Cisplatin Response Research Articles

KIAA1522 potentiates TNF\u03b1-NF\u03baB signaling to antagonize platinum-based chemotherapy in lung adenocarcinoma

BackgroundThe platinum-based chemotherapy is the first-line regimen for the treatment of Non-small cell lung cancer (NSCLC). However, the therapeutic efficiency is largely limited by tenacious chemo-insensitivity that results in inferior prognosis in a cohort of patients. It has been known that KIAA1522 is aberrantly expressed and implicated in several types of solid tumors including NSCLC. Nowadays, knowledge about this gene is quite limited. Here, we aimed to identify the role of KIAA1522 in lung adenocarcinomas, and the molecular events that underlie KIAA1522-mediated chemoresistance to the platinum.MethodsImmunohistochemistry were used to detect KIAA1522 expression in clinical NSCLC samples. Then, the survival analyses were performed to assess the link between KIAA1522 expression and overall survival or therapeutic outcome. In vivo depletion of KIAA1522 in adenocarcinoma cells were achieved by adeno-associated virus-mediated sgRNA/Cre delivery into the conditional KrasG12D/Cas9 expressed mice, which were designated to identify the roles of KIAA1522 in tumorigenesis and/or chemotherapy responses. The effects of KIAA1522 and downstream molecular events were studied by pharmacology in mice model and assays using in vitro cultured cells. The clinical relevance of our findings was examined by data-mining of online datasets from multiple cohorts.ResultsThe clinical evidences reveal that KIAA1522 independently predicts both the overall survival and the outcome of platinum-based chemotherapy in lung adenocarcinomas. By using a KrasG12D-driven murine lung adenocarcinoma model and performing in vitro assays, we demonstrated that KIAA1522 is a critical positive regulator of lung adenocarcinoma and a modulator of cisplatin response. KIAA1522 potentiates the TNFα-TNFR2-NFκB signaling which in turn intensifies recalcitrance to cisplatin treatment. These results were further manifested by integrative bioinformatic analyses of independent datasets, in which KIAA1522 is tightly associated with the activity of TNFα-NFκB pathway and the cisplatin-resistant gene signatures. More strikingly, overexpression of KIAA1522 counteracts the cisplatin-induced tumor growth arrest in vivo, and this effect can be remarkably diminished by the disruption of NFκB activity.ConclusionHigh expression of KIAA1522 is turned out to be an indicator of dismal effectiveness of platinum-based therapy in lung adenocarcinomas. KIAA1522 hyperactivates TNFα-NFκB signaling to facilitate resistance to platinum reagents. Targeting NFκB signaling through small molecule inhibitors may be a rational strategy to conquer chemoresistance and synergize platinum-based chemotherapy in KIAA1522 overexpressed lung adenocarcinomas.

Impact of ERCC1, XPF and DNA Polymerase β Expression on Platinum Response in Patient-Derived Ovarian Cancer Xenografts.

Platinum resistance is an unmet medical need in ovarian carcinoma. Molecular biomarkers to predict the response to platinum-based therapy could allow patient stratification and alternative therapeutic strategies early in clinical management. Sensitivity and resistance to platinum therapy are partially determined by the tumor’s intrinsic DNA repair activities, including nucleotide excision repair (NER) and base excision repair (BER). We investigated the role of the NER proteins—ERCC1, XPF, ERCC1/XPF complex—and of the BER protein DNA polymerase β, as possible biomarkers of cisplatin (DDP) response in a platform of recently established patient-derived ovarian carcinoma xenografts (OC-PDXs). ERCC1 and DNA polymerase β protein expressions were measured by immunohistochemistry, the ERCC1/XPF foci number was detected by proximity ligation assay (PLA) and their mRNA levels by real-time PCR. We then correlated the proteins, gene expression and ERCC1/XPF complexes with OC-PDXs’ response to platinum. To the best of our knowledge, this is the first investigation of the role of the ERCC1/XPF complex, detected by PLA, in relation to the response to DDP in ovarian carcinoma. None of the proteins in the BER and NER pathways studied predicted platinum activity in this panel of OC-PDXs, nor did the ERCC1/XPF foci number. These results were partially explained by the experimental evidence that the ERCC1/XPF complex increases after DDP treatment and this possibly better associates with the cancer cells’ abilities to activate the NER pathway to repair platinum-induced damage than its basal level. Our findings highlight the need for DNA functional assays to predict the response to platinum-based therapy.

Skipping Exon-v6 from CD44v6-Containing Isoforms Influences Chemotherapy Response and Self-Renewal Capacity of Gastric Cancer Cells.

De novo expressed CD44 isoforms containing exon-v6 are frequently associated with gastric cancer (GC) aggressiveness, and may predict chemotherapy response in vitro. Whether exon-v6 itself is responsible for conferring these properties to CD44v6-containing isoforms remains to be elucidated. CRISPR/Cas9 and Phosphorodiamidate Morpholino oligomers (PMOs) were used to induce specific exon-v6 skipping, maintaining the CD44 reading frame, in two GC cell lines endogenously expressing CD44v6. Cisplatin and 5-fluorouracil treatment response, and self-renewal ability was compared between CRISPR/Cas9-edited, CD44v6 knockdown and mock cells. We obtained homozygous genome-edited cell lines with exon-v6 deletion. Edited cells transcribed CD44v isoforms presenting in frame v5–v7 splicing, mimicking exon-v6 skipping. Results showed that removing specifically exon-v6 sensitizes cells to cisplatin and impairs cells’ self-renewal ability, similarly to CD44v6 knockdown. In parallel, we also tested a clinically feasible approach for transient exon-v6 skipping with a PMO-based strategy. We demonstrate that exon-v6 specific removal from CD44v isoforms increases cell sensitivity to cisplatin and impairs GC cells self-renewal. We trust that a PMO approach designed towards CD44v6 overexpressing GC cells may be a suitable approach to sensitize tumor cells for conventional therapy.

Expression and roles of system L amino acid transporters in human embryonal carcinoma cells.

Testicular germ cell tumors (TGCTs) are the most common malignant cancer in young men. Although TGCTs are generally responsive to platinum-based chemotherapy particularly cisplatin, acquired resistance in patients with metastasis still occurs resulting in poor prognosis. Specifically, differentiation of embryonal carcinoma (EC) cells, the stem cells of TGCTs, can lead to the reduction of cisplatin responsiveness. Therefore, novel therapeutic strategies for TGCTs are needed. System L amino acid transporters have been reported to be up-regulated and to play an important role in tumorigenesis. However, expression and role of system L amino acid transporters in TGCTs remain elusive. Expression of system L amino acid transporters was analyzed in TGCT samples from The Cancer Genome Atlas (TCGA). Expression of LAT1, LAT2, and 4F2hc was examined in human embryonal carcinoma cell line NTERA2. Roles of system L amino acid transporters on NTERA2 cell survival, cell proliferation, pluripotency, and cisplatin sensitivity were evaluated. Based upon TCGA datasets, we found that two isoforms of system L (LAT1 and LAT2) and their chaperone protein 4F2hc are highly expressed in EC samples compared with other groups. Treatment with the system L inhibitor BCH significantly suppressed leucine uptake into the pluripotent EC cell line NTERA2. The malignant phenotypes including cell viability, cell proliferation, and clonal ability were decreased following BCH treatment. Nonetheless, system L inhibition did not alter expression of stemness genes in NTERA2 cells. After NTERA2 differentiation, expressions of LAT1 and LAT2 were decreased. Finally, co-administration of BCH enhanced cisplatin sensitivity in both undifferentiated and differentiated cells. These effects were associated with the reduction in p70S6K phosphorylation. Taken together, these results shed light on the roles of system L amino acid transporters in TGCTs. Therefore, system L amino acid transporters could provide novel therapeutic targets for treatment against TGCTs.

Enhancing chemotherapy response through augmented synthetic lethality by co-targeting nucleotide excision repair and cell-cycle checkpoints

In response to DNA damage, a synthetic lethal relationship exists between the cell cycle checkpoint kinase MK2 and the tumor suppressor p53. Here, we describe the concept of augmented synthetic lethality (ASL): depletion of a third gene product enhances a pre-existing synthetic lethal combination. We show that loss of the DNA repair protein XPA markedly augments the synthetic lethality between MK2 and p53, enhancing anti-tumor responses alone and in combination with cisplatin chemotherapy. Delivery of siRNA-peptide nanoplexes co-targeting MK2 and XPA to pre-existing p53-deficient tumors in a highly aggressive, immunocompetent mouse model of lung adenocarcinoma improves long-term survival and cisplatin response beyond those of the synthetic lethal p53 mutant/MK2 combination alone. These findings establish a mechanism for co-targeting DNA damage-induced cell cycle checkpoints in combination with repair of cisplatin-DNA lesions in vivo using RNAi nanocarriers, and motivate further exploration of ASL as a generalized strategy to improve cancer treatment.

Abstract 3985: GDF15 expression level impacts the canonical pathways of the tumor microenvironment and ovarian tumors with wild-type p53 in response to chemotherapy

Abstract Background The standard treatment for ovarian cancer is surgery, followed by chemotherapy with a platinum agent, such as cisplatin or carboplatin, and a taxane, such as paclitaxel. We previously observed that patients with ovarian cancer that was wild-type for p53 had a lower survival rate than did those with p53 mutations. A better understanding of the molecular changes that occur in epithelial ovarian cancer cells with wild-type p53 in response to cisplatin treatment could reveal a novel mechanism of chemoresistance. Therefore, we might be able to develop novel strategy to overcome chemoresistance. Methods Gene expression profiling was performed on the wild-type p53 ovarian cancer cell line A2780 to identify genes that are highly induced by cisplatin. Growth differentiation factor 15 (GDF15) was identified as being highly induced by cisplatin or carboplatin. This finding was validated in a panel of wild-type and mutant p53 ovarian cancer cell lines. Mouse orthotopic models were used to further investigate the induction of GDF15 by cisplatin in vivo and the effect of GDF15 knockdown (GDF15-KD) on tumor growth and cisplatin response. A novel analysis was performed of RNAseq data generated from tumors formed in cisplatin-treated orthotopic mouse models to understand the tumor and stromal cell signaling changes. Results The induction of GDF15 was demonstrated in vivo, and the serum level of GDF15 after cisplatin treatment was also found to be significantly up-regulated in an A2780 ovarian cancer orthotopic mouse model. More importantly, cisplatin treatment was more effective in shrinking the mouse tumors developed from GDF15-KD A2780 cell line than did control tumors. Histopathological and novel RNAseq analyses of mouse tumors revealed that GDF15-KD tumors had a lower stromal component than did control tumors after cisplatin treatment. GDF15-KD tumors also had a different repertoire of activated and inhibited canonical pathways in their stromal cell and cancer cell components than did control tumors in response to cisplatin treatment. Conclusions GDF15 expression in wild-type p53 ovarian cancer cells can modulate the canonical pathways in the tumor microenvironment in response to cisplatin and may be a mechanism of chemoresistance. Targeting GDF15 signaling could be a novel strategy to enhance the effectiveness of chemotherapy. Citation Format: Daisy I. Izaguirre, Yvonne TM Tsang, Eucharist H. Kun, David M. Gershenson, Kwong-Kwok Wong. GDF15 expression level impacts the canonical pathways of the tumor microenvironment and ovarian tumors with wild-type p53 in response to chemotherapy [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 3985.

Abstract 1496: Paired, single-cell profiling of circulating tumor cell-derived xenograft models of small cell lung cancer reveals intratumoral heterogeneity and emergence of new cell clusters following treatment relapse

Abstract Small cell lung cancer (SCLC), the most aggressive form of lung cancer, is notable for early dissemination and impressive, yet transient, responses to frontline chemotherapy that are rapidly undone by refractory relapses. To gain a better understanding of resistance mechanisms, we generated circulating tumor cell-derived xenograft (CDX) models from liquid biopsies of SCLC patients and treated them with chemotherapy or targeted agents. As expected, CDXs demonstrate similar chemotherapy response to the patient from whom they were derived. We have previously established that baseline chemosensitive CDXs are molecularly homogeneous at the single cell level, while chemoresistant CDXs exhibit increased intratumoral heterogeneity (ITH) with distinct variations in gene expression between cancer cell populations. We hypothesize that paired analyses of chemosensitive CDXs will demonstrate increased ITH after developing acquired resistance and are likely to offer more specific insights into resistance mechanisms than prior work with unrelated CDX models. Platinum-sensitive CDXs were treated with cisplatin or DNA damage response targeted therapies (PARP inhibitor or CHK inhibitor) continuously until tumor progression was observed. To identify transcriptional changes associated with onset of resistance, single-cell RNAseq analysis was performed on vehicle-treated and relapsed CDX tumors. We found globally increased ITH including heterogeneous expression of therapeutic targets and potential resistance pathways, such as EMT, between cellular subpopulations following treatment-resistance. Relapse was consistently associated with increased ITH score (P<0.001) and cell cluster number. To determine whether transcriptional diversity in either therapeutic targets (ex., MYC, DLL3, TOP2A, PARP1, CHEK1, EZH2, etc.) or EMT genes (ex., ZEB1, ZEB2, TWIST1, VIM, AXL) was detectable between the paired tumors after onset of resistance, we compared transcriptional differences between clusters present in vehicle-treated versus cisplatin or targeted therapy-treated tumors. We discovered the emergence of new cell clusters in relapsed tumors in all CDX models (4 CDXs, 3 distinct treatments), including clusters expressing EMT or NOTCH signaling genes. These data suggest that, in response to treatment, SCLC develops increasing transcriptional ITH marked by concurrent, diverse resistant cell clusters. Clinically, these data underscore the importance of maximizing and maintaining the initial response in platinum-sensitive SCLC tumors and highlight the intrinsic transcriptional fluidity underlying SCLC's profound treatment resistance following initial therapy. Citation Format: C. Allison Stewart, Carl M. Gay, Yuanxin Xi, Santhosh Sivajothi, Junya Fujimoto, Patrice M. Hartsfield, Hai Tran, Stephen G. Swisher, Jack A. Roth, Jianjun Zhang, Bonnie Glisson, John V. Heymach, Ignacio Wistuba, Paul Robson, Jing Wang, Lauren A. Byers. Paired, single-cell profiling of circulating tumor cell-derived xenograft models of small cell lung cancer reveals intratumoral heterogeneity and emergence of new cell clusters following treatment relapse [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 1496.

MiR-302b as a Combinatorial Therapeutic Approach to Improve Cisplatin Chemotherapy Efficacy in Human Triple-Negative Breast Cancer.

Introduction: Chemotherapy is still the standard of care for triple-negative breast cancers (TNBCs). Here, we investigated miR-302b as a therapeutic tool to enhance cisplatin sensitivity in vivo and unraveled the molecular mechanism. Materials and Methods: TNBC-xenografted mice were treated with miR-302b or control, alone or with cisplatin. Genome-wide transcriptome analysis and independent-validation of Integrin Subunit Alpha 6 (ITGA6) expression was assessed on mice tumor samples. Silencing of ITGA6 was performed to evaluate cisplatin response in vitro. Further, potential transcription factors of ITGA6 (E2F transcription facor 1 (E2F1), E2F transcription factor 2 (E2F2), and Yin Yang 1 (YY1)) were explored to define the miRNA molecular mechanism. The miR-302b expression was also assessed in TNBC patients treated with chemotherapy. Results: The miR–302b-cisplatin combination significantly impaired tumor growth versus the control through indirect ITGA6 downregulation. Indeed, ITGA6 was downmodulated in mice treated with miR-302b–cisplatin, and ITGA6 silencing increased drug sensitivity in TNBC cells. In silico analyses and preclinical assays pointed out the regulatory role of the E2F family and YY1 on ITGA6 expression under miR-302b–cisplatin treatment. Finally, miR-302b enrichment correlated with better overall survival in 118 TNBC patients. Conclusion: MiR-302b can be exploited as a new therapeutic tool to improve the response to chemotherapy, modulating the E2F family, YY1, and ITGA6 expression. Moreover, miR-302b could be defined as a new prognostic factor in TNBC patients.

Abstract B69: Gut microbiome attenuates epithelial ovarian cancer growth and sensitivity to cisplatin: New opportunities for ovarian cancer treatments

Abstract Epithelial ovarian cancer (EOC) is the second most common gynecologic malignancy in the United States, but the leading cause of gynecologic cancer death. Despite many women achieving remission with first-line therapy, which includes cytoreductive surgery and platinum-taxane chemotherapy, up to 80% of patients will recur and require additional treatment. The interval from last platinum chemotherapy to recurrence has important prognostic and therapeutic implications for patients. Patients with platinum-resistant EOC have fewer treatment options, worse prognosis, and their disease is seldom curable. Antibiotic therapy is frequently used during cancer treatments in patients with EOC for both prophylaxis and treatment of infections after surgery or through chemotherapy. While critical for the care of the patient, we now appreciate that the microbiome has many roles in maintenance of human health and is increasingly linked with many disease states including obesity, cardiovascular disease, and cancer. Recent evidence suggests the gut microbiome may modulate responses to cancer treatment, including traditional chemotherapy and immunotherapy. Given that antibiotics may lead to microbiome disruption for care of EOC patients and lack of understanding of the impact of these antibiotics on tumor progression, in preclinical models, we assessed the impact of microbiome disruption on EOC progression and cisplatin sensitivity. We established a necessary role for the microbiome in suppression of tumor growth and cisplatin response in two mouse models of EOC. Following treatment with antibiotics (ampicillin, neomycin, vancomycin, and metronidazole), murine ID8 or ID8-VEGF EOC that were injected intraperitoneally into C57Bl/6J mice exhibited accelerated tumor growth compared to non-antibiotic treated controls. Tumor growth was monitored by ultrasound weekly. ID8 and ID8-VEGF tumors in antibiotic-treated mice exhibited reduced sensitivity to cisplatin compared to non-antibiotic controls. Mice treated with antibiotics had significantly worse survival compared to non-antibiotic controls. We confirmed depletion of the gut microbiome based on dilated cecum and reduced microbial 16S rRNA concentration in stool of antibiotic-treated compared to control mice. The accelerated tumor growth and cisplatin resistance was not dependent on an intact immune system as we replicated the effect in immune-deficient mice. In mechanistic studies we determined that disruption of the microbiome led to increased cancer stem cells that was further augmented by cisplatin treatment. Collectively, these studies indicate an intact microbiome provides a tumor-suppressive microenvironment and enhances sensitivity to cisplatin. Future studies will assess whether individual microbial communities are sufficient to reverse the accelerated tumor growth and resistance to cisplatin observed in antibiotic-treated mice and use of selective antibiotics to target pathogenic bacteria while sparing beneficial microbes. Citation Format: Laura Chambers, Emily Esakov, Chad Braley, Leila Edelman, Roberto Vargas, Justin Lathia, Chad Michener, Ofer Reizes. Gut microbiome attenuates epithelial ovarian cancer growth and sensitivity to cisplatin: New opportunities for ovarian cancer treatments [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 B69.

Acquisition of Cisplatin Resistance Shifts Head and Neck Squamous Cell Carcinoma Metabolism toward Neutralization of Oxidative Stress.

Background: Cisplatin (CDDP) is commonly utilized in the treatment of advanced solid tumors including head and neck squamous cell carcinoma (HNSCC). Cisplatin response remains highly variable among individual tumors and development of cisplatin resistance is common. We hypothesized that development of cisplatin resistance is partially driven by metabolic reprogramming. Methods: Using a pre-clinical HNSCC model and an integrated approach to steady state metabolomics, metabolic flux and gene expression data we characterized the interaction between cisplatin resistance and metabolic reprogramming. Results: Cisplatin toxicity in HNSCC was driven by generation of intra-cellular oxidative stress. This was validated by demonstrating that acquisition of cisplatin resistance generates cross-resistance to ferroptosis agonists despite the fact that cisplatin itself does not trigger ferroptosis. Acquisition of cisplatin resistance dysregulated the expression of genes involved in amino acid, fatty acid metabolism and central carbon catabolic pathways, enhanced glucose catabolism and serine synthesis. Acute cisplatin exposure increased intra-tumoral levels of S-methyl-5-thiadenosine (MTA) precursors and metabotoxins indicative of generalized oxidative stress. Conclusions: Acquisition of cisplatin resistance is linked to metabolic recovery from oxidative stress. Although this portends poor effectiveness for directed metabolic targeting, it supports the potential for biomarker development of cisplatin effectiveness using an integrated approach.

Abstract PR08: Inhibition of ATR as a therapeutic strategy to overcome cisplatin resistance in HNSCC

Abstract Background: Standard treatment for head and neck squamous cell carcinoma (HNSCC) includes platinum-based therapy given in combination with radiation. Although platinum has been a key drug of HNSCC treatment for decades, many patients develop resistance and the prognosis is unsatisfactory, and therefore development of novel treatment strategies is needed. Targeting ATR has been shown to be effective as a monotherapy in various type of cancers. Thus, inhibition of ATR could be a new approach when combined with DNA damaging agents to kill cancer cells. Here, we hypothesize that addition of ATR inhibitor AZD6738 can enhance the antitumor effects of cisplatin in HNSCC cells and result in better survival in preclinical models of HNSCC. Materials and Methods: Clonogenic survival assays, reverse phase protein array (RPPA), Western blotting, cell cycle analysis, and apoptosis assay were used to evaluate the in vitro sensitivity of HNSCC cells to AZD6738 and cisplatin and the molecular mechanisms associated with antitumor response. An orthotopic mouse model of oral cancer was used to explore in vivo efficacy of the combination treatment. Results: HNSCC cells treated with various concentrations of AZD6738 showed an IC50 value of 0.24-3.00 μmol/L and no differential sensitivity was observed among cells with different TP53 mutational status. Treatment with constant drug concentration ratios of AZD6738 and cisplatin (2:1) resulted in a combination index of <1, indicating synergistic effects. RPPA pointed out proteins involved in DNA repair, DNA replication, and cell cycle to be predominantly changed in the combination groups compared to single treatments. Among these, the replication factors Cdc6 and Cdt1 were both reduced with the combination treatment. Furthermore, Western blot results revealed that combination treatments lead to enhanced DNA damage indicated by increased phosphorylation of H2AX and induction of replication stress marker (decrease of phospho-CDK1 and phospho-CHK1). Moreover, levels of RRM2 and Rad51 were diminished, suggesting insufficient nucleotide supply and impaired homologous replication. Combining both drugs also resulted in a time-dependent increase of cleaved PARP. This apoptotic death was further confirmed by Annexin V/PI assay. Cell cycle analysis and live cell imaging revealed that an accumulation of cells in the S phase was more than doubled after 12 and 48 hours, indicating replicative stress. In a pilot in vivo study, cisplatin combined with AZD6738 reduced tumor growth in mice bearing oral tumors. Conclusions: Addition of AZD6738 synergizes with cisplatin in vitro and potentiates cisplatin response in vivo. Mechanistically, combination of cisplatin and AZD6738 induces DNA damage and replication stress, resulting in apoptotic cell death. Interestingly, proteins essential for the origin licensing as part of the DNA replication initiation machinery were decreased following combination treatment, offering a potential mechanistic insight and warranting further investigation. Citation Format: Antje Lindemann, Hideaki Takahashi, Ameeta A. Patel, Walter N. Hittelman, Abdullah A. Osman, Jeffrey N. Myers. Inhibition of ATR as a therapeutic strategy to overcome cisplatin resistance in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr PR08.

Mitochondrial DNA alterations may influence the cisplatin responsiveness of oral squamous cell carcinoma

Cisplatin is the first-line chemotherapeutic agent for the treatment of oral squamous cell carcinoma (OSCC). However, the intrinsic or acquired resistance against cisplatin remains a major obstacle to treatment efficacy in OSCC. Recently, mitochondrial DNA (mtDNA) alterations have been reported in a variety of cancers. However, the role of mtDNA alterations in OSCC has not been comprehensively studied. In this study, we evaluated the correlation between mtDNA alterations (mtDNA content, point mutations, large-scale deletions, and methylation status) and cisplatin sensitivity using two OSCC cell lines, namely SAS and H103, and stem cell-like tumour spheres derived from SAS. By microarray analysis, we found that the tumour spheres profited from aberrant lipid and glucose metabolism and became resistant to cisplatin. By qPCR analysis, we found that the cells with less mtDNA were less responsive to cisplatin (H103 and the tumour spheres). Based on the findings, we theorised that the metabolic changes in the tumour spheres probably resulted in mtDNA depletion, as the cells suppressed mitochondrial respiration and switched to an alternative mode of energy production, i.e. glycolysis. Then, to ascertain the origin of the variation in mtDNA content, we used MinION, a nanopore sequencer, to sequence the mitochondrial genomes of H103, SAS, and the tumour spheres. We found that the lower cisplatin sensitivity of H103 could have been caused by a constellation of genetic and epigenetic changes in its mitochondrial genome. Future work may look into how changes in mtDNA translate into an impact on cell function and therefore cisplatin response.

Loss of AKR1C1 is a good prognostic factor in advanced NPC cases and increases chemosensitivity to cisplatin in NPC cells.

Cisplatin resistance is one of the main obstacles in the treatment of advanced nasopharyngeal carcinoma (NPC). AKR1C1 is a member of the Aldo‐keto reductase superfamily (AKRs), which converts aldehydes and ketones to their corresponding alcohols and has been reported to be involved in chemotherapeutic resistance of multiple drugs. The expression and function of AKR1C1 in NPC have not been reported until now. The aim of this research was to investigate the expression of AKR1C1 and it is role in cisplatin resistance in NPC. AKR1C1 protein expression was detected by immunohistochemistry in human NPC tissues and by Western blot assays in NPC and immortalized nasopharyngeal epithelial cells. The effects of AKR1C1 knock‐down by siRNA on proliferation, migration and invasion in NPC cells were evaluated by CCK8, wound healing and transwell assays. To evaluate the effects of AKR1C1 silencing on cisplatin sensitivity in NPC cells, CCK8 assays were used to detect cell proliferation, flow cytometry was used to detect cell cycle distribution, and flow cytometry and DAPI staining were used to detect cell apoptosis. AKR1C1 down‐regulation was associated with advanced clinicopathological characters such as larger tumor size, more lymphatic nodes involvement, with metastasis and later clinical stages, while AKR1C1 down‐regulation was a good prognostic factor for overall survival (OS) in NPC patients. In vitro study showed that AKR1C1 was not directly involved in the malignant biological behaviours such as proliferation, cell cycle progression and migration of NPC cells, whereas AKR1C1 knock‐down could enhance cisplatin sensitivity of NPC cells. These results suggest that AKR1C1 is a potential marker for predicting cisplatin response and could serve as a molecular target to increase cisplatin sensitivity in NPC.

Expression of CD44v6-Containing Isoforms Influences Cisplatin Response in Gastric Cancer Cells.

CD44v6-containing isoforms are frequently de novo expressed in gastric cancer (GC). Whether CD44v6 has a central role in GC transformation and/or progression, whether it conditions response to therapy or whether it is only a bystander marker is still not known. Therefore, we aimed to clarify the role of CD44v6 in GC. We generated GC isogenic cell lines stably expressing CD44s or CD44v6 and tested them for different cancer hallmarks and response to cisplatin, and we further confirmed our findings in cells that endogenously express CD44v6. No correlation between overexpression of CD44v6 and the tested cancer hallmarks was observed, suggesting CD44v6 is not a driver of GC progression. Upon cisplatin treatment, CD44v6+ cells survive better and have lower apoptosis levels than CD44v6− cells, possibly due to concomitant activation of STAT3 and P38. In co-culture experiments, we discovered that CD44v6+ cells are involved in GC cell overgrowth after cisplatin treatment. In conclusion, we show that CD44v6 expression increases cell survival in response to cisplatin treatment in GC cells and that these cells override CD44v6-negative cells after cisplatin-treatment. This suggests that tumor expression of CD44v6-containing variants may condition the outcome of GC patients treated with chemotherapy.

Expression of large tumour suppressor (LATS) kinases modulates chemotherapy response in advanced non-small cell lung cancer.

BackgroundThe Hippo signalling pathway plays an important role in regulating organ size and cell proliferation. Down-regulation of large tumour suppressor (LATS) protein homologs LATS1 or LATS2 has been found in lung cancer. LATS1 and LATS2 are the core components of the Hippo signalling pathway. LATS1 and LATS2 share some conserved structural features and exhibit redundant biological functions. The aim of this study was to dissect the interaction between these two homologs.MethodsIn lung adenocarcinoma (AD) cells, protein expression of LATS1 and LATS2 were determined by western blotting; cell viability and apoptosis were measured by MTT and annexin V staining after treatment with cisplatin; subcellular distributions of LATS proteins were determined by immunofluorescence microscopy; LATS2 expression was modulated by shRNA-mediated knockdown or ectopic expression in cancer cell lines.ResultsManipulation of the expression of these two LATS kinases influenced cisplatin response in advanced lung AD cell lines. High LATS2-to-LATS1 ratio in H2023 cells was associated with cisplatin resistance, while low LATS2-to-LATS1 ratio in CL1-0 and CL83 cells was associated with sensitivity to cisplatin. Manipulating the LATS2-to-LATS1 ratio by LATS2 over-expression in CL1-0 and CL83 rendered them resistant to cisplatin treatment, whereas LATS2 knockdown in H2023 alleviated the LATS2-to-LATS1 ratio and sensitized cancer cells to cisplatin exposure.ConclusionsOur data suggested that the ratio of expression of LATS kinases played a role in the modulation of cisplatin sensitivity in advanced lung AD, and targeting of LATS proteins as a novel therapeutic strategy for lung AD deserves further investigation.

Ovarian cancer-derived copy number alterations signatures are prognostic in chemoradiotherapy-treated head and neck squamous cell carcinoma.

DNA copy number alterations (CNAs) are frequent in cancer, and recently developed CNA signatures revealed their value in molecular tumor stratification for patient prognosis and platinum resistance prediction in ovarian cancer. Head and neck squamous cell carcinoma (HNSCC) is also characterized by high CNAs. In this study, we determined CNA in 173 human papilloma virus‐negative HNSCC from a Dutch multicenter cohort by low‐coverage whole genome sequencing and tested the prognostic value of seven cancer‐derived CNA signatures for these cisplatin‐ and radiotherapy‐treated patients. We find that a high CNA signature 1 (s1) score is associated with low values for all other signatures and better patient outcomes in the Dutch cohorts and The Cancer Genome Atlas HNSCC data set. High s5 and s7 scores are associated with increased distant metastasis rates and high s6 scores with poor overall survival. High cumulative cisplatin doses result in improved outcomes in chemoradiotherapy‐treated HNSCC patients. Here we find that tumors high in s1 or low in s6 are most responsive to a change in cisplatin dose. High s5 values, however, significantly increase the risk for metastasis in patients with low cumulative cisplatin doses. Together this suggests that the processes causing these CNA signatures affect cisplatin response in HNSCC. In conclusion, CNA signatures derived from a different cancer type were prognostic and associated with cisplatin response in HNSCC, suggesting they represent underlying molecular processes that define patient outcome.

Long Non-coding RNA CCAT1 Sponges miR-454 to Promote Chemoresistance of Ovarian Cancer Cells to Cisplatin by Regulation of Surviving.

PurposeColon cancer-associated transcript 1 (CCAT1) was identified as an oncogenic long non-coding RNA (lncRNA) in a variety of cancers. However, there was a lack of understanding of the mechanism by which CCAT1 conferred cisplatin (also known as DDP) resistance in ovarian cancer cells.Materials and MethodsCell viability of A2780, SKOV3, A2780/DDP, and SKOV3/DDP cells upon cisplatin treatment was monitored by MTT assay. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) detected the expression levels of CCAT1 and miR-454. The effect of sh-CCAT1 on cisplatin response was investigated in xenografts study. Bioinformatic analysis, luciferase reporter assay and qRT-PCR were conducted to validate the direct interaction among CCAT1, miR-454, and survivin. Apoptosis was determined by flow cytometry after dual staining of Annexin-V-FITC/propidium iodide, and the expression of apoptosis-related proteins Bcl-2, Bax and survivin were detected by qRT-PCR and Western blotting. Xenograft study was conducted to monitor in vivo tumor formation.ResultsCCAT1 was highly expressed in cisplatin-resistant ovarian cancer cell line A2780/DDP and SKOV3/DDP. Knockdown of CCAT1 restored sensitivity to cisplatin in vitro and in vivo. Our data revealed that silencing of CCAT1 promoted cisplatin-induced apoptosis via modulating the expression of pro- or anti-apoptotic proteins Bax, Bcl-2, and survivin. CCAT1 directly interacted with miR-454, and miR-454 overexpression potentiated cisplatin-induced apoptosis. Survivin was identified as a functional target of miR-454, restoration of survivin attenuated the effect of miR-454 on cisplatin response. In addition, miR-454 inhibitor or overexpression of survivin was found to abolish sh-CCAT1–induced apoptosis upon cisplatin treatment.ConclusionCCAT1/miR-454/survivin axis conferred cisplatin resistance in ovarian cancer cells.

Truncated Bid Regulates Cisplatin Response via Activation of Mitochondrial Apoptosis Pathway in Ovarian Cancer.

Refractoriness to conventional chemotherapy is a major challenge in the treatment of advanced ovarian cancer (OC). There is increasing evidence that mitochondrial priming correlates with cisplatin response in various cancers. Notably, Bim and Bid, two of the proapoptotic BH3-only proteins, are recognized as the most effective inducers of mitochondrial priming in OC. In this study, we constructed two tumor-specific oncolytic adenoviruses (Ads) coding for Bim (Ad-Bim) or truncated Bid (Ad-tBid), respectively, and performed gain-of-function assays in nine OC cell lines. Ad-tBid exhibited significant antitumor efficacy than the controls. On addition of Ad-tBid pretreatment, mito-primed cells displayed more sensitivity to cisplatin both in vitro and ex vivo. We also found that Ad-tBid induced mitochondrial apoptosis in a Bak-dependent manner. Furthermore, a combined cisplatin plus Ad-tBid therapy markedly inhibited tumor growth in a subcutaneous xenotransplanted tumor model. In mice bearing peritoneal disseminated OC, intraperitoneal administration of Ad-tBid potentiated the antitumor effect of cisplatin. Our findings suggest that Ad-tBid enhances cisplatin response in OC cells, establishing the potential treatment of advanced OC via a combination of cisplatin and Ad-tBid.

Genomic Characteristics of Triple-Negative Breast Cancer Nominate Molecular Subtypes That Predict Chemotherapy Response.

The heterogeneity of triple-negative breast cancer (TNBC) poses difficulties for suitable treatment and leads to poor outcome. This study aimed to define a consensus molecular subtype (CMS) of TNBC and thus elucidate genomic characteristics and relevant therapy. We integrated the expression profiles of 957 TNBC samples from published datasets. We identified genomic characteristics of subtype by exploring the pathway activity, microenvironment, and clinical relevance. In addition, drug response (DR) scores (n = 181) were computationally investigated using chemical perturbation gene signatures and validated in our own patient with TNBC (n = 38) who received chemotherapy and organoid biobank data (n = 64). Subsequently, cooperative functions with drugs were also explored. Finally, we classified TNBC into four CMSs: stem-like; mesenchymal-like; immunomodulatory; luminal-androgen receptor. CMSs also elucidated distinct tumor-associated microenvironment and pathway activities. Furthermore, we discovered metastasis-promoting genes, such as secreted phosphoprotein 1 by comparing with primary. Computational DR scores associated with CMS revealed drug candidates (n = 18), and it was successfully evaluated in cisplatin response of both patients and organoids. Our CMS recapitulated in-depth functional and cellular heterogeneity encompassing primary and metastatic TNBC. We suggest DR scores to predict CMS-specific DRs and to be successfully validated. Finally, our approach systemically proposes a relevant therapeutic prediction model as well as prognostic markers for TNBC. IMPLICATIONS: We delineated the genomic characteristic and computational DR prediction for TNBC CMS from gene expression profile. Our systematic approach provides diagnostic markers for subtype and metastasis verified by machine-learning and novel therapeutic candidates for patients with TNBC.

Targeting of EGFR by a combination of antibodies mediates unconventional EGFR trafficking and degradation

Antibody combinations targeting cell surface receptors are a new modality of cancer therapy. The trafficking and signalling mechanisms regulated by such therapeutics are not fully understood but could underlie differential tumour responses. We explored EGFR trafficking upon treatment with the antibody combination Sym004 which has shown promise clinically. Sym004 promoted EGFR endocytosis distinctly from EGF: it was asynchronous, not accompanied by canonical signalling events and involved EGFR clustering within detergent-insoluble plasma mebrane-associated tubules. Sym004 induced lysosomal degradation independently of EGFR ubiquitylation but dependent upon Hrs/Tsg101 that are required for the formation of intraluminal vesicles (ILVs) within late endosomes. We propose Sym004 cross-links EGFR physically triggering EGFR endocytosis and incorporation onto ILVs and so Sym004 sensitivity correlates with EGFR numbers available for binding, rather than specific signalling events. Consistently Sym004 efficacy and potentiation of cisplatin responses correlated with EGFR surface expression in head and neck cancer cells. These findings will have implications in understanding the mode of action of this new class of cancer therapeutics.