-C3(1)-TAg female animals, mammary glands were palpated weekly to establish tumor latency and examined after necropsy to record tumor incidence. Statistical difference in median tumor latency and tumor incidence between experimental groups was analyzed by log-rank test and logistic mixed-effects model, respectively. Results We report that ID injection of 70% ethanol effectively ablates the mammary epithelia with limited collateral damage to surrounding stroma and vasculature in the murine ductal tree. ID injection of 70% ethanol into the mammary glands of the C3(1)-TAg multifocal breast cancer model significantly delayed tumor formation (median latency of 150 days in the untreated control group [n = 25] vs. 217 days in the ethanol-treated group [n = 13], p value < 0.0001) and reduced tumor incidence (34% of glands with tumors [85 of 250] in the untreated control group vs. 7.3% of glands with tumor [7 of 95] in the ethanol-treated group, risk ratio = 4.76 [95% CI 1.89 to 11.97, p value < 0.0001]). Conclusions This preclinical study demonstrates the feasibility of local ductal tree ablation as a novel strategy for primary prevention of breast cancer. Given the existing clinical uses of ethanol, ethanol-based ablation protocols could be readily implemented in first-in-human clinical trials for high-risk individuals.

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Efficacy, toxicity, and applicability of high-dose sequential chemotherapy as adjuvant treatment in operable breast cancer with 10 or more involved axillary nodes: five-year results.

Journal of Clinical Oncology ◽

10.1200/jco.1997.15.6.2312 ◽

1997 ◽

Vol 15 (6) ◽

pp. 2312-2321 ◽

Cited By ~ 130

Author(s):

A M Gianni ◽

S Siena ◽

M Bregni ◽

M Di Nicola ◽

S Orefice ◽

...

Keyword(s):

Breast Cancer ◽

Standard Dose ◽

Treated Group ◽

Multicenter Trial ◽

Control Group ◽

High Dose ◽

Axillary Nodes ◽

Major Toxicity ◽

Nonhematologic Toxicity

PURPOSE To assess the efficacy, toxicity, and applicability of high-dose therapy administered as adjuvant initial treatment to women with breast cancer with extensive nodal involvement. PATIENTS AND METHODS Sixty-seven patients with stage II to III breast cancer involving > or = 10 axillary nodes received a novel high-dose sequential (HDS) regimen, including the high-dose administration of three non-cross-resistant drugs (cyclophosphamide, methotrexate, and melphalan) given within the shortest interval of time as possible with hematologic and nonhematologic toxicity. RESULTS Sixty-three patients completed the program as planned, one patient died of acute toxicity, and three patients were switched to standard-dose adjuvant therapy. After a median follow-up duration of 48.5 months and a lead follow-up of 78 months, actuarial relapse-free survival for all 67 registered patients is 57% and overall survival is 70%, respectively. Comparison with a historical control group of 58 consecutive patients showed a significantly superior rate of freedom from relapse for the HDS-treated group (57% v 41%, respectively), in particular when two subgroups of patients, more homogeneous for their number of involved nodes, were compared (65% v 42%). Overall, treatment was of short duration (median, 70 days), required a median of 32 days of hospital stay, and was associated with only a few severe side effects (the most distressing being oral mucositis after melphalan therapy). CONCLUSION HDS therapy emerges as an effective and applicable regimen, whose major toxicity was occasional. Final assessment of its value in a randomized, multicenter trial is presently underway.

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Cytotoxicity Assessment of Quassinoids Neosergeolide and Isobrucein B toward Hematopoietic and Solid Malignancies Identifies STAT3 Activation To Be Predictive of Antitumor Response.

Blood ◽

10.1182/blood.v110.11.1394.1394 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1394-1394

Author(s):

Mitsuteru Hiwatari ◽

Jingqiu Dai ◽

Wei Liu ◽

Yu-Dong Zhou ◽

Dale G. Nagle ◽

...

Keyword(s):

Breast Cancer ◽

Cytotoxic Activity ◽

Cell Line ◽

Cell Lines ◽

Strong Support ◽

Luciferase Reporter ◽

Control Group ◽

Antitumor Effects ◽

Protein Levels ◽

Ic50 Values

Abstract Quassinoids are natural product compounds known to possess tumor cytotoxicity and antimalarial activity. Neosergiolide and isobrucein B are two quassinoids previously isolated from roots and stems of Picrolemma sprucei. In screening studies to identify inhibitors that target STAT3, we discovered neosergeolide and isobrucein B as active compounds. Approximately 5000 plant-derived extracts were screened using a cell line that stably expresses a STAT3-dependent luciferase reporter and NPM-ALK, which constitutively induces STAT3 transcriptional activity. Of 25 total hits, a P. sprucei extract was potent and selective for STAT3 inhibition, and bioassay-guided isolation identified neosergeolide and isobrucein B as the inhibitory compounds. Western blot analysis confirmed that neosergeolide and isobrucein B not only inhibit the tyrosine phosphorylation and activation of STAT3 but also decrease total STAT3 protein levels via a mechanism due in part to enhanced proteasome-mediated degradation. Small-molecule proteasome inhibitors such as MG132 and ALLN reversed the ability of the two quassinoids to decrease STAT3 protein levels; furthermore, simultaneous incubation of various hematopoietic malignancy cell lines with either neosergeolide or isobrucein B and MG132 or ALLN antagonized the cytotoxic activity of the quassinoids. Assessment of neosergiolide and isobrucein B antitumor effects using an XTT assay revealed both compounds to possess potent cytotoxic activity across a broad spectrum of hematopoietic malignancies, with T-leukemias/lymphomas being especially responsive. For example, mycosis fungoides (MF)- and Sezary syndrome (SS)-derived cell lines, as well as non-MF/SS cutaneous T-cell lymphoma (CTCL) lines, were potently inhibited by both quassinoids (neosergiolide IC50 values: MAC-1, 11.6 nM; MAC-2A, 6.9 nM; Hut-78, 6.6 nM; HH, 4.3 nM; MJ, 7.0 nM; isobrucein B IC50 values: MAC-1, 31.9 nM; MAC-2A, 72.3 nM; Hut-78, 23.5 nM; HH; 20.3 nM; MJ, 13.5 nM). Non-hematopoietic cell lines representing various solid tumors also exhibited potent cytotoxic responses to the quassinoids (e.g., gastric carcinoma line AGS [neosergiolide IC50: 16.9 nM; isobrucein B IC50: 114.9 nM]). With rare exceptions, the cytotoxicity of the quassinoids against a specific tumor cell line correlated with STAT3 activation status; for example, breast cancer line MCF7 with inactive STAT3 was resistant to both quassinoids even at the maximum concentration tested (6.25 μM), whereas breast cancer lines MDA-MB-468 and MDA-MB-435s with activated STAT3 were inhibited by both compounds at low concentrations (neosergiolide IC50: MDA-MB-435s, 31.3 nM; MDA-MB-468, 29.9 nM; isobrucein B IC50: MDA-MB-435s, 209.3 nM; MDA-MB-468, 356.8 nM). The in vitro antitumor activity of the two quassinoids could also be demonstrated in vivo. For example, isobrucein B (1.0 mg/kg IP once q 3d x 5 doses) could be safely administered and potently inhibited the growth in SCID mice of the CD30+ primary CTCL MAC-1 cell line; mice at treatment day 16 showed average subcutaneous tumor volumes of 3839 ± 863 (s.e.) mm3 in the vehicle-control group and 913 ± 349 (s.e.) mm3 in the isobrucein B group (P=0.008, t-test). These results provide strong support for STAT3 targeting in antitumor drug discovery and suggest that quassinoids may have utility in such an approach.

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Potassium Channels as Novel Pharmacological Targets in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v112.11.4034.4034 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4034-4034 ◽

Cited By ~ 1

Author(s):

Emanuele De Lorenzo ◽

Serena Pillozzi ◽

Marika Masselli ◽

Olivia Crociani ◽

Andrea Becchetti ◽

...

Keyword(s):

Ion Channels ◽

Cell Line ◽

Intracellular Signaling ◽

Molecular Mechanisms ◽

Treated Group ◽

Scid Mice ◽

Control Group ◽

Cell Physiology ◽

Vascular Area

Abstract Targeted therapies are considerably changing the treatment and prognosis of hematologic malignancies. The progressive elucidation of the molecular mechanisms that regulate establishment and progression of tumours is leading to more specific and efficacious pharmacological approaches. In this picture, ion channels represent a relatively unexpected, but very promising players. In particular hERG1 channel expression is altered in many primary leukemias and frequently turn out to exert pleiotropic effects on cancer cell physiology, interaction with the external matrix and stimulation of angiogenesis. hERG1 channels can also trigger intracellular signaling cascades by forming protein complexes with integrins as well as other membrane proteins. These results convey the hypothesis that drugs acting on ion channels could have therapeutic value in the treatment of cancers. Recent evidence suggests that, in certain tumours, application of channel inhibitors does in fact impair cell growth both in vitro and in vivo. A major objection to such a pharmacological approach is the presence of serious side effects, particularly cardiac arrhythmias, especially in the case of hERG1 blockers. This flaw is now being overcome by different approaches, ie the identification of non-arrhythmogenic compounds or calibration of treatment by exploitation of drug selectivity for specific channel states. We tested this possibility in a preclinical model represented by NOD-SCID mice injected with acute leukemia cells and treated with hERG1 blockers. Previous experiments, using NOD/SCID mice injected with AML cells, had shown that herg1 over-expression confers a greater malignancy (Pillozzi S et al, Blood110:1238–50, 2007). The treatment of mice injected with AML cells with specific hERG1 blockers as well as with anti-hERG1 mAb, led to a significant decrease of AML engraftment into the BM and migration into the PB and peripheral organs (Pillozzi S et al, Blood ASH110: 877, 2007). We recently extended our work to an AML cell line stably transfected with the herg1 cDNA (HL60-hERG1), as well as to a ALL cell line (697), which endogenously shows a high herg1 expression. Three groups of treatment were established: control group, E4031-treated group (i.p. starting 1 week after inoculum, 20 mg/kg, daily for 2 weeks) and E4031-treated group (as above, daily until the end of experiment). Various morphometric characteristics of microvessels (density, total vascular area, several size- and shape-related parameters), highlighted through anti-CD34 staining, were quantitated in the BM. Overall, the group of mice treated with hERG1 inhibitors had decreased number of microvessels, decreased total vascular area and size-related parameters. Moreover, E4031 treated mice showed a longer survival compared to the untreated ones. Finally, we evaluated cardiac toxicity in vivo of E4031: no significant variation in ECG parameters were detected, nor gross morphological alterations. Nevertheless, we are also testing different pharmacological categories of hERG1 blockers, such the anti-psychotic drug sertindole, proven to be avoid of any cardiac side effect, despite a strong block of hERG1.

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1Hz and 100mT Electromagnetic Field Induces Apoptosis in Breast Cancer Cells Through Up-Regulation of P38 and P21

10.30699/acadpub.mci.4.1.23 ◽

2020 ◽

Vol 4 (1) ◽

pp. 23-29

Author(s):

Akram Sajadian ◽

Elham Razmpoosh ◽

Farshid Alaeddini ◽

Maryam Bassiri

Keyword(s):

Breast Cancer ◽

Electromagnetic Field ◽

Mrna Expression ◽

Cell Line ◽

Cancer Cell Line ◽

Underlying Mechanism ◽

Expression Level ◽

Control Group ◽

Mrna Expression Level ◽

Sham Exposure

Introduction: Breast cancer is the most common cause of cancer-related death among women. Recently, extremely low-frequency electromagnetic field (ELF-EMF) has been proposed as a new interfering agent with future therapeutic potentials. Many studies have revealed that cellular processes such as apoptosis in breast cancer are affected by ELF-EMFs. However, more researches are needed to clarify the underlying mechanism of action for these fields. In this study, the apoptotic effect of ELF-EMF on the MC4L2 cell line was examined and the mRNA expression level of the P21 and P38 genes were further investigated. Methods: A triple-positive mouse breast cancer cell line (MC4L2) was purchased from the Genetic Resource Center (Iran). This study was performed on two groups of ELF-EMF exposure (100mT/1 Hz for 5 days, 120 min each day) and sham exposure. Cell viability and apoptosis rate of both the exposure and sham exposure groups weredetermined by flow cytometry. Alterations in the P21 and P38 mRNAs expression levelswere investigated; using real-time PCR. Results: ELF-EMF exposure induced 30% apoptosis in MC4L2 cells compared with the control group. The mRNA expression level of P38 and P21 was significantly increased after ELF-EMF exposure compared to the control group. Conclusions: ELF-EMF induces apoptosis in the MC4L2 triple-positive cell line. Furthermore, this exposure affects important gene expression involved in the cell cycle. Our data propose that ELF-EMF in a specific time, intensity, and frequency could be beneficial for breast cancer treatment. However, more studies are required to confirm our findings.

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Metformin suppresses the proliferation and invasion through NF-kB and MMPs in MCF-7 cell line

Turkish Journal of Biochemistry ◽

10.1515/tjb-2019-0197 ◽

2019 ◽

Vol 45 (3) ◽

pp. 295-304

Author(s):

Nail Besli ◽

Guven Yenmis ◽

Matem Tunçdemir ◽

Elif Yaprak Sarac ◽

Sibel Doğan ◽

...

Keyword(s):

Breast Cancer ◽

Cell Proliferation ◽

Cell Line ◽

Cancer Cell Line ◽

Immunocytochemical Staining ◽

Breast Adenocarcinoma ◽

Control Group ◽

Expression Levels ◽

Er Positive ◽

Mcf 7

AbstractObjectiveMCF-7 cells, a breast cancer cell line, are used for experiments of estrogen receptor (ER)-positive breast cancer and many sub-clones representing different classes of ER-positive tumors. We aimed to determine the efficacy of metformin, a potential anti-cancer agent, on the cell proliferation, and the expressions of NF-kB (p65), MMP-2 and MMP-9 in MCF-7 cell line.Materials and methodsMCF-7 cells (human breast adenocarcinoma) were treated with elevating doses of metformin (0–50 mM) for 24 h. The anti-proliferative effect of metformin was studied by BrdU proliferation assay, and the expression levels of NF-kB (p65), MMP-2 and MMP-9 were analyzed by immunocytochemical staining.ResultsThe percentage of cell proliferation was reduced significantly by 10 and 50 mM doses of metformin (p < 0.001). The expression levels of nuclear NF-kB (p65), MMP-9 and MMP-2 were considerably reduced in 50 mM metformin treated cells while the expression of cytoplasmic NF-kB (p65) elevated compared to control group (p < 0.05). Ten millimolar metformin also reduced expression of MMP-9 significantly (p < 0.05).ConclusionMetformin may act on the proliferation, and the processes of invasion and metastasis of MCF-7 cells through blocking NF-kB, which is intensely expressed in breast cancer cells, and through diminishing the expression of MMP-2 and MMP-9 significantly.

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Early efficacy analysis of the AE37 vaccine in patients with HER2 low-expressing and triple-negative breast cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.27_suppl.109 ◽

2012 ◽

Vol 30 (27_suppl) ◽

pp. 109-109 ◽

Cited By ~ 3

Author(s):

Elizabeth Ann Mittendorf ◽

Sonia A. Perez ◽

Diane F. Hale ◽

Timothy J. Vreeland ◽

Alan K. Sears ◽

...

Keyword(s):

Breast Cancer ◽

Risk Reduction ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Phase Iii ◽

Control Group ◽

Her2 Expression ◽

Gm Csf ◽

Helper Epitope ◽

Disease Free

109 Background: Peptide vaccines comprised of HLA class II epitopes, which elicit CD4+ T cell responses, play a critical role in potentiating immune responses. We are conducting a randomized phase II trial of AE37, a hybrid peptide created by the addition of the Ii-Key moiety (LRMK) to the HER2 helper epitope, AE36 (HER2 aa776-790). Here, we present efficacy data focusing on outcomes in patients with low HER2 (IHC 1+ or 2+) expression and triple negative breast cancer (TNBC). Methods: The trial is enrolling node positive or high risk node negative breast cancer patients with any degree of HER2 expression (IHC 1+, 2+ or 3+ or FISH > 1.2) rendered disease-free following standard of care therapy. Patients are randomized to receive either AE37+GM-CSF or GM-CSF alone in 6 monthly intradermal inoculations followed by booster inoculations administered every 6 months. Results: The trial has enrolled 254 patients; 105 in the vaccine group (VG) and 149 in the control group (CG). After a median follow-up of 22.3 months, the disease-free survival (DFS) rate in the VG is 90.3% vs 81.1% in the CG (p=.46), a 49% risk reduction. Evaluating patients with low HER2 expression (IHC 1+ or 2+), there are 53 VG patients and 77 CG patients. The groups are well-matched with respect to the percentage of patients with high grade tumors, tumors > 2cm, the rate of node positivity and ER/PR status (all p>.5). The DFS rate in the VG of low HER2 expressers is 89.8% vs 68.2% in the CG (p=.12), a 68% risk reduction. When limiting analyses to patients with TNBC (ER/PR negative, HER2 1+ or 2+), there are 13 VG patients and 23 CG patients. The groups are again well-matched with the exception of control patients having a larger percentage of tumors > 2 cm (70% vs 31%; p=.02). The DFS rate in the VG of TNBC patients is 83.3% vs 47.6% in the CG (p=.23), a 68% risk reduction. Conclusions: Early analyses suggest clinical benefit to vaccination with AE37, particularly in patients with low HER2-expressing tumors. Importantly, the benefit appears to persist in TNBC patients. Patients will continue to be followed per protocol for 5 years; however, these data suggest that a subsequent phase III trial should evaluate the vaccine in patients with low HER2-expressing disease to include TNBC.

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