Bone Imaging in Metastatic Breast Cancer HANIAOKA, Tsuyoshi; MADEWELL, John E; PODOLOFF, Donald A ...
Journal of clinical oncology,
07/2004, Letnik:
22, Številka:
14
Journal Article
Recenzirano
Bone is the most common site to which breast cancer metastasizes. Imaging-by skeletal scintigraphy, plain radiography, computed tomography, or magnetic resonance imaging-is an essential part, and ...positron emission tomography or single-photon emission computed tomography have a potential of evaluating bone metastases, but no consensus exists as to the best modality for diagnosing the lesion and for assessing its response to treatment. Imaging bone metastases is problematic because the lesions can be osteolytic, osteoblastic, or mixed, and imaging modalities are based on either direct anatomic visualization of the bone or tumor or indirect measurements of bone or tumor metabolism. Although bone metastases can be treated, their response to treatment is considered "unmeasurable" according to existing response criteria. Therefore, the process by which oncologists and radiologists diagnose and monitor the response of bone metastases needs revision, and the current inability to assess the response of bone metastases excludes patients with breast cancer and bone disease from participating in clinical trials of new treatments for breast cancer. In this review of the MEDLINE literature, we discuss the pros and cons of each modality for diagnosing bone metastases and for assessing their response to treatment and we present a practical approach for diagnosis and assessment of bone metastasis.
Paclitaxel stabilizes microtubules, causing mitotic arrest and activating the spindle assembly checkpoint. We determined whether suppression of the checkpoint genes Mad2 and BubR1 affects paclitaxel ...resistance and whether overexpression of Mad2 protein in checkpoint-defective cells enhances paclitaxel sensitivity. Suppression of Mad2 and BubR1 in paclitaxel-treated cancer cells abolished checkpoint function, resulting in paclitaxel resistance that correlated with suppression of cyclin-dependent kinase-1 activity. In contrast, overexpression of Mad2 in cells with a checkpoint defect attributable to low Mad2 expression restored checkpoint function, resulting in enhanced paclitaxel sensitivity that correlated with enhanced cyclin-dependent kinase-1 activity. However, overexpression of Mad2 failed to enhance paclitaxel sensitivity via checkpoint activation in Mad2-independent checkpoint-defective and -intact cells. Thus, checkpoint function is required for paclitaxel sensitivity. These findings show that any molecules that could interfere with the spindle assembly checkpoint could generate paclitaxel resistance in any patient.
Abstract
BACKGROUND: CDK4/6 regulates the G1-S phase transition by phosphorylating the retinoblastoma protein (Rb). Given their potent clinical efficacy, CDK4/6 inhibitors used in combination with ...hormone receptor (HR) blockade (with an aromatase inhibitor or fulvestrant) are emerging as the standard of care for patients with metastatic HR-positive breast cancers. The CDK4/6 inhibitors palbociclib and ribociclib are FDA-approved for use in HR-positive breast cancer patients, and abemaciclib is currently in phase III trials. We observed that approximately 74% (25/34) of breast cancer cell lines had high phosphorylated Rb (phospho-Rb) expression levels and that triple-negative breast cancer (TNBC) cell lines often expressed phospho-Rb, suggesting that targeting phospho-Rb via CDK4/6 inhibition may be effective against TNBC. The histone deacetylase (HDAC) inhibitors increase p21Cip1 levels, promoting proteasomal degradation of cyclin B1 and resulting in G2/M arrest. Entinostat is an oral, class 1, selective HDAC inhibitor currently in phase III testing in HR-positive breast cancer. Preclinical and clinical data demonstrate that entinostat, in combination with HR blockade, has anticancer activity. Our group recently reported that entinostat combined with other anticancer drugs induced apoptosis via induction of proapoptotic proteins such as Noxa and Bim in breast cancer cell lines. Based on these findings, we hypothesized that entinostat-induced apoptosis and palbociclib-induced cell cycle arrest synergize to produce enhanced antitumor effects in estrogen receptor (ER)-positive breast cancer and TNBC cell lines with high phospho-Rb expression levels.
METHODS: We assessed the combination antitumor effects and their mechanisms via CellTiter Blue and sulforhodamine B assays, flow cytometry, apoptosis (caspase 3/7) assays, anchorage-independent growth assays, Western blotting, reverse phase protein array (RPPA), and mammary fat pad xenograft mouse models.
RESULTS: RPPA data showed that ER-positive and TNBC cell lines more often expressed phospho-Rb than did other breast cancer cell subtypes (7/10 and 8/17 cell lines, respectively). We found that the combination of entinostat and palbociclib synergistically inhibited tumor cell proliferation (combinational index less than 1.0), reduced in vitro colony formation (P < 0.05), inhibited in vivo tumor growth in ER-positive MCF-7 breast cancer cells (P < 0.05), and inhibited tumor growth in TNBC xenograft mouse models (MDA-MB-231) more effectively than did either drug alone.
CONCLUSION: Taken together, our data provide evidence that combining entinostat with palbociclib enhances the antitumor effects of these drugs. Along with our continued effort to determine predictive biomarkers, our findings justify conducting a clinical trial of combination treatment with entinostat and palbociclib in patients with ER-positive breast cancer or TNBC.
Citation Format: Lee J, Lim B, Pearson T, Tripathy D, Ordentlich P, Ueno NT. The synergistic antitumor activity of entinostat (MS-275) in combination with palbociclib (PD 0332991) in estrogen receptor-positive and triple-negative breast cancer abstract. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-15.
Abstract
Inflammatory breast cancer (IBC) accounts for 2% of breast cancers but 10% of breast cancer-related deaths in the US. Clinical hallmarks of IBC are tumor cell emboli in lymphatic vessels and ...overexpression of E-cadherin, which promotes cell clustering. Given these hallmarks, IBC is thought to spread via collective invasion and cell clusters. However, we showed that IBC cells underwent epithelial-to-mesenchymal transition (EMT) and metastasized through EMT. Thus, there are two contradictory theories of IBC metastasis. The objectives of this study were 1) to propose a model that reconciles these two models, and 2) to identify target molecules for inhibition of IBC metastasis. Methods: We previously showed that Matrigel culture induced EMT-like changes in SUM149 IBC cells. To test if this transformation from epithelial (E) to mesenchymal (M) in Matrigel culture is unique to IBC cells, a panel of breast cancer cells was cultured in both monolayer and Matrigel-coated plates. The cells were IBC (SUM149, SUM190, KPL4, IBC3), triple-negative breast cancer (TNBC) (MDA-MB-231, MDA-MB-468), and ER+ (MCF7) cells. Phenotypic changes in morphology and expression of EMT markers (E-cadherin, vimentin) were captured with bright field and immunofluorescent (IF) images, respectively. For genome-wide and targeted transcriptional analysis, SUM149 cells cultured in monolayer and Matrigel were processed using DNA microarrays and Taqman qRT-PCR. To correlate the Matrigel gene signature with M features in human breast cancer, a human breast cancer data set was hierarchically clustered with the Matrigel gene signature. Results: SUM149 cells showed a remarkable phenotypic change from E in monolayer culture to M in Matrigel. IF analysis confirmed induction of vimentin expression in Matrigel but stable expression of E-cadherin (thus, we refer to this state as E/M hybrid). This trend was also observed with SUM190 cells. Using qRT-PCR, we confirmed downregulation of E-cadherin and upregulation of M markers (vimentin, Twist1, Snail1, ZEB2) in Matrigel-cultured SUM149 and SUM190 cells compared to monolayer-cultured cells. DNA microarray transcriptional analysis confirmed this trend in SUM149 cells. TNBC has more M-like features than other breast cancer subtypes. Given this evidence, we clustered human breast cancer data using overexpressed genes in Matrigel-cultured SUM149 cells. We identified a cluster of 20 genes in TNBC samples and, assuming that these genes are drivers of E to E/M transition, chose the inflammation-related gene CSF1 as a candidate. The CSF1/CSF1R axis was inhibited by a CSF1R inhibitor, BLZ945; moreover, treatment with BLZ945 reversed the EMT changes in cells in Matrigel culture. Treatment with 5 μM BLZ945 re-induced E-cadherin expression and suppressed Snail1 and Twist1 expression in Matrigel-cultured SUM149 cells. Conclusion: IBC cells are more prone to undergo transition from E to E/M phenotype in Matrigel culture than are cells of other breast cancer subtypes. The CSF1/CSF1R axis plays a role in this E to E/M transition, thus warranting testing its significance using an in vivo IBC model. Phenotypic transition and reversion between E and E/M phenotypes could be a new paradigm that reconciles two contradictory models of IBC metastasis.
Citation Format: Kai K, Iwamoto T, Zhang D, Rao AUK, Thompson A, Sen S, Ueno NT. CSF1/CSF1R axis reprograms epithelial-to-mesenchymal phenotypes in inflammatory breast cancer abstract. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-14-02.
Trastuzumab (Herceptin) is a recombinant humanized monoclonal antibody directed against HER-2. The objective response rate to trastuzumab monotherapy is 12-34% for a median duration of 9 months, by ...which point most patients become resistant to treatment. We created two trastuzumab-resistant (TR) pools from the SKBR3 HER-2-overexpressing breast cancer cell line to study the mechanisms by which breast cancer cells escape trastuzumab-mediated growth inhibition. Both pools maintained her-2 gene amplification and protein overexpression. Resistant cells demonstrated a higher S-phase fraction by flow cytometry and a faster doubling time of 24-36 h compared with 72 h for parental cells. The cyclin-dependent kinase inhibitor p27(kip1) was decreased in TR cells, and cyclin-dependent kinase 2 activity was increased. Importantly, exogenous addition of p27(kip1) increased trastuzumab sensitivity. Additionally, resistant cells displayed heightened sensitivity to the proteasome inhibitor MG132, which induced p27(kip1) expression. Thus, we propose that trastuzumab resistance may be associated with decreased p27(kip1) levels and may be susceptible to treatments that induce p27(kip1) expression.
Abstract
Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer and there are no approved targeted therapies specifically for IBC. Targeting the ...epidermal growth factor receptor (EGFR) pathway is a promising therapeutic target for patients with triple-negative IBC (TN-IBC) with a reported pathological complete response rate of 42% (JAMA Oncology, 2018). The tumor microenvironment (TME) is a critical contributor to the aggressiveness of IBC. Delineating cross-talk between EGFR-targeted therapies and TME components, which define IBC, could inform more efficient combination regimens and novel clinical trial designs for IBC. However, such studies have not been conducted due to the lack of a syngeneic IBC mouse model. Here we report the establishment of an IBC immunocompetent mouse model and the effects of panitumumab (PmAb) on IBC tumor growth and the TME.
Methods: TN-IBC cell lines, SUM149 or FC-IBC-02, were mixed with 50% Matrigel and inoculated into mammary fat pads of hu-NSG-SGM3 mice engrafted with hematopoietic stem cells (The Jackson Laboratory). SUM149 tumor growth in hu-NSG-SGM3 mice treated with either IgG2 (isotype control, 4 mg/kg) or PmAb (1 mg/kg and 4 mg/kg) was measured. The percentages of TME components, including human CD4+ T, CD8+ T, regulatory T (Tregs), and natural killer (NK) cells, and M1 or M2 macrophages, in the peripheral blood and tumor tissues treated with IgG2 and PmAb for 7 weeks were measured by flow cytometry.
Results: Hu-NSG-SGM3 mice supported the growth of TN-IBC SUM149 and FC-IBC-02 xenografts. These humanized mouse models were named SUM149-huSGM3 and FC-IBC-02-huSGM3, respectively. Analysis of the blood cells showed that SUM149-huSGM3 mice display human CD4+ T, CD8+ T, Tregs, M1 and M2 macrophages. T cell infiltration and M1 and M2 macrophages were also detected in SUM149-huSGM3 tumors. NK cells were not detected in both peripheral blood and tumors. PmAb treated SUM149-huSGM3 mice had significantly reduced SUM149 tumor growth, compared with mice that received the IgG2 control. PmAb treatment increased the percentage of CD8+ T cells and reduced the percentage of Tregs in peripheral blood. A similar analysis of tumor infiltrating lymphocytes isolated from each group showed an increase in percent CD8+ T cells in mice treated with PmAb. There were no significant changes of M1 or M2 macrophages following PmAb treatment. These results suggest that the increase in percentage of CD8+ T cells in peripheral blood and IBC tumors, and the decrease in percentage of Tregs in peripheral blood may contribute to the therapeutic efficacy of PmAb.
Conclusion: We established the first immunocompetent mouse model to study the TME and immune response in IBC, which provides the premise for conducting a diversity of novel preclinical therapeutic studies. The mechanism of how immune responses of TN-IBC xenografts mediates the therapeutic efficacy of PmAb in IBC tumors needs to be further investigated. Our study also suggests that combination therapy with immune checkpoint inhibitors may potentiate the efficacy of anti-EGFR therapy in IBC. The therapeutic efficacy of PmAb and anti-PD-L1 combination in SUM149 humanized mice is in progress.
Citation Format: Wang X, Shao S, Pearson T, Cheng Y, Reuben JM, Tripathy D, Ueno NT. Immune modulation with humanized anti-EGFR antibody panitumumab in an immunocompetent mouse model for inflammatory breast cancer abstract. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-17-06.
Abstract
Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive type of breast cancer; it accounts for 2-4% of breast cancer cases but causes 8-10% of breast cancer deaths. ...Novel targeted therapy to improve the outcomes of patients with IBC is urgently needed. The receptor tyrosine kinase AXL is a driver for metastasis and drug resistance in various cancers, including breast cancer. Our previous work showed that AXL signaling contributes to the aggressiveness of IBC. In addition, emerging evidence indicates that the tumor microenvironment components, particularly tumor-associated macrophages, are critical drivers of the IBC clinical phenotype and promote IBC metastasis. AXL signaling has been shown to modulate the tumor microenvironment. In the present study, we investigated the impact of TP-0903, a small-molecule AXL kinase inhibitor, with additional activity against Aurora B and Janus kinase 2 (JAK2), on IBC cells and macrophage polarization.
Methods: The effects of TP-0903 on IBC cell proliferation, migration/invasion, and mammosphere formation were analyzed. The effects of TP-0903 on the polarization of human monocytic cells THP-1 were tested in vitro. In addition, the signaling pathways involved in TP-0903-regulated M2 macrophage polarization were investigated using Western blotting.
Results: The half-maximal inhibitory concentration (IC50) of TP-0903 in an array of IBC cells (including SUM149, SUM190, BCX010, FC-IBC-02, MDA-IBC-3, and KPL4) ranged from 66 nM to 346 nM, suggesting a strong cell growth inhibitory effect. TP-0903 treatment decreased the migration, invasion, and mammosphere formation of IBC cells. In addition, TP-0903 inhibited both AXL signaling and Aurora B activation, which induced a G2/M cell cycle arrest in IBC cells. Based on the importance of AXL and JAK2 in the regulation of the tumor microenvironment, we showed that TP-0903 decreased expression of CD163/CD206 and the CCL17/CCL18 cytokine, and markers of M2 macrophages, suggesting that TP-0903 treatment inhibits the polarization of THP-1 cells to M2 macrophages in vitro. We also found that TP-0903 treatment decreased the phosphorylation of STAT6, a critical molecule in M2 polarization, and that knockdown of STAT6 expression decreased M2 macrophage polarization, indicating that TP-0903 may regulate macrophage polarization via STAT6 signaling.
Conclusion: Our results demonstrated the dual functions of TP-0903 targeting of both IBC cells and macrophages, possibly via the targeting of multiple kinases, including AXL and Aurora B. Examinations of the impact of TP-0903 on the cross-talk between IBC cells and macrophages in vitro and in vivo and the related mechanisms are ongoing and will be presented at the meeting.
Citation Format: Cheng Y, Funakoshi Y, Wang X, Warner SL, Bearss DJ, Ueno NT. TP-0903, an AXL kinase inhibitor, reduces inflammatory breast cancer aggressiveness and macrophage polarization through additional mechanisms that may include JAK2 and Aurora B abstract. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-05.
Abstract
Background: Although tremendous progress has been achieved with targeted therapy for HER2-positive (HER2+) metastatic breast cancer, most advanced tumors eventually develop resistance. ...Improving our understanding of mechanisms of resistance to anti-HER2 therapy is needed to develop new therapeutic approaches. The purpose of this study was to identify the mechanisms of resistance to treatment with ado-trastuzumab emtansine (T-DM1) and/or taxane/pertuzumab/trastuzumab (TPH).
Methods: In our preclinical analysis, HER2+ cell lines resistant to treatment with T-DM1 (n=5), and pertuzumab/trastuzumab (n=3) were generated. HER2 expression in the original and resistant cell lines was compared using Western blot, and HER2 gene amplification was compared in them using fluorescence in situ hybridization (FISH) and a Droplet Digital Polymerase Chain Reaction HER2 copy-number-validation assay. In our clinical analysis, nine patients with HER2+ metastatic breast cancer who had progressed on T-DM1 and/or TPH were enrolled. Patients underwent biopsies following treatment with T-DM1 and/or TPH. Targeted next-generation sequencing was performed using the FoundationOne® assay (Foundation Medicine, Inc.) to identify gene alterations. Also, the HER2 expression before and after the therapy was compared using immunohistochemistry and/or FISH.
Results: In preclinical analysis, HER2 expression/amplification by Western blot and gene copy-number analysis was significantly decreased in T-DM1–resistant cell lines (four of five cell lines; P < 0.01) but not in pertuzumab/trastuzumab-resistant cell lines (none of three cell lines). In clinical analysis, the patients' median age was 54 years (range, 45-77 years), and five patients (56%) were ER+. Five patients (56%) received first-line anti-HER2 therapy, and four patients (44%) received two lines of anti-HER2 therapy prior to enrollment. We observed loss of HER2 expression in four of nine patients (44%) after undergoing anti-HER2 therapy. After receiving TPH, one of eight patients (13%) lost HER2 positivity according to FISH. In contrast, after T-DM1, three of four tested patients (75%) lost HER2 amplification by FISH. As for next-generation sequencing, we analyzed seven samples: three after treatment with TPH and four after treatment with T-DM1. In four of these samples (57%), we observed loss of HER2 amplification: one after treatment with TPH and three after treatment with T-DM1. TP53 mutations were seen in all patients. Additionally, we observed TOP2A and MCL1 amplification in two patients with ERBB2 amplificationand AKT1 amplification in one patient with ERBB2 amplification loss.
Conclusions: We show for the first time that T-DM1–resistant breast cancer cells lose HER2 expression and amplification. Additionally, we observed loss of HER2 expression in patient samples following treatment with HER2 targeted therapy. Further study of resistant tumor samples is required to understand the impact of HER2 loss on outcomes. For the time being, repeating biopsy analysis of a metastatic site after treatment with T-DM1 to determine the HER2 expression status is reasonable, and it may increase the efficacy of future anti-HER2 therapy.
Citation Format: Kida K, Lee J, Liu H, Lim B, Murthy RK, Sahin AA, Tripathy D, Ueno NT. Changes in the expression of HER2 and other genes in HER2-positive metastatic breast cancer induced by treatment with ado-trastuzumab emtansine and/or pertuzumab/trastuzumab abstract. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-23.
Abstract
Background: Neratinib, a potent irreversible pan-ErbB tyrosine kinase inhibitor that blocks signal transduction through HER1, HER2, and HER4, has demonstrated activity against metastatic ...HER2-positive breast cancer (HER2+ BC) in patients pretreated with trastuzumab. The FDA recently approved neratinib as an adjuvant treatment for HER2+ BC patients who have received trastuzumab for at least 1 year. Preclinical data demonstrate that trastuzumab-resistant BC cells remain sensitive to neratinib. Also, neratinib enhances responses to trastuzumab possibly by increasing trastuzumab's internalization, immune-mediated action, and other mechanisms. Taken together, these findings provide the rationale for adding neratinib to the standard of care combination of trastuzumab and pertuzumab with paclitaxel to enhance anti-HER2 efficacy in advanced HER2+ BC. Here, we report on the phase Ib portion of an ongoing phase Ib/II trial of this drug combination.
Trial Design: Patients with metastatic or locally advanced HER2+ BC will be enrolled in the phase Ib portion of the trial. Neratinib is given orally in 3-week cycles. The initial neratinib dose of 80 mg daily is increased to 120, 160, and 200 mg daily after safety assessments of each dose level. Other agents are administered as per the standard of care. Patients continue therapy with per-protocol dose escalation and de-escalation according to toxicity until the maximum tolerated dose (MTD) of neratinib is reached. The target maximum dose-limiting toxicity rate is 20%. All patients receive 4 cycles of the combination therapy. If patients do not have disease progression or excessive toxicity, they may receive 2-4 additional cycles at the treating physician's discretion. During therapy, patients undergo blood tests every week and have clinical visits and restaging scans every 3 weeks. Because gastrointestinal toxicity, mainly diarrhea, is anticipated, patients receive prophylactic antidiarrheal medication (e.g., loperamide, budesonide) beginning with the first dose of neratinib.
Eligibility Criteria: Eligible patients must have histologically confirmed metastatic or locally advanced HER2+ BC (BC may be inflammatory or non-inflammatory and have any hormone receptor status); an ECOG performance status score of 0 or 1; and adequate hematologic and organ function, including adequate cardiac function (as indicated by a left ventricle ejection fraction of ≥50%).
Specific Aims:
1- To determine the MTD of neratinib in combination with paclitaxel, pertuzumab, and trastuzumab.
2- Pharmacodynamic markers will be measured on biologic specimens. Neratinib-induced changes in pEGFR and/or HER2 expression will be analyzed and compared between dose levels.
Statistical Methods: The Bayesian modified toxicity probability interval is used to determine dose adjustment.
Accrual: The target enrollment for the phase Ib cohort is 20 patients. The trial has enrolled 3 patients since its activation in January 2018. This trial is supported by Puma Biotechnology, Aggressive Breast Cancer Research Program Grant.
Citation Format: Al-Awadhi A, Kono M, Marx A, Moseley T, Willey J, Sun H, Fu M, Whitman GJ, Valero V, Ueno NT, Lim B. A phase Ib study of neratinib, pertuzumab, and trastuzumab with paclitaxel in patients with metastatic and locally advanced breast cancer abstract. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT2-07-09.
Abstract
Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by a high rate of metastatic recurrence and poor prognosis. Molecular mechanism ...underlying the metastatic behavior of TNBC has not been well elucidated, and newer approaches addressing drivers of metastasis are crucial to improving patient outcomes. PEA15 (Phosphoprotein enriched in astrocytes-15) regulates cell proliferation, apoptosis, and autophagy. In breast cancer, PEA15 expression inhibits invasion by binding to ERK and preventing its nuclear translocation. The biological function of PEA15 is tightly regulated by its phosphorylation at Ser104 and Ser116. However, the effect of PEA15 phosphorylation status on TNBC remains unknown. In this study, we tested the hypothesis that unphosphorylated PEA15 will prevent metastasis in TNBC through inhibition of the epithelial-to-mesenchymal transition (EMT).
Method: We established stable cells overexpressing unphosphorylatable (PEA15-AA) and phospho-mimetic (PEA15-DD) PEA15 mutants in MDA-MB-468 cells. To dissect specific Cellular Mechanisms regulated by PEA15 phosphorylation, we performed RT-PCR immune and metastasis arrays. In vivo mouse models were used to see effects of PEA15 phosphorylation on tumor growth.
Results: The clonogenic growth of PEA15-AA–expressing cells was significantly reduced by 80% compared with empty vector-transfected cells (PEA15-V). Anchorage-independent growth, an indicator of in vivo tumorigenicity, was inhibited in cells expressing PEA15-AA by 60% compared with PEA15-V. PEA15-AA upregulated the expression of E-cadherin and decreased the expression of mesenchymal markers, suggesting that PEA15-AA reverses EMT. Compared with PEA15-V, migration and invasion of cells expressing PEA15-AA were reduced by 65% and 72%, respectively. In contrast, PEA15-DD promoted migration, invasion, and expression of mesenchymal markers. To determine the in vivo effect of PEA15-AA, we injected stable PEA15 transfectants of MDA-MB-468 cells into the mammary fat pad of NOD/SCID mice. The PEA15-DD–injected group showed greater tumor volumes than PEA15-V and PEA15-AA groups, suggesting that PEA15-AA has antitumor effects both in vitro and in vivo. From the immune and metastasis arrays, we found that expression level of IL-8, which is known to induce EMT, was greatly decreased by PEA15-AA, while IL-8 was highly expressed in PEA15-DD cells. Addition of recombinant IL-8 to the cells expressing PEA15-AA partially rescued mesenchymal characteristics, increasing migration and expression of mesenchymal markers. By contrast, IL-8 knockdown in PEA15-DD–expressing cells decreased the mesenchymal phenotype. These findings indicate that IL-8 may play an important role as a mediator of phosphorylation of PEA-15 in breast cancer cell migration and invasion and suggest that PEA15-AA inhibits the expression of IL-8, thereby reversing EMT.
Conclusion: Taken together, our results show that PEA15 phosphorylation serves as an important regulator, having a dual role as an oncogene or tumor suppressor. Further studies are warranted to evaluate the impact of PEA15 phosphorylation status on metastasis in vivo. These findings support the development of PEA15-AA as a potential therapeutic strategy for TNBC.
Citation Format: Park J, Chauhan G, Cohen EN, Ueno NT, Battula VL, Tripathy D, Reuben JM, Bartholomeusz C. PEA15-AA, an unphosphorylatable mutant of PEA15, as a novel therapeutic gene for triple-negative breast cancer abstract. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-22.
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