Andrographolide sensitizes KRAS-mutant colorectal cancer cells to cetuximab by inhibiting the EGFR/AKT and PDGFRß/AKT signaling pathways.

BACKGROUND: Cetuximab, an inhibitor targeting EGFR, is widely applied in clinical management of colorectal cancer (CRC). Nevertheless, drug resistance induced by KRAS-mutations limits cetuximab's anti-cancer effectiveness. Furthermore, the persistent activation of EGFR-independent AKT is another significant factor in cetuximab resistance. Nevertheless, the mechanism that EGFR-independent AKT drives cetuximab resistance remains unclear. Thus, highlighting the need to optimize therapies to overcome cetuximab resistance and also to explore the underlying mechanism. PURPOSE: This work aimed to investigate whether and how andrographolide enhance the therapeutic efficacy of cetuximab in KRAS-mutant CRC cells by modulating AKT. METHODS: The viabilities of CRC cell lines were analyzed by CCK-8. The intracellular proteins phosphorylation levels were investigated by Human Phospho-kinase Antibody Array analysis. Knockdown and transfection of PDGFRß were used to evaluate the role of andrographolide on PDGFRß. The western blotting was used to investigate Wnt/ß-catenin pathways, PI3K/AKT, and EMT in KRAS-mutant CRC cells. The animal models including subcutaneous tumor and lung metastasis were performed to assess tumor response to therapy in vivo. RESULTS: Andrographolide was demonstrated to decrease the expression of PI3K and AKT through targeting PDGFRß and EGFR, and it enhanced cetuximab effect on KRAS-mutant CRC cells by this mechanism. Meanwhile, andrographolide helped cetuximab to inhibit Wnt/ß-catenin, CRC cell migration and reduced Vimentin expression, while increasing that of E-cadherin. Lastly, co-treatment with cetuximab and andrographolide reduced the growth of KRAS-mutant tumors and pulmonary metastases in vivo. CONCLUSIONS: Our findings suggest that andrographolide can overcome the KRAS-mutant CRC cells' resistance to cetuximab through inhibiting the EGFR/PI3K/AKT and PDGFRß /AKT signaling pathways. This research provided a possible theory that andrographolide sensitizes KRAS-mutant tumor to EGFR TKI.

Divarasib plus cetuximab in KRAS G12C-positive colorectal cancer: a phase 1b trial.

KRAS G12C mutation is prevalent in ~4% of colorectal cancer (CRC) and is associated with poor prognosis. Divarasib, a KRAS G12C inhibitor, has shown modest activity as a single agent in KRAS G12C-positive CRC at 400 mg. Epidermal growth factor receptor has been recognized as a major upstream activator of RAS-MAPK signaling, a proposed key mechanism of resistance to KRAS G12C inhibition in CRC. Here, we report on divarasib plus cetuximab (epidermal growth factor receptor inhibitor) in patients with KRAS G12C-positive CRC (n = 29) from arm C of an ongoing phase 1b trial. The primary objective was to evaluate safety. Secondary objectives included preliminary antitumor activity. The safety profile of this combination was consistent with those of single-agent divarasib and cetuximab. Treatment-related adverse events led to divarasib dose reductions in four patients (13.8%); there were no treatment withdrawals. The objective response rate was 62.5% (95% confidence interval: 40.6%, 81.2%) in KRAS G12C inhibitor-naive patients (n = 24). The median duration of response was 6.9 months. The median progression-free survival was 8.1 months (95% confidence interval: 5.5, 12.3). As an exploratory objective, we observed a decline in KRAS G12C variant allele frequency associated with response and identified acquired genomic alterations at disease progression that may be associated with resistance. The manageable safety profile and encouraging antitumor activity of divarasib plus cetuximab support the further investigation of this combination in KRAS G12C-positive CRC.ClinicalTrials.gov identifier: NCT04449874.

Selecting Optimal First-Line Treatment for Microsatellite Stable and Non-Mutated RAS/BRAF Metastatic Colorectal Cancer.

OPINION STATEMENT: Standard frontline treatment of metastatic colorectal cancer (CRC) is cytotoxic chemotherapy plus a biologic agent such as an anti-EGFR monoclonal antibody (cetuximab or panitumumab) or anti-VEGF antibody (bevacizumab). Predictive biomarkers include mismatch repair (MMR) status, and RAS and BRAF mutation status; and important factors in treatment selection include primary tumor location, intent of therapy, and potential toxicity, as well as patient age, comorbidities, and patient preference. To date, single-, double-, or triple-agent cytotoxic chemotherapy all have important roles in appropriately selected patients, with the addition of anti-VEGF or anti-EGFR antibody therapy based on the relevant predictive biomarker. Data indicate that patients with proficient MMR, RAS/BRAF wt mCRC are candidates for an anti-EGFR antibody plus doublet chemotherapy if they have a left-sided primary tumor, or for anti-VEGF (bevacizumab) plus doublet or triplet chemotherapy if they have a right-sided primary tumor. Future studies may provide more predictive biomarkers to further personalize therapy for this heterogeneous disease.

Treatment of BRAF-V600E mutant metastatic colorectal cancer: new insights and biomarkers.

INTRODUCTION: The presence of a BRAF-V600E mutation in metastatic colorectal cancer (mCRC) is observed in approximately 12% of cases and is associated with poor prognosis and aggressive disease. Unlike melanoma, the development of successful BRAF blockade in colorectal cancer has been complex. The phase III BEACON trial made significant progress in the development of BRAF inhibitors by establishing encorafenib-cetuximab as the new standard of care for patients with mCRC who have progressed to one or two previous lines of treatment. Nonetheless, not all patients respond to encorafenib-based combinations, and some responses are short-lived. Identifying new strategies to boost antitumor activity and improve survival is paramount. AREAS COVERED: The development of targeted therapy for BRAF-V600E mCRC starting with BRAF inhibitors as monotherapy through novel combinations with anti-VEGF or anti-PD1 agents to enhance antitumor activity is reviewed, with a particular focus on the development of predictive and prognostic biomarkers. EXPERT OPINION: There is a crucial need to better understand tumor biology and develop accurate and reliable biomarkers to enhance the antitumor activity of encorafenib-based combinations. The RNF43 mutation is an accurate and reliable predictive biomarker of response, and combinations that target crosstalk between the MAPK pathway, the immune system, and WNT pathways seem promising.

Cetuximab enhances radiosensitivity of esophageal squamous cell carcinoma cells by G2/M cycle arrest and DNA repair delay through inhibiting p-EGFR and p-ERK.

BACKGROUND: Although radiotherapy has improved local control in esophageal squamous cell carcinoma (ESCC), a considerable number of patients still experience relapse due to resistance. In this study, we aimed to evaluate the effects of cetuximab on radiosensitivity in two ESCC cell lines (ECA109 and TE-13) and to investigate their underlying mechanisms. METHODS: Cells were pretreated with or without cetuximab before irradiation. The MTT assay and clonogenic survival assay were performed to evaluate cell viability and radiosensitivity. Flow cytometry was performed to analyze cell cycle distribution and apoptosis. The γH2AX foci were counted to determine cellular DNA-repairing capacity via immunofluorescence assay. The phosphorylation of key molecules involved in the epidermal growth factor receptor (EGFR) signaling pathway and DNA double-strand break (DSB) repair were measured by western blot. RESULTS: Cetuximab alone was not sufficient to suppress cell viability, but significantly enhanced radiation-induced inhibition of clonogenic survival in ECA109 and TE-13. The radiation sensitivity enhancement ratio (SER) was 1.341 and 1.237 for ECA109 and TE-13, respectively. ESCC cells treated with cetuximab were arrested at the G2/M phase in response to radiation. No significant increase in apoptotic rate was observed in irradiated cells that were treated with cetuximab. The average number of γH2AX foci increased in the combination group (cetuximab and radiation). Cetuximab suppressed phosphorylation of EGFR and downstream ERK, but had no significant effect on AKT. CONCLUSIONS: These results indicate the potential for use of cetuximab as an effective radiosensitizer in ESCC. Cetuximab promotes G2/M cycle arrest and reduces DSB repair, as well as inhibiting EGFR and downstream ERK pathways in ESCC.

Conversion Surgery After Encorafenib Plus Cetuximab for Chemorefractory BRAF V600E-mutated Colorectal Cancer With Para-aortic Lymph Node Metastases.

BACKGROUND/AIM: Mutant BRAF V600E colorectal cancer accounts for 5% of metastatic colorectal cancers, and it has a poor response to systemic chemotherapy and a poor prognosis. However, combination treatment involving MAPK pathway blockade is effective for this cancer. Herein, we report a case of a patient who underwent conversion surgery after encorafenib plus cetuximab for chemorefractory BRAF V600E-mutated colorectal cancer with para-aortic lymph node metastases. CASE REPORT: A 68-year-old woman was diagnosed with ascending colon cancer and multiple para-aortic lymph node metastases. After primary tumor resection, molecular genetic testing of the primary tumor revealed a BRAF V600E mutation. She was treated with FOLFOXIRI plus bevacizumab as first-line chemotherapy. After disease progression, the regimen was changed to encorafenib plus cetuximab, and the metastatic lesions shrank. She underwent para-aortic lymph node dissection as conversion surgery, and pathology revealed complete response of the lymph nodes. She achieved long-term survival. CONCLUSION: The development of new treatments for BRAF V600E-mutated metastatic colorectal cancer will increase opportunities for conversion therapy.

Therapeutic landscape and future direction of metastatic colorectal cancer.

In the era of targeted therapy based on genomic alterations, the treatment strategy for metastatic colorectal cancer (mCRC) has been changing. Before systemic treatment initiation, determination of tumour genomic status for KRAS and NRAS, BRAFV600E mutations, ERBB2, and microsatellite instability and/or mismatch repair (MMR) status is recommended. In patients with deficient MMR and BRAFV600E mCRC, randomized phase III trials have established the efficacy of pembrolizumab as first-line therapy and the combination of encorafenib and cetuximab as second-line or third-line therapy. In addition, new agents have been actively developed in other rare molecular fractions such as ERBB2 alterations and KRASG12C mutations. In March 2022, the combination of pertuzumab and trastuzumab for ERBB2-positive mCRC was approved in Japan, thereby combining real-world evidence from the SCRUM-Japan Registry. As the populations are highly fragmented owing to rare genomic alterations, various strategies in clinical development are expected. Clinical development of a tumour-agnostic approach, such as NTRK fusion and tumour mutational burden, has successfully introduced corresponding drugs to clinical practice. Considering the difficulty of randomized trials owing to cost-benefit and rarity, a promising solution could be real-world evidence utilized as an external control from the molecular-based disease registry.

A Novel EGFR Targeted Immunotoxin Based on Cetuximab and Type 1 RIP Quinoin Overcomes the Cetuximab Resistance in Colorectal Cancer Cells.

Cetuximab is a monoclonal antibody blocking the epidermal growth factor receptor (EGFR) in metastatic colorectal cancer (mCRC). However, cetuximab treatment has no clinical benefits in patients affected by mCRC with KRAS mutation or in the presence of constitutive activation of signalling pathways acting downstream of the EGFR. The aim of this study was to improve cetuximab's therapeutic action by conjugating cetuximab with the type 1 ribosome inactivating protein (RIP) quinoin isolated from quinoa seeds. A chemical conjugation strategy based on the use of heterobifunctional reagent succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was applied to obtain the antibody-type 1 RIP chimeric immunoconjugate. The immunotoxin was then purified by chromatographic technique, and its enzymatic action was evaluated compared to quinoin alone. Functional assays were performed to test the cytotoxic action of the quinoin cetuximab immunoconjugate against the cetuximab-resistant GEO-CR cells. The novel quinoin cetuximab immunoconjugate showed a significant dose-dependent cytotoxicity towards GEO-CR cells, achieving IC50 values of 27.7 nM (~5.0 µg/mL) at 72 h compared to cetuximab (IC50 = 176.7 nM) or quinoin (IC50 = 149.3 nM) alone assayed in equimolar amounts. These results support the therapeutic potential of quinoin cetuximab immunoconjugate for the EGFR targeted therapy, providing a promising candidate for further development towards clinical use in the treatment of cetuximab-resistant metastatic colorectal cancer.

Promotion or remission: a role of noncoding RNAs in colorectal cancer resistance to anti-EGFR therapy.

Anti-epidermal-growth-factor-receptor (EGFR) monoclonal antibodies (mAbs) are of great significance for RAS and BRAF wild-type metastatic colorectal cancer (mCRC) patients. However, the generation of primary and secondary resistance to anti-EGFR mAbs has become an important factor restricting its efficacy. Recent studies have revealed that non-coding RNAs (ncRNAs), especially long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs), are implicated in anti-EGFR antibodies resistance, affecting the sensitivity of CRC cells to Cetuximab and Panitumumab. This paper briefly reviewed the research advance of the expression, signaling network and functional mechanism of ncRNAs related to anti-EGFR mAbs resistance in CRC, as well as their relationship with clinical prognosis and the possibility of therapeutic targets. In addition, some ncRNAs that are involved in the regulation of signaling pathways or genes related to anti-EGFR resistance, but need to be further verified by resistance experiments were also included in this review, thereby providing more ideas and basis for ncRNAs as CRC prognostic markers and anti-EGFR therapy sensitizers. Video Abstract.

The Evolving Treatment Landscape in BRAF-V600E-Mutated Metastatic Colorectal Cancer.

Between 8% and 12% of patients with metastatic colorectal cancer (mCRC) harbor a BRAF-V600E mutation in their tumors, which is associated with a poor response to standard chemotherapy and short overall survival. Moreover, nearly 30% of BRAF-V600E mCRC tumors also have microsatellite instability. Transcriptomic signatures suggest a strong immunogenic biologic background for most of these tumors. In contrast to the melanoma context, single-agent BRAF inhibition does not achieve clinical benefit in BRAF-V600E mCRC. Different preclinical/translational studies have elucidated that, in this context, upon BRAF inhibition, there is immediate signal upregulation via the EGFR, and therefore an anti-EGFR treatment should be added to the BRAF inhibitor. Several phase II studies have confirmed the activity of BRAF inhibitors combined with EGFR-directed monoclonal antibodies in patients with BRAF-V600E mCRC. The role of other mitogen-activated protein kinase inhibitors, such as mitogen-activated protein kinase kinase or PI3K inhibitors, remains unclear. The phase III BEACON clinical trial confirmed the BRAF/EGFR inhibitor combination of encorafenib/cetuximab as the new standard of care for BRAF-V600E mCRC after at least one previous line of systemic therapy. Novel approaches for managing BRAF-V600E mCRC include, among others, triple combinations of BRAF inhibitors and anti-EGFR antibodies combined with immune checkpoint inhibitors in the microsatellite instability population and evaluation of the encorafenib/cetuximab treatment in combination with standard chemotherapy with bevacizumab in the first-line setting.

Interaction of lncRNA MIR100HG with hnRNPA2B1 facilitates m6A-dependent stabilization of TCF7L2 mRNA and colorectal cancer progression.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is a process linked to metastasis and drug resistance with non-coding RNAs (ncRNAs) playing pivotal roles. We previously showed that miR-100 and miR-125b, embedded within the third intron of the ncRNA host gene MIR100HG, confer resistance to cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, in colorectal cancer (CRC). However, whether the MIR100HG transcript itself has a role in cetuximab resistance or EMT is unknown. METHODS: The correlation between MIR100HG and EMT was analyzed by curating public CRC data repositories. The biological roles of MIR100HG in EMT, metastasis and cetuximab resistance in CRC were determined both in vitro and in vivo. The expression patterns of MIR100HG, hnRNPA2B1 and TCF7L2 in CRC specimens from patients who progressed on cetuximab and patients with metastatic disease were analyzed by RNAscope and immunohistochemical staining. RESULTS: The expression of MIR100HG was strongly correlated with EMT markers and acted as a positive regulator of EMT. MIR100HG sustained cetuximab resistance and facilitated invasion and metastasis in CRC cells both in vitro and in vivo. hnRNPA2B1 was identified as a binding partner of MIR100HG. Mechanistically, MIR100HG maintained mRNA stability of TCF7L2, a major transcriptional coactivator of the Wnt/ß-catenin signaling, by interacting with hnRNPA2B1. hnRNPA2B1 recognized the N6-methyladenosine (m6A) site of TCF7L2 mRNA in the presence of MIR100HG. TCF7L2, in turn, activated MIR100HG transcription, forming a feed forward regulatory loop. The MIR100HG/hnRNPA2B1/TCF7L2 axis was augmented in specimens from CRC patients who either developed local or distant metastasis or had disease progression that was associated with cetuximab resistance. CONCLUSIONS: MIR100HG and hnRNPA2B1 interact to control the transcriptional activity of Wnt signaling in CRC via regulation of TCF7L2 mRNA stability. Our findings identified MIR100HG as a potent EMT inducer in CRC that may contribute to cetuximab resistance and metastasis by activation of a MIR100HG/hnRNPA2B1/TCF7L2 feedback loop.

Targeted Therapy for Colorectal Cancer.

Metastatic colorectal cancer (mCRC) is incurable in patients with unresectable disease. For most patients, the primary treatment is palliative systemic chemotherapy. Genomic profiling is used to detect specific genetic mutations that may offer selected patients a modest survival benefit with targeted therapy. Patients with mCRC with KRAS/NRAS/BRAF wild-type left-sided tumors may benefit from epidermal growth factor receptor (EGFR) inhibition with either cetuximab or panitumumab, in conjunction with chemotherapy. EGFR inhibitors can extend survival by 6 months compared with chemotherapy alone. The vascular endothelial growth factor (VEGF) inhibitor bevacizumab can serve as an alternative to EGFR inhibitors in right-sided tumors or second-line therapy. Many patients will have RAS mutations, and targeted therapies will not provide any benefit. The PRIME trial demonstrated that the addition of panitumumab to FOLFOX was associated with reduced overall survival. Patients with BRAF mutations do not benefit from targeted therapy unless a BRAF inhibitor supplements treatment. Triple combination therapy with cetuximab, the BRAF inhibitor encorafenib, and the MEK kinase inhibitor binimetinib has extended overall survival by about 3 months compared with chemotherapy alone. Finally, for the minority patients with microsatellite instability (MSI) high/mismatch repair (MMR) deficient tumors, either due to Lynch syndrome or sporadic mutations, immunotherapy is recommended as first-line treatment. The KEYNOTE-177 trial demonstrated that therapy with single-agent pembrolizumab improved progression-free survival by 8 months compared with FOLFOX or FOLFIRI and with or without EGFR inhibition. At this time, targeted therapy should only be used in patients with unresectable metastatic disease.

Correlative N-glycan and charge variant analysis of cetuximab expressed in murine, chinese hamster and human expression systems.

A well-defined and controlled glycosylation pattern is important to maintain quality and safety of therapeutic proteins. Glycosylation is strongly dependent on the host cell line used for recombinant protein expression. Cetuximab, which is produced in mouse myeloma cells has been shown to harbour Fab glycans, which contain non-human like features and hence, can potentially cause an immunogenic response in patients. In light of the advent of biosimilar and biobetter development, we produced cetuximab variants in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. A combination of orthogonal chromatographic modes such as hydrophilic interaction, size exclusion and strong cation exchange chromatography with various detection strategies was employed to characterise the three different cetuximab variants and to compare the in-house produced HEK and CHO variants with the reference drug product. While Fc galactosylation and sialic acid content of the drug product and the HEK variant were highly similar, the CHO product showed lower galactosylation on Fc glycans and a comparatively low sialic acid content in the Fab region. The elevated high-mannose content of CHO cetuximab also suggests potential rapid clearence from circulation. The combination of multiple chromatographic separation modes has proven powerful for the characterisation of expression system dependent protein quality attributes such as N-glycosylation.

The prognostic role of miR-31 in colorectal cancer: the results of a meta-analysis of 4720 patients.

Aims: To study the association between miR-31 expression and clinical outcomes in colorectal cancer. Methods: A systematic search was performed and 16 studies were found eligible. To calculate the combined hazard ratio (HR), the DerSimonian and Laird random-effects model was used. Results: Pooled analysis revealed significant associations between high miR-31 expression and poor overall (HR: 0.68; 95% CI: 0.47-0.97; I2: 68.6%) and progression-free survival (HR: 0.49; 95% CI: 0.33-0.73; I2: 81.1%). High expressers were more likely to have a BRAF mutation. Therapeutic regimen and the mutational status significantly affected the observed associations. Conclusion: We identified that high miR-31 expression is associated with poor overall survival and progression-free survival and has a significant predictive value for anti-EGFR response.

Lay abstract We aim to investigate whether the molecular marker miR-31 is useful in predicting clinical outcomes in colorectal cancer (CRC). We conducted a systematic search in the major scientific databases was performed and 16 studies were found eligible for data extraction.The analysis revealed that high levels of miR-31 in CRC patients are indicative of a shorter survival time. Patients with high miR-31 levels were also more likely to have a mutation in BRAF, an important gene in the pathogenesis and response to treatment of CRC patients. We showed that high levels of miR-31 are associated with shorter survival with a significant predictive value for anti-EGFR response.

Affinity Maturation of B7-H6 Translates into Enhanced NK Cell-Mediated Tumor Cell Lysis and Improved Proinflammatory Cytokine Release of Bispecific Immunoligands via NKp30 Engagement.

Activating NK cell receptors represent promising target structures to elicit potent antitumor immune responses. In this study, novel immunoligands were generated that bridge the activating NK cell receptor NKp30 on NK cells with epidermal growth factor receptor (EGFR) on tumor cells in a bispecific IgG-like format based on affinity-optimized versions of B7-H6 and the Fab arm derived from cetuximab. To enhance NKp30 binding, the solitary N-terminal IgV domain of B7-H6 (ΔB7-H6) was affinity matured by an evolutionary library approach combined with yeast surface display. Biochemical and functional characterization of 36 of these novel ΔB7-H6-derived NK cell engagers revealed an up to 45-fold-enhanced affinity for NKp30 and significantly improved NK cell-mediated, EGFR-dependent killing of tumor cells compared with the NK cell engager based on the wild-type ΔB7-H6 domain. In this regard, potencies (EC50 killing) of the best immunoligands were substantially improved by up to 87-fold. Moreover, release of IFN-γ and TNF-α was significantly increased. Importantly, equipment of the ΔB7-H6-based NK cell engagers with a human IgG1 Fc part competent in Fc receptor binding resulted in an almost 10-fold superior killing of EGFR-overexpressing tumor cells compared with molecules either triggering FcγRIIIa or NKp30. Additionally, INF-γ and TNF-α release was increased compared with molecules solely triggering FcγRIIIa, including the clinically approved Ab cetuximab. Thus, incorporating affinity-matured ligands for NK cell-activating receptors might represent an effective strategy for the generation of potent novel therapeutic agents with unique effector functions in cancer immunotherapy.

Model-based analysis of response and resistance factors of cetuximab treatment in gastric cancer cell lines.

Targeted cancer therapies are powerful alternatives to chemotherapies or can be used complementary to these. Yet, the response to targeted treatments depends on a variety of factors, including mutations and expression levels, and therefore their outcome is difficult to predict. Here, we develop a mechanistic model of gastric cancer to study response and resistance factors for cetuximab treatment. The model captures the EGFR, ERK and AKT signaling pathways in two gastric cancer cell lines with different mutation patterns. We train the model using a comprehensive selection of time and dose response measurements, and provide an assessment of parameter and prediction uncertainties. We demonstrate that the proposed model facilitates the identification of causal differences between the cell lines. Furthermore, our study shows that the model provides predictions for the responses to different perturbations, such as knockdown and knockout experiments. Among other results, the model predicted the effect of MET mutations on cetuximab sensitivity. These predictive capabilities render the model a basis for the assessment of gastric cancer signaling and possibly for the development and discovery of predictive biomarkers.

Production and introduction of a novel immunotoxin based on engineered RNase A for inducing death to Her1-positive cell lines.

The present study was performed to design an immunotoxin consisting of engineered RNase A and scFv of Cetuximab. To accomplish this study goal, at first to evade RNase A from its inhibitors in the cytoplasm, six amino acids of RNase A were substituted, then the physicochemical features of engineered RNase A were assessed. To investigate the interaction between the engineered RNase A and the ribonuclease inhibitor, protein-protein docking was performed. After engineering the RNase A, it was theoretically conjugated with scFv of Cetuximab using a cleavable linker to produce scFv-engineered RNase A. Then, wild-RNase A (14 kD), engineered RNase A (14 kD) and scFv-engineered RNase A (42 kDa) were expressed in the BL21 (DE3) strain of Escherichia coli and purified by Ni-NTA columns. To confirm the expressed proteins, western blot analysis was performed. The functioning of wild-RNase A and engineered RNase A were investigated by RNA fragmentation assay. Finally, to evaluate the cytotoxicity of scFv-engineered RNase A, a dose-response cytotoxicity assay was performed on Her1-positive and Her1-negative cell lines. The results showed that engineered RNase A could maintain its structure and disulfide bonds and evade its inhibitor. Expression and purification were successfully conducted and both enzymes could degrade yeast RNA. The result of cytotoxicity showed that the engineered immunotoxin could induce cell death to Her1-positive cell lines with an IC50 of 50 nM. It appears that scFv-engineered RNase A can be a promising molecule for use.

miR-302a Inhibits Metastasis and Cetuximab Resistance in Colorectal Cancer by Targeting NFIB and CD44.

Introduction: Metastasis and drug resistance contribute substantially to the poor prognosis of colorectal cancer (CRC) patients. However, the epigenetic regulatory mechanisms by which CRC develops metastatic and drug-resistant characteristics remain unclear. This study aimed to investigate the role of miR-302a in the metastasis and molecular-targeted drug resistance of CRC and elucidate the underlying molecular mechanisms. Methods: miR-302a expression in CRC cell lines and patient tissue microarrays was analyzed by qPCR and fluorescence in situ hybridization. The roles of miR-302a in metastasis and cetuximab (CTX) resistance were evaluated both in vitro and in vivo. Bioinformatic prediction algorithms and luciferase reporter assays were performed to identify the miR-302a binding regions in the NFIB and CD44 3'-UTRs. A chromatin immunoprecipitation assay was performed to examine NFIB occupancy in the ITGA6 promoter region. Immunoblotting was performed to identify the EGFR-mediated pathways altered by miR-302a. Results: miR-302a expression was frequently reduced in CRC cells and tissues, especially in CTX-resistant cells and patient-derived xenografts. The decreased miR-302a levels correlated with poor overall CRC patient survival. miR-302a overexpression inhibited metastasis and restored CTX responsiveness in CRC cells, whereas miR-302a silencing exerted the opposite effects. NFIB and CD44 were identified as novel targets of miR-302a. miR-302a inhibited the metastasis-promoting effect of NFIB that physiologically activates ITGA6 transcription. miR-302a restored CTX responsiveness by suppressing CD44-induced cancer stem cell-like properties and EGFR-mediated MAPK and AKT signaling. These results are consistent with clinical observations indicating that miR-302a expression is inversely correlated with the expression of its targets in CRC specimens. Conclusions: Our findings show that miR-302a acts as a multifaceted regulator of CRC metastasis and CTX resistance by targeting NFIB and CD44, respectively. Our study implicates miR-302a as a candidate prognostic predictor and a therapeutic agent in CRC.

The influence of FCGR2A and FCGR3A polymorphisms on the survival of patients with recurrent or metastatic squamous cell head and neck cancer treated with cetuximab.

FCGR2A-H131R and FCGR3A-V157F are single-nucleotide polymorphisms known to influence the outcome of patients treated with rituximab, cetuximab and trastuzumab. We investigated the impact of these polymorphisms on the clinical outcome of 103 patients with recurrent or metastatic squamous cell carcinoma of the head and neck treated with a platinum compound, fluorouracil and cetuximab as palliative first-line therapy. The survival of patients with FCGR2A-131H/H and/or FCGR3A-157V/V genotypes was significantly longer compared with patients carrying 131R and 157F alleles (median progression-free survival (PFS): 5.5 vs 4.1 months, P=0.02; median overall survival: 10.2 vs 7.2 months, P=0.04). In multivariate analysis, the FCGR2A and 3A genotypes as well as the time between initial diagnosis and relapse of disease not amenable to curative therapy remained the only independent prognostic factors for PFS. The results are in line with previous reports in colorectal cancer patients and confirm the possible value of genetic polymorphisms of immunocompetent cells for the success of cetuximab treatment.

Natural and non-natural amino-acid side-chain substitutions: affinity and diffraction studies of meditope-Fab complexes.

Herein, multiple crystal structures of meditope peptide derivatives incorporating natural and unnatural amino acids bound to the cetuximab Fab domain are presented. The affinity of each derivative was determined by surface plasmon resonance and correlated to the atomic structure. Overall, it was observed that the hydrophobic residues in the meditope peptide, Phe3, Leu5 and Leu10, could accommodate a number of moderate substitutions, but these invariably reduced the overall affinity and half-life of the interaction. In one case, the substitution of Phe3 by histidine led to a change in the rotamer conformation, in which the imidazole ring flipped to a solvent-exposed position. Based on this observation, Phe3 was substituted by diphenylalanine and it was found that the phenyl rings in this variant mimic the superposition of the Phe3 and His3 structures, producing a moderate increase, of 1.4-fold, in the half-life of the complex. In addition, it was observed that substitution of Leu5 by tyrosine and glutamate strongly reduced the affinity, whereas the substitution of Leu5 by diphenylalanine moderately reduced the half-life (by approximately fivefold). Finally, it was observed that substitution of Arg8 and Arg9 by citrulline dramatically reduced the overall affinity, presumably owing to lost electrostatic interactions. Taken together, these studies provide insight into the meditope-cetuximab interaction at the atomic level.