Inhibition of chemotherapy resistant breast cancer stem cells by a ROR1 specific antibody - PubMed (original) (raw)

. 2019 Jan 22;116(4):1370-1377.

doi: 10.1073/pnas.1816262116. Epub 2019 Jan 8.

Han Zhang 2, Emanuela M Ghia 2, Jiajia Huang 3, Liufeng Wu 1, Jianchao Zhang 1, Sharon Lam 2, Yang Lei 1, Jinsong He 4, Bing Cui 2, George F Widhopf 2nd 2, Jian Yu 2, Richard Schwab 2, Karen Messer 2 5, Wenqi Jiang 3, Barbara A Parker 2, Dennis A Carson 6 2, Thomas J Kipps 6 2

Affiliations

Inhibition of chemotherapy resistant breast cancer stem cells by a ROR1 specific antibody

Suping Zhang et al. Proc Natl Acad Sci U S A. 2019.

Abstract

Breast cancers enduring treatment with chemotherapy may be enriched for cancer stem cells or tumor-initiating cells, which have an enhanced capacity for self-renewal, tumor initiation, and/or metastasis. Breast cancer cells that express the type I tyrosine kinaselike orphan receptor ROR1 also may have such features. Here we find that the expression of ROR1 increased in breast cancer cells following treatment with chemotherapy, which also enhanced expression of genes induced by the activation of Rho-GTPases, Hippo-YAP/TAZ, or B lymphoma Mo-MLV insertion region 1 homolog (BMI1). Expression of ROR1 also enhanced the capacity of breast cancer cells to invade Matrigel, form spheroids, engraft in Rag2-/-[Formula: see text] mice, or survive treatment with paclitaxel. Treatment of mice bearing breast cancer patient-derived xenografts (PDXs) with the humanized anti-ROR1 monoclonal antibody cirmtuzumab repressed expression of genes associated with breast cancer stemness, reduced activation of Rho-GTPases, Hippo-YAP/TAZ, or BMI1, and impaired the capacity of breast cancer PDXs to metastasize or reengraft Rag2-/-[Formula: see text] mice. Finally, treatment of PDX-bearing mice with cirmtuzumab and paclitaxel was more effective than treatment with either alone in eradicating breast cancer PDXs. These results indicate that targeting ROR1 may improve the response to chemotherapy of patients with breast cancer.

Keywords: ROR1; ROR1-signaling; breast-cancer stem cells; chemotherapy; cirmtuzumab.

Copyright © 2019 the Author(s). Published by PNAS.

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Conflict of interest statement

Conflict of interest statement: Cirmtuzumab was developed by T.J.K. in the T.J.K. laboratory and licensed by the University of California to Onternal Therapeutics, Inc., which provided stock options and research funding to the T.K.J. laboratory.

Figures

Fig. 1.

Fig. 1.

Chemotherapy enhances breast cancer expression of ROR1 and stemness. (A) Immunohistochemical staining of ROR1 in breast biopsy specimens obtained from patients (n = 22) before (Pre) or after (Post) therapy with docetaxel/epirubicin ± cyclophosphamide. (Scale bar: 25 µM.) The table to the Right shows the elevation of ROR1 on the breast cancer clinical specimens obtained from patients after chemotherapy treatment as assessed by Fisher’s exact test. (B) The graph depicts the mean tumor growth of breast cancer PDXs in animals that did not receive treatment (black line, n = 7) or had received paclitaxel (red line, n = 5) on the days indicated by the red arrows. (C) Lysates from PDX4 or PDX5 isolated from untreated mice (−) or mice treated with paclitaxel (+) were examined for ROR1 or β-actin, which served to monitor the amount of protein per lane. The numbers below each lane are the ratios of band densities of ROR1 to β-actin normalized to that for the PDXs isolated from untreated mice. (D and E) The histograms depict the average numbers of spheroids (D) or invading cells (E) from PDX4 or PDX5 excised from mice that did not receive treatment (open bars, n = 3) or that were treated with paclitaxel (black bars, n = 3) ± standard error of the mean (SEM). Representative photomicrographs to the Left of the histograms depict the spheroids or invasive cells of PDXs excised from untreated mice or from mice that received paclitaxel. (Scale bar: 100 µm.) (F) Tumor cells were isolated from PDX4 or PDX5 mice that did not receive treatment (Untreated) or were treated with paclitaxel (Paclitaxel) as indicated on the Left. Isolated tumor cells from each PDX were reimplanted into mice (n = 5), and the tumor incidence was recorded. The Left provides representative photographs for tumors isolated from untreated mice or from mice that received paclitaxel. The frequency of tumorigenic cells and the probability estimates are shown on the Right using the extreme limiting dilution analysis (ELDA) software.

Fig. 2.

Fig. 2.

Wnt5a induces ROR1-dependent activation of Rho-GTPases, YAP/TAZ, and BMI1. (A) Immunoblot analyses for proteins as indicated on the right using lysates prepared from Hs578T cells (Parental) or Hs578T knocked out for ROR1 (ROR1−/−) that were stimulated with Wnt5a for the times indicated. The numbers below each lane are the ratios of the band densities of activated versus total GTPase, normalized with respect to cells treated without Wnt5a. (B) Immunoblot analyses for proteins indicated on the right using lysates from parental or ROR1−/− Hs578T that were treated without (−) or with (+) Wnt5a as indicated. The numbers below each lane represent the ratios of the band densities for each protein relative to that of β-actin, normalized with respect to cells treated without Wnt5a. (C) Photomicrographs of parental (Top row) or ROR1−/− Hs578T cells (Bottom row) that were treated without or with Wnt5a as indicated and then stained for YAP/TAZ and 4′,6-diamidino-2-phenylindole (DAPI) as indicated and then examined using confocal microscopy. (Scale bar: 20 μm.) The histogram to the Right of the photomicrographs provides the average percentages of YAP/TAZ located within the nuclei of the cells in each field (n = 10, ±SEM). (D) Immunoblot analyses for proteins indicated on the right using lysates of parental or ROR1−/− Hs578T (as indicated on the Bottom) that were treated with Wnt5a for the times indicated. The numbers below each lane are the ratios of band densities of BMI1 versus β-actin or pAKT versus total AKT normalized to that of the sample collected at time 0. (E) Immunoblot analyses for proteins indicated on the right using lysates of Hs578T that had been treated with control small interfering RNA (siRNA) or AKT-specific siRNA (AKT-siRNA) as indicated. The numbers below each lane are as in Fig. 3_D_. (F and G) The histograms depict the average numbers of spheroids (F) or invasive cells (G) from parental or ROR1−/− Hs578T that were treated without or with Wnt5a in triplicate ±SEM.

Fig. 3.

Fig. 3.

Cirmtuzumab inhibits Wnt5a-induced ROR1-dependent activation of Rho-GTPases, YAP/TAZ, and BMI1. (A) Immunoblot analyses for proteins indicated on the right using lysates of Hs578T that had been treated with a control hIgG or cirmtuzumab and then stimulated with Wnt5a for the times indicated. The numbers below each lane are the ratios of the band densities of activated versus total GTPase, normalized with respect to the hIgG-treated cells without Wnt5a. (B) Immunoblot analyses for proteins indicated on the right using lysates prepared from Hs578T cultured with cirmtuzumab or hIgG and then treated without or with Wnt5a as indicated. The numbers below each lane represent the ratios of the band densities for each protein relative to that of β-actin, normalized with respect to the hIgG-treated cells without Wnt5a. (C) Photomicrographs of Hs578T cultured overnight with cirmtuzumab or hIgG (as indicted on the Left), then treated without or with Wnt5a for 4 h (as indicated), then stained for YAP/TAZ and DAPI (as indicated), and then examined via confocal microscopy. (Scale bar: 20 μm.) The histogram to the Right of the photomicrographs provides the average percentages of YAP/TAZ located within the nuclei of the cells in each field (n = 10, ±SEM). (D) Immunoblot analyses for proteins indicated on the right using lysates of Hs578T that had been treated overnight with hIgG or cirmtuzumab and then treated without or with Wnt5a as indicated. The numbers below each lane are the ratios of band densities of pAKT versus total AKT, normalized with respect to the hIgG-treated cells without Wnt5a. (E) Immunoblot analyses for proteins indicated on the right using lysates of Hs578T that had been treated overnight with hIgG or cirmtuzumab and then treated with Wnt5a for the times indicated. The numbers below each lane are the ratios of band densities of BMI1 versus β-actin, normalized with respect to the hIgG-treated cells without Wnt5a. (F and G) The bar graph depicts the average numbers of spheroids (F) or invasive cells (G) from Hs578T cells that were incubated with hIgG or cirmtuzumab overnight and then treated with or without Wnt5a in three separate culture wells ± SEM. The open bars indicate the number of spheroids detected during the first passage, whereas the closed bars provide those of the second passage in three separate culture wells ± SEM.

Fig. 4.

Fig. 4.

Cirmtuzumab inhibits stemness of breast cancer PDXs. (A) The line graph depicts the mean tumor growth of each of PDX3, PDX4, and PDX5 over time (±SEM, n = 6–8) for animals that did not receive treatment (black line) or that were treated with cirmtuzumab (red line) on the days indicated by the black arrows. One asterisk indicates P < 0.05, and two asterisks indicate P < 0.01 using Student’s t Test. (B) Hematoxylin and eosin staining of lung tissue from a representative tumor-bearing mouse engrafted with cells of PDX5 and treated with control hIgG or cirmtuzumab as indicated. A dashed-lined circle highlights metastatic foci. (Scale bar: 100 µm.) The scatter plot shows the average numbers of metastatic foci that were found in the lungs of each animal by the treatment group (±SEM, n = 6). (C) Enrichment plots of genes associated with the activation of Rho-GTPases, Hippo-YAP, BMI1, or gene signature common on both CD44+/CD24Low cancer stem cells and MS-forming cells in PDXs derived from PDX4 in mice treated with control hIgG versus cirmtuzumab as assessed via RNA sequencing (RNA-seq) (GSE108632). (D) Immunoblot analyses for proteins indicated on the right using lysates prepared from PDX4 or PDX5 (as indicated on the Bottom) that were extirpated from mice treated with control hIgG or cirmtuzumab as indicated. The numbers below each row are the ratios of band densities of activated versus total GTPase, pAKT versus total AKT, BMI1, TAZ/YAP versus β-actin, or ROR1 versus β-actin, normalized to that of the first control sample. (E) Table providing the numbers of mice that developed tumors (numerator) versus the numbers of mice implanted (denominator) with cells from PDX3, PDX4, or PDX5 (as indicated in the left column), which were removed from mice treated with either hIgG or cirmtuzumab (as indicated in the second column). For these experiments, mice were given varying numbers of tumor cells (as indicated in the row below “cell number”). The frequencies of tumorigenic cells computed using ELDA software are provided in the penultimate right column. The P values indicate the significance of the difference between the tumorigenic frequencies of tumor cells recovered from hIgG- versus cirmtuzumab-treated mice.

Fig. 5.

Fig. 5.

Cirmtuzumab and paclitaxel have complementary antitumor activity. (A) The line graph depicts the mean tumor growth of PDX4 or PDX5 over time (±SEM, n = 8–10) in mice that were untreated (Control, black line) or treated with cirmtuzumab (purple line), paclitaxel (green line), or both (blue line). (B) Immunoblot analyses for proteins indicated on the right using lysates prepared from PDX4 of mice that were not treated (Control) or treated with cirmtuzumab, paclitaxel, or both as indicated. The numbers below each row are the ratios of band densities as in Fig. 4_E_. (C) Table providing the numbers of mice that developed PDXs (numerator) versus the numbers of mice engrafted (denominator) with cells from either PDX4 or PDX5 (as indicated in the top row) that were removed from mice treated with hIgG, cirmtuzumab, paclitaxel, or cirmtuzumab and paclitaxel (as indicated in the far left column). The mice were given varying numbers of tumor cells as indicated in the row below the PDX designation. The Bottom provides a photograph of representative tumors that developed in mice engrafted with tumor cells from PDX4 of mice that were untreated or treated with cirmtuzumab and/or paclitaxel as indicated.

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