Promotes Drug Resistance Research Articles

Angiotensin-Converting Enzyme Inhibitors Have Adverse Effects in Anti-Angiogenesis Therapy for Hepatocellular Carcinoma

Background: Currently, most clinicians prescribe angiotensin-converting enzyme inhibitors (ACEIs) to alleviate proteinuria caused by anti-angiogenic drugs (AADs) according to diabetic nephropathy guidelines or expert recommendations. However, the efficacy ACEIs and potential cancer promoting effect are controversial. This study aimed at confirming whether ACEIs are beneficial in anti-angiogenesis therapy for hepatocellular carcinoma. Methods: In clinic, we observed the impact of ACEIs on AADs during the treatment of hepatocellular carcinoma. Next, we established different tumor-bearing mouse models to confirm that whether ACEIs have any effect on the anti-cancer efficacy and proteinuria side effects of AADs. Further, we confirmed the relevant mechanism in vivo. Findings: Our clinical data have shown that some hepatocellular carcinoma (HCC) patients experienced tumor progression by ACEIs administration for the treatment of hypertension or proteinuria caused by AADs. We have confirmed that in different tumor-bearing mouse models, ACEIs did not delay the appearance of proteinuria or alleviate proteinuria caused by AADs but compromised the anticancer efficacy of AADs. This effect is unrelated to a change in the VEGF signaling pathway. Our data showed that the combination of ACEIs and AADs flared the production of kidney-derived erythropoietin (EPO). In turn, EPO compromises the anti-angiogenic effects of AADs and decreases antitumor activity. Interpretation: For the treatment of proteinuria caused by AADs, ACEIs have no efficacy but promote drug resistance. Kidney-derived EPO is mainly responsible for ACEIs induced anti-angiogenesis resistance. Funding Statement: This study was supported by the National Natural Science Foundation of China (No. 81672884) and the National Science and Technology Major Project of China (No. 2017ZX10203207-004-005). Declaration of Interests: The authors have declared that no conflict of interest exists. Ethics Approval Statement: All human studies were approved by the Clinical Research Ethics Committee of Tianjin Medical University Cancer Institute. All animal experiments were carried out in accordance with a protocol approved by the ethics committee of the Institutional Animal Care of Tianjin Medical University Cancer Institute and Hospital.

Glutathione S-transferases P1-mediated interleukin-6 in tumor-associated macrophages augments drug-resistance in MCF-7 breast cancer

Glutathione S-transferase P1 (GSTP1), a phase II detoxifying enzyme, is overexpressed and plays an important role during breast cancer drug resistance. Tumor-associated macrophages (TAMs), representing most of the leukocyte population in solid tumors, are involved in cancer cell resistance to chemotherapy. Although GSTP1 exists in TAMs, whether GSTP1 in TAMs promotes drug resistance is still unclear. In the current study, we found a novel mechanism that GSTP1 in TAMs contributes breast cancer cell drug resistance. GSTP1 is aberrantly expressed in TAMs from breast cancer tissues of patients after chemotherapy than that without chemotherapy. Adriamycin (ADR) time-dependently induced the expression of GSTP1 in TAMs in vitro. Conditional medium of TAMs significantly inhibited ADR-induced cell death of MCF-7 breast cancer cells. Meanwhile, overexpression of GSTP1 in TAMs promoted the expression and release of interleukin-6 (IL-6) associated with reduced ADR-induced breast cell death, which was reversed by IL-6 antibody. Mechanistically, GSTP1 interacted with inhibitor of nuclear factor κB kinase β (IKKβ) to activate nuclear factor-κB (NF-κB) to induced the expression and release of IL-6 in TAMs. Moreover, IL-6 further upregulated GSTP1 through c-Jun, and ultimately mediated drug resistance in MCF-7 cells. Taken together, our data demonstrated for the first time that GSTP1 in TAMs promoted ADR-resistance in breast cancer by regulating interleukin-6 release.

Tac2-N serves an oncogenic role and promotes drug resistance in human gastric cancer cells.

Gastric cancer is one of the most common types of malignancy worldwide. Tac2-N (TC2N) has been reported to serve as either an oncogene or tumor suppressor in numerous different types of cancer; however, the role of TC2N in gastric cancer remains poorly understood. The present study aimed to investigate the role of TC2N in gastric cancer and reveal its regulatory mechanism. A Cell Counting Kit-8 assay was used to analyze the cell proliferation rate, while wound healing and Transwell Matrigel assays were performed to determine the cell migratory and invasive abilities, respectively. Cell cycle distribution was determined by flow cytometric analysis, and the expression levels of TC2N, P-glycoprotein (P-gp), cyclin D1, CDK4, cyclin E1, MMP2, MMP9 and N-Myc downstream regulated gene 1 were analyzed using reverse transcription-quantitative PCR or western blotting. Bioinformatics analysis revealed a high expression of TC2N in patients with gastric cancer. The experimental results revealed that TC2N expression levels were significantly unregulated in gastric cancer cell lines. The knockdown of TC2N in AGS cells significantly inhibited the cell proliferation rate and induced cell cycle arrest at the G0/G1 phase, while downregulating cyclin E1, cyclin D1 and CDK4 expression levels. The knockdown of TC2N also inhibited cell migration and invasion. Furthermore, the knockdown of TC2N improved the sensitivity of AGS cells to cisplatin, paclitaxel and 5-fluorouracil, and downregulated the protein expression levels of P-gp. By contrast, TC2N overexpression exerted the opposite effects in AGS cells. In conclusion, the findings of the present study indicated that the genetic knockdown of TC2N may inhibit cell proliferation, migration and invasion, while inducing cell cycle arrest in the G1/S phase and reversing the drug resistance of AGS cells, which may be partly through inhibiting P-gp expression levels. Thus, TC2N may serve as a novel diagnostic marker and therapeutic target for patients with gastric cancer.

Normoxic Tumour Extracellular Vesicles Modulate the Response of Hypoxic Cancer and Stromal Cells to Doxorubicin In Vitro.

Extracellular vesicles (EV) secreted in the tumour microenvironment (TME) are emerging as major antagonists of anticancer therapies by orchestrating the therapeutic outcome through altering the behaviour of recipient cells. Recent evidence suggested that chemotherapeutic drugs could be responsible for the EV-mediated tumour–stroma crosstalk associated with cancer cell drug resistance. Here, we investigated the capacity of tumour EV (TEV) secreted by normoxic and hypoxic (1% oxygen) C26 cancer cells after doxorubicin (DOX) treatment to alter the response of naïve C26 cells and RAW 264.7 macrophages to DOX. We observed that C26 cells were less responsive to DOX treatment under normoxia compared to hypoxia, and a minimally cytotoxic DOX concentration that mounted distinct effects on cell viability was selected for TEV harvesting. Homotypic and heterotypic pretreatment of naïve hypoxic cancer and macrophage-like cells with normoxic DOX-elicited TEV rendered these cells slightly less responsive to DOX treatment. The observed effects were associated with strong hypoxia-inducible factor 1-alpha (HIF-1α) induction and B-cell lymphoma–extra-large anti-apoptotic protein (Bcl-xL)-mediated anti-apoptotic response in normoxic DOX-treated TEV donor cells, being also tightly connected to the DOX-TEV-mediated HIF-1α induction, as well as Bcl-xL levels increasing in recipient cells. Altogether, our results could open new perspectives for investigating the role of chemotherapy-elicited TEV in the colorectal cancer TME and their modulatory actions on promoting drug resistance.

Role of Transmembrane 4 L Six Family 1 in the Development and Progression of Cancer.

Transmembrane 4 L six family 1 (TM4SF1) is a protein with four transmembrane domains that belongs to the transmembrane 4 L six family members (TM4SFs). Structurally, TM4SF1 consists of four transmembrane domains (TM1–4), N- and C-terminal intracellular domains, two extracellular domains, a smaller domain between TM1 and TM2, and a larger domain between TM3 and TM4. Within the cell, TM4SF1 is located at the cell surface where it transmits extracellular signals into the cytoplasm. TM4SF1 interacts with tetraspanins, integrin, receptor tyrosine kinases, and other proteins to form tetraspanin-enriched microdomains. This interaction affects the pro-migratory activity of the cells, and thus it plays important roles in the development and progression of cancer. TM4SF1 has been shown to be overexpressed in many malignant tumors, including gliomas; malignant melanomas; and liver, prostate, breast, pancreatic, bladder, colon, lung, gastric, ovarian, and thyroid cancers. TM4SF1 promotes the migration and invasion of cancer cells by inducing epithelial-mesenchymal transition, self-renewal ability, tumor angiogenesis, invadopodia formation, and regulating the related signaling pathway. TM4SF1 is an independent prognostic indicator and biomarker in several cancers. It also promotes drug resistance, which is a major cause of therapeutic failure. These characteristics make TM4SF1 an attractive target for antibody-based immunotherapy. Here, we review the many functions of TM4SF1 in malignant tumors, with the aim to understand the interaction between its expression and the biological behaviors of cancer and to supply a basis for exploring new therapeutic targets.

AT101 [(-)-Gossypol] Selectively Inhibits MCL1 and Sensitizes Carcinoma to BH3 Mimetics by Inducing and Stabilizing NOXA.

Anti-apoptotic BCL2 proteins are important targets for cancer therapy as cancers depend on their activity for survival. Direct inhibitors of MCL1 have entered clinical trials, although their efficacy may be limited by toxicity. An alternative approach may be to induce the pro-apoptotic protein NOXA which selectively inhibits MCL1 in cells. Many compounds originally proposed as inhibitors of the BCL2 family were subsequently found to induce the pro-apoptotic protein NOXA through the unfolded protein response. In the present study, we compared various putative BH3 mimetics across a panel of carcinoma cell lines and measured expression of NOXA protein and mRNA, as well as the kinetics of NOXA induction. We found that AT101 [(-)-gossypol] induces high levels of NOXA in carcinoma cell lines yet cells survive. When combined with an appropriate BCL2 or BCL-XL inhibitor, NOXA-dependent sensitization occurs. NOXA protein continues to accumulate for many hours after AT101 is removed, providing a window for administering these combinations. As MCL1 promotes drug resistance and overall survival, we propose that NOXA induction is an alternative therapeutic strategy to target MCL1 and either kill cancer cells that are dependent on MCL1 or sensitize cancer cells to other BCL2 inhibitors.

TNFAIP8 promotes AML chemoresistance by activating ERK signaling pathway through interaction with Rac1

BackgroundChemoresistance is emerging as a major barrier to successful treatment in acute myeloid leukemia (AML), and evasion of apoptosis is among the fundamental underlying mechanisms. Therefore, unraveling molecular networks that drive this process constitutes an urgent unmet need. Herein, we aim to characterize the role and molecular mechanism of the tumor necrosis factor ɑ-induced protein 8 (TNFAIP8), a novel anti-apoptotic molecule, in AML chemoresistance.MethodsThe expression levels of TNFAIP8 were assessed in AML patients and cell lines by RT-qPCR and western blots. The transcriptional regulation of TNFAIP8 was analyzed with luciferase reporter assay and ChIP followed by RT-qPCR. Functional experiments were conducted to evaluate the effects of TNFAIP8 on apoptosis, drug sensitivity and proliferation of AML cells. Potential effects of TNFAIP8 on the activation of extracellular signal-regulated kinase (ERK) pathway were detected by western blots. CoIP and P21-activated kinase (PAK) pull-down assay were performed to ascertain the upstream target. The overall effects of TNFAIP8 on AML were examined in murine models.ResultsUpregulated TNFAIP8 expression was first confirmed in human AML patients and cell lines. E74 like ETS transcription factor 1 (ELF1) was then identified to contribute to its aberrant expression. Through manipulating TNFAIP8 expression, we described its role in protecting AML cells from apoptosis induced by chemotherapeutic agents and in promoting drug resistance. Notably, the leukemia-promoting action of TNFAIP8 was mediated by sustaining activity of the ERK signaling pathway, through an interaction with Rac family small GTPase 1 (Rac1). In addition, in vivo experiments confirmed that TNFAIP8 suppression lowered leukemia infiltration and improved survival.ConclusionOur data provide a molecular basis for the role of TNFAIP8 in chemoresistance and progression of AML and highlight the unique function of TNFAIP8 as an attractive therapeutic target.

Abstract 1485: E7011, a novel anti-angiogenesis inhibitor targeting Notch4 receptor signal pathway

Abstract Notch4 is selectively expressed on tumor endothelial cells and hypothesized to contribute to cancer malignancy through mechanisms including tumor angiogenesis regulation and promoting drug resistance. We developed E7011, a humanized IgG2κ antibody targeting Notch4 receptor signaling. To obtain anti-human Notch4 monoclonal antibody, human Notch4 3EGFR (epidermal growth factor repeat) domain - NRR (negative regulatory region) - SEAP (secreted alkaline phosphatase) - His protein was immunized to mice and 6698 hybridomas were obtained. By screening these clones with the Notch4 binding assay and Notch4-specific gal4-fusion luciferase reporter assay, mouse anti-human Notch4 mAb clone (6-3-A6) was selected. The CDRs (complementarity determining regions) of 6-3-A6 were grafted into the variable regions of human IgG2κ, and E7011, novel humanized anti-human Notch4 monoclonal antibody, was developed by screening in assays above. The biomolecular interaction analysis showed that E7011 is able to bind to human and mouse Notch4-NRR domain-SEAP-His, and inhibit human Notch4 receptor signaling in an antibody-dose dependent manner as well as 6-3-A6. IHC analysis showed that Notch4 was expressed on CD31 positive endothelial cells in Calu6 human non-small cell lung carcinoma xenograft tumor. Intravenous administration of E7011 at 1, 3, 10 mg/kg in twice a week significantly decreased tumor growth compared with control IgG treatment without severe body weight loss. We also investigated the functional tumor angiogenesis by hoechest dye-perfusion and staining with anti-CD31 antibody in the Calu6 xenograft model. E7011 treatment significantly decreased hoechst-positive perfusion area compared with vehicle treatment. In conclusion, E7011 is a novel antitumor agent that is a specific signal inhibitor targeting the Notch4 receptor and suppresses tumor growth through decreasing tumor vessel function in based on preclinical studies. Citation Format: Yusuke Adachi, Yoshimasa Sakamoto, Youya Nakazawa, Sho Tachino, Ken Ito, Takanori Abe, Yukinori Minoshima, Kana Hoshino-Negishi, Hideaki Ogasawara, Tomomi Kawakatsu, Miyuki Nishimura, Masashi Shimizu, Kazuhiro Tahara, Toshitaka Sato, Katsuhisa Suzuki, Kishan Agarwala, Masao Iwata, Yasuhiro Funahashi, Kenichi Nomoto, Yoichi Ozawa, Junji Matsui, Toshio Imai, Yu Kato. E7011, a novel anti-angiogenesis inhibitor targeting Notch4 receptor signal pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1485.

Abstract 4088: The functional mechanism of Clk2 supporting the development of chemoresistance in non-BRCA1 deficient ovarian cancer

Abstract Background: Platinum is the most commonly used drug for the treatment of ovarian cancer. However, resistance to platinum has also become a serious clinical problem. The mutation of BRCA (BRCA1/2) in ovarian cancer, is the major cause of the occurrence of tumors. Our previous microarray data showed that expression level of CDC-like kinase (Clk2) was higher in ovarian cancer tissues than the compared normal tissues. As a protein kinase, Clk2 phosphorylates and activates BRCA1. Methods: 1.Quantitative PCR and IHC were conducted to verify the expression of Clk2 in normal ovarian and tumor tissues.2.The WB was used to examine the correlation between Clk2 protein level and the time and concentration of cisplatin, as well as the protein expression of Clk2 in the platinum sensitive and resistant cell strains of ovarian cancer.3.After knocking down Clk2, the IC50 of cisplatin, the DNA damage level and the apoptosis ratio of drug-resistant cell were examined.4.Tumor formation model was made in nude mice to test the tumor size of drug-resistant cell.5.Protein profiling was analysed to screen proteins that bind to endogenous Clk2 in ovarian cancer cells. Co-IP was used to prove the binding of Clk2 to BRCA1 and to detect the relationship between their binding levels and the time and concentration gradient of platinum.6.The effect of Clk2 overexpression on the activation of DNA damage-related proteins was detected by WB.7.IHC was used to observe of changes of p-BRCA1 and formation of DNA damage repair focus after knockout of Clk2.8.The correlation between p-BRCA1 expression and Clk2 in ovarian cancer was demonstrated via IHC.9.The recombinant kinase experiment was conducted to analyze the ability of the Clk2 protein kinase to activate BRCA1.10.The related pathway inhibitors were used to preliminarily screen for the signaling pathways in which cisplatin induced upregulation of Clk2 and validate.11.The "synergistic lethal effect" of the combination of Clk2 with PARP inhibitors was explored preliminarily. Conclusions: 1.Compared with normal ovarian tissues, Clk2 is up-regulated in ovarian cancer tissue, especially in chemo-resistant ovarian cancer tissues, and high expression of Clk2 indicates poor prognosis. 2.Clk2 enhances DNA damage repair and promotes drug resistance in ovarian cancer. 3.Clk2 activates BRCA1 in chemo-resistant ovarian cancer cells. 4.Platinum activates p38 signal, which contributes to the phosphorylation of T343 in Clk2 Activation Loop, then Clk2 protein is stabilized. And Clk2 can activate BRCA1 in chemo-resistant ovarian cancer cells. 5.A preliminary study shows that combining Clk2 and PARP inhibitors can form a "synergistic lethal effect" and increase DNA damage in ovarian cancer platinum-resistant cells. Citation Format: Shuting Huang, Yinan Jiang, Min Zheng. The functional mechanism of Clk2 supporting the development of chemoresistance in non-BRCA1 deficient ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4088.

Abstract 3008: A new mechanism of drug resistance in cancer: Extracellular ATP-induced resistance through ATP internalization and upregulation of ATP-binding cassette multidrug transporters

Abstract Cancer is the second leading cause of death in the US. Multidrug resistance (MDR) and the resulting ineffectiveness of the drug treatment are responsible for most relapses of cancer and cancer-related death. One major mechanism of MDR is the efflux of multiple drugs mediated by ATP-binding Cassette (ABC) multidrug transporters. ABC transporters pump drugs with different structures and mechanisms of action out of cancer cells using ATP-provided energy. And ABC activities is also intimately linked to epithelial mesenchymal transition and metastasis. The opportunistic uptake of extracellular molecules has been named as a key emerging hallmark of cancer metabolism. Intratumoral extracellular ATP levels of various cancer types are 103 to 104 times higher than those in their corresponding normal tissues. Our previous studies have shown that extracellular ATP is internalized by cancer cell via macropinocytosis, and promotes cancer drug resistance by directly fueling ABC transporters and increase its drug efflux activities. However, whether extracellular ATP can induce change in the expression of ABC transporters and the underlying mechanisms remain unclear. Our results from quantitative reverse transcription PCR and flow cytometry show that extracellular ATP significantly alters mRNA levels and cell surface expression of ABC transporters. These results suggest that extracellular ATP has profound regulatory effects on ABC transporters and thus promotes drug resistance, linking the ATP-rich tumor microenvironment with cancer drug resistance. On-going studies include identification of mechanisms for the extracellular ATP-induced drug resistance via ABC transporters. The completion of the study will expand our understanding of the roles of extracellular ATP in cancer, and reveal new therapeutic targets for reducing drug resistance and enhancing anticancer efficacy of chemotherapeutics and targeted drugs. Citation Format: Haiyun Zhang, Xuan Wang, Xiaozhuo Chen. A new mechanism of drug resistance in cancer: Extracellular ATP-induced resistance through ATP internalization and upregulation of ATP-binding cassette multidrug transporters [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3008.

Pretreatment and Acquired Antiretroviral Drug Resistance Among Persons Living With HIV in Four African Countries.

BackgroundEmerging HIV drug resistance (HIVDR) could jeopardize the success of standardized HIV management protocols in resource-limited settings. We characterized HIVDR among antiretroviral therapy (ART)-naive and experienced participants in the African Cohort Study (AFRICOS).MethodsFrom January 2013 to April 2019, adults with HIV-1 RNA >1000 copies/mL underwent ART history review and HIVDR testing upon enrollment at 12 clinics in Uganda, Kenya, Tanzania, and Nigeria. We calculated resistance scores for specific drugs and tallied major mutations to non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), and protease inhibitors (PIs) using Stanford HIVDB 8.8 and SmartGene IDNS software. For ART-naive participants, World Health Organization surveillance drug resistance mutations (SDRMs) were noted.ResultsHIVDR testing was performed on 972 participants with median age 35.7 (interquartile range [IQR] 29.7–42.7) years and median CD4 295 (IQR 148–478) cells/mm3. Among 801 ART-naive participants, the prevalence of SDRMs was 11.0%, NNRTI mutations 8.2%, NRTI mutations 4.7%, and PI mutations 0.4%. Among 171 viremic ART-experienced participants, NNRTI mutation prevalence was 83.6%, NRTI 67.8%, and PI 1.8%. There were 90 ART-experienced participants with resistance to both efavirenz and lamivudine, 33 (36.7%) of whom were still prescribed these drugs. There were 10 with resistance to both tenofovir and lamivudine, 8 (80.0%) of whom were prescribed these drugs.ConclusionsParticipants on failing ART regimens had a high burden of HIVDR that potentially limited the efficacy of standardized first- and second-line regimens. Management strategies that emphasize adherence counseling while delaying ART switch may promote drug resistance and should be reconsidered.

Cancer-Associated Fibroblasts in Mycosis Fungoides Promote Tumor Cell Migration and Drug Resistance through CXCL12/CXCR4

Cancer cells are known to reprogram normal fibroblasts into cancer-associated fibroblasts (CAFs) to act as tumor supporters. The presence and role of CAFs in mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma, are unknown. This study sought to characterize CAFs in MF and their cross talk with the lymphoma cells using primary fibroblast cultures from punch biopsies of patients with early-stage MF and healthy subjects. MF cultures yielded significantly increased levels of FAPα, a CAF marker, and CAF-associated genes and proteins: CXCL12 (ligand of CXCR4 expressed on MF cells), collagen XI, and matrix metalloproteinase 2. Cultured MF fibroblasts showed greater proliferation than normal fibroblasts in exvivo experiments. A coculture with MyLa cells (MF cell line) increased normal fibroblast growth, reduced the sensitivity of MyLa cells to doxorubicin, and enhanced their migration. Inhibiting the CXCL12/CXCR4 axis increased doxorubicin-induced apoptosis of MyLa cells and reduced MyLa cell motility. Our data suggest that the fibroblasts in MF lesions are more proliferative than fibroblasts in normal skin and that CAFs protect MF cells from doxorubicin-induced cell death and increase their migration through the secretion of CXCL12. Reversing the CAF-mediated tumor microenvironment in MF may improve the efficiency of anticancer therapy.

ABL1-dependent OTULIN phosphorylation promotes genotoxic Wnt/\u03b2-catenin activation to enhance drug resistance in breast cancers

Dysregulated Wnt/β-catenin activation plays a critical role in cancer progression, metastasis, and drug resistance. Genotoxic agents such as radiation and chemotherapeutics have been shown to activate the Wnt/β-catenin signaling although the underlying mechanism remains incompletely understood. Here, we show that genotoxic agent-activated Wnt/β-catenin signaling is independent of the FZD/LRP heterodimeric receptors and Wnt ligands. OTULIN, a linear linkage-specific deubiquitinase, is essential for the DNA damage-induced β-catenin activation. OTULIN inhibits linear ubiquitination of β-catenin, which attenuates its Lys48-linked ubiquitination and proteasomal degradation upon DNA damage. The association with β-catenin is enhanced by OTULIN Tyr56 phosphorylation, which depends on genotoxic stress-activated ABL1/c-Abl. Inhibiting OTULIN or Wnt/β-catenin sensitizes triple-negative breast cancer xenograft tumors to chemotherapeutics and reduces metastasis. Increased OTULIN levels are associated with aggressive molecular subtypes and poor survival in breast cancer patients. Thus, OTULIN-mediated Wnt/β-catenin activation upon genotoxic treatments promotes drug resistance and metastasis in breast cancers.

HSP70 regulates Eg5 distribution within the mitotic spindle and modulates the cytotoxicity of Eg5 inhibitors

The heat shock protein 70 (HSP70) is a conserved molecular chaperone and proteostasis regulator that protects cells from pharmacological stress and promotes drug resistance in cancer cells. In this study, we found that HSP70 may promote resistance to anticancer drugs that target the mitotic kinesin, Eg5, which is essential for assembly and maintenance of the mitotic spindle and cell proliferation. Our data show that loss of HSP70 activity enhances Eg5 inhibitor-induced cytotoxicity and spindle abnormalities. Furthermore, HSP70 colocalizes with Eg5 in the mitotic spindle, and inhibition of HSP70 disrupts this colocalization. Inhibition or depletion of HSP70 also causes Eg5 to accumulate at the spindle pole, altering microtubule dynamics and leading to chromosome misalignment. Using ground state depletion microscopy followed by individual molecule return (GSDIM), we found that HSP70 inhibition reduces the size of Eg5 ensembles and prevents their localization to the inter-polar region of the spindle. In addition, bis(maleimido)hexane-mediated protein-protein crosslinking and proximity ligation assays revealed that HSP70 inhibition deregulates the interaction between Eg5 tetramers and TPX2 at the spindle pole, leading to their accumulation in high-molecular-weight complexes. Finally, we showed that the passive substrate-binding activity of HSP70 is required for appropriate Eg5 distribution and function. Together, our results show that HSP70 substrate-binding activity may regulate proper assembly of Eg5 ensembles and Eg5-TPX2 complexes to modulate mitotic distribution/function of Eg5. Thus, HSP70 inhibition may sensitize cancer cells to Eg5 inhibitor-induced cytotoxicity.

3108 – MOLECULAR MECHANISMS OF LENALIDOMIDE RESISTANCE IN DEL(5Q) MYELODYSPLASTIC SYNDROMES

Interstitial deletion of the long arm of chromosome 5 (del(5q)) is the commonest structural genomic variant in myelodysplastic syndromes (MDS). Lenalidomide (LEN) is the treatment of choice for patients with del(5q) MDS. Half of the responding patients become resistant within two years of which around 20% present TP53 mutations at the time of LEN resistance. To identify novel genetic determinants of LEN resistance, we compared protein coding variants from paired samples from 29 del(5q) patients who responded to LEN and eventually became resistant to treatment. Our analysis identified, in addition to TP53, recurrent RUNX1 alterations in relapsed patients. Exposure of del(5q) cell lines to LEN showed upregulation of RUNX1 protein levels and function in a CRBN and TP53 dependent manner. RUNX1 deletion, shRNA-mediated downregulation or RUNX1 mutant expression rendered sensitive cells unresponsive to LEN. LEN-induced IKZF1 degradation released the transcriptional activity of a RUNX1/GATA2 complex to drive megakaryocytic differentiation of del(5q) cells, which was required for Calpain-dependent del(5q) cell death. We confirmed these findings in human CD34+ cells expressing a shRNA targeting CSNK1A1 expression, which reproduce del(5q) cell LEN sensitivity. RUNX1 and TP53 loss-of-function in del(5q) cells blocked LEN induced megakaryocytic differentiation and subsequent cell death. Transcriptome analysis from paired del(5q) MDS patient samples showed loss of megakaryocytic gene signatures at relapse. Our results suggest that GATA2, RUNX1 and TP53 cooperate to drive megakaryocytic differentiation after LEN-mediated degradation of IKZF1. Mutations blocking LEN-induced megakaryocytic differentiation promote drug resistance and activating GATA2 may re-sensitize therapy-resistant del(5q) cells.

Inhibition of TLR4 Signaling Affects Mitochondrial Fitness and Overcomes Bortezomib Resistance in Myeloma Plasma Cells.

Multiple myeloma (MM) is a B-cell malignancy requiring inflammatory microenvironment signals for cell survival and proliferation. Despite improvements in pharmacological tools, MM remains incurable mainly because of drug resistance. The present study aimed to investigate the implication of Toll-like receptor 4 (TLR4) as the potential mechanism of bortezomib (BTZ) resistance. We found that TLR4 activation induced mitochondrial biogenesis and increased mitochondrial mass in human MM cell lines. Moreover, TLR4 signaling was activated after BTZ exposure and was increased in BTZ-resistant U266 (U266-R) cells. A combination of BTZ with TAK-242, a selective TLR4 inhibitor, overcame drug resistance through the generation of higher and extended oxidative stress, strong mitochondrial depolarization and severe impairment of mitochondrial fitness which in turn caused cell energy crisis and activated mitophagy and apoptosis. We further confirmed the efficacy of a TAK-242/BTZ combination in plasma cells from refractory myeloma patients. Consistently, inhibition of TLR4 increased BTZ-induced mitochondrial depolarization, restoring pharmacological response. Taken together, these findings indicate that TLR4 signaling acts as a stress-responsive mechanism protecting mitochondria during BTZ exposure, sustaining mitochondrial metabolism and promoting drug resistance. Inhibition of TLR4 could be therefore be a possible target in patients with refractory MM to overcome BTZ resistance.

Intercellular Mitochondrial Transfer in the Tumor Microenvironment.

Cell-to-cell communication is a fundamental process in every multicellular organism. In addition to membrane-bound and released factors, the sharing of cytosolic components represents a new, poorly explored signaling route. An extraordinary example of this communication channel is the direct transport of mitochondria between cells. In this review, we discuss how intercellular mitochondrial transfer can be used by cancer cells to sustain their high metabolic requirements and promote drug resistance and describe relevant molecular players in the context of current and future cancer therapy.

Abstract A55: DCLK1 mediates tumor stemness and platinum resistance in high-grade serous epithelial ovarian cancer

Abstract Background: Ovarian cancer is the fifth leading cause of cancer death in the United States, with an anticipated 13,000 deaths in the United States in 2017. High-grade serous (HGSOC) sub-type is both the most common and the most lethal histopathology. As more than 85% of patients present with metastatic disease, the 5-year overall survival remains dismal at only 30%. It is known that human cancers are initiated and maintained by self-renewing stem cells. Cancer stem cell related protein Doublecortin-like kinase 1 (DCLK1) is a major regulator of stemness and EMT, promoting drug resistance, tumor dormancy, progression, and metastasis in solid tumors. The molecular mechanisms of DCLK1 in tumorigenesis and drug resistance remain limited but DCLK1 expression is increased in cancer, especially in the drug-resistant setting. Results: Our lab has made the discovery that DCLK1 is overexpressed in HGSOC where it regulates tumor progression, tumor stemness, and EMT via noncoding RNA regulation of downstream oncogenic pathways. Our study involved assessment of DCLK1 expression in tissue culture and xenografts created from primary patient-derived cell lines (PDACs). Expression of the DCLK1 protein was assessed by immunoblot analysis in lysates of cell lines representing HGSOC and PDACs obtained from the ascites of patients at the University of Oklahoma at the time of diagnosis, prior to initiation of chemotherapy. FTE-187, FTE-188, and OSE cells were used as normal controls. Elevated levels of DCLK1 expression were observed in the panel of HGSOC cell lines and in PDAC lines. Our in vitro studies confirmed DCLK1 upregulation in the tissue and stroma of PDAC lines and our in vivo studies with PDAC xenografts revealed that siRNA knockdown of DCLK1 negatively regulated tumor suppressor miRNAs including mi-143/145, let-7a, and miR-200a-c resulting in downregulation of pluripotency, EMT, and proangiogenic markers including OCT4, SOX2, KLF4, NANOG, LIN28B, NOTCH, KRAS, and c-MYC. This was most pronounced in the setting of platinum resistance. For this, lysates from paired cells lines with known platinum-sensitive and platinum-resistant variants were assessed (Tyk-nu/Tyk-nu CPR and A2780/A2780.cp). An increase in DCLK1 expression was observed in both of the platinum-resistant lines compared to the platinum-sensitive lines. This was confirmed in PDACs where clinical platinum status correlated with DCLK1 expression, suggesting the role of DCLK1 in inherent platinum-resistant tumor clones. Conclusions: Our data implicate DCLK1 in ovarian cancer pathogenesis and platinum resistance and indicate that DCLK1 knockdown via may suppress malignant progression and resensitize tumors to platinum chemotherapy. These data could have a significant impact on future therapeutic strategies involving DCLK1 inhibitors in treating platinum-resistant patients. Given the high recurrence rate and inevitable development of platinum-resistant disease, such compounds have high potential to impact clinical outcomes in this lethal malignancy. Citation Format: Katherine M. Moxley, JiHee Ha, Daniel Andrade, Courtney Houchen, Danny Dhanasekaran. DCLK1 mediates tumor stemness and platinum resistance in high-grade serous epithelial ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A55.

MiR-145 Regulates the Chemoresistance of Hepatic Carcinoma Cells Against 5-Fluorouracil by Targeting Toll-Like Receptor 4.

Background5-fluorouracil (5-FU) is a common drug for hepatic carcinoma (HCC), but the drug resistance of clinical chemotherapy restricts its use. Studies have demonstrated that miRNA molecules can act as a chemoresistance regulator in drug resistance of tumors, whereas the role of miR-145 in the 5-FU-resistant HCC remains unclear.ObjectiveTo explore the prognostic value of miR-145 in HCC and its molecular mechanism in 5-FU-resistant HCC cells.MethodsA qRT-PCR assay was conducted to quantify miR-145 in HCC tissues and 5-FU-resistant HCC cells. The Cell Counting Kit-8 (CCK-8) and flow cytometry were adopted to analyze the proliferation and apoptosis of 5-FU-resistant HCC cells. The Western blot was adopted to quantify toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and apoptosis-related proteins. Moreover, an in vivo tumor xenotransplantation of nude mice was conducted to determine the effect of miR-145 on 5-FU-resistant HCC cells.ResultsMiR-145 was expressed lowly in HCC tissues and cells, and linked to high TNM staging and lymph node metastasis of HCC patients. Down-regulation of miR-145 indicated a poorer prognosis and it promoted drug resistance of HCC cells and inhibited cell apoptosis. In contrast, miR-145 overexpression improved the sensitivity of HCC cells to 5-FU and enhanced the inhibition of 5-FU on tumor growth. The luciferase reporter gene assay showed that TLR4 was the direct target of miR-145, and the Western blot assay revealed that overexpression of TLR4 reversed the inhibitory effect of miR-145 overexpression on TLR4 and MyD88 protein and the effects of it on apoptosis-related proteins.ConclusionMiR-145 is an inhibiting factor in HCC and can target TLR4 to mediate the chemoresistance of HCC, which may provide novel ideas for treating HCC.

ACLY: A biomarker of recurrence in breast cancer

ObjectiveACLY is a cytoplasmic metabolic enzyme involved in lipid synthesis. It also affects proliferation and metastasis of breast cancer. However, the correlation of ACLY expression with breast cancer recurrence is unclear. MethodsThe Oncomine and TCGA databases were used to investigate the mRNA expression of ACLY in breast cancer. Immunohistochemistry (IHC) was used to evaluate ACLY expression level in tumor tissues and normal tissues from 127 breast cancer patients. Next, the prognostic role of ACLY was explored by analyzing the clinicopathological features and prognosis during follow-up. The role of ACLY in breast cancer cells drug resistance was further detected by CCK-8 assays and quantitative real-time polymerase chain reaction (qRT-PCR). ResultsACLY mRNA and protein expression was significantly increased in the breast cancer tissues compared to normal tissues. Clinically, high ACLY levels were associated with ER status, PR status, tumor size, TNM stage, and lymph node invasion. Upregulated ACLY predicted worse tumor relapse-free survival (RFS) of breast cancer patients in univariate analyses and in multivariate models. In subgroup analysis, patients with high ACLY expression showed worse RFS in the TNM III or ER positive subgroups. Moreover, ACLY over-expression induced the resistance of breast cancer cells to docetaxel and promoted the expression of multi-drug resistant protein ABCB1/ABCG2. ConclusionsOur study highlights the possibility of ACLY as a potential and independent biomarker for the recurrence prediction in breast cancer patients. It may be related to ACLY promoting drug resistance in breast cancer cells.