Expression dynamics of periodic transcripts during cancer cell cycle progression and their correlation with anticancer drug sensitivity.

BACKGROUND: The cell cycle is at the center of cellular activities and is orchestrated by complex regulatory mechanisms, among which transcriptional regulation is one of the most important components. Alternative splicing dramatically expands the regulatory network by producing transcript isoforms of genes to exquisitely control the cell cycle. However, the patterns of transcript isoform expression in the cell cycle are unclear. Therapies targeting cell cycle checkpoints are commonly used as anticancer therapies, but none of them have been designed or evaluated at the alternative splicing transcript level. The utility of these transcripts as markers of cell cycle-related drug sensitivity is still unknown, and studies on the expression patterns of cell cycle-targeting drug-related transcripts are also rare. METHODS: To explore alternative splicing patterns during cell cycle progression, we performed sequential transcriptomic assays following cell cycle synchronization in colon cancer HCT116 and breast cancer MDA-MB-231 cell lines, using flow cytometry and reference cell cycle transcripts to confirm the cell cycle phases of samples, and we developed a new algorithm to describe the periodic patterns of transcripts fluctuating during the cell cycle. Genomics of Drug Sensitivity in Cancer (GDSC) drug sensitivity datasets and Cancer Cell Line Encyclopedia (CCLE) transcript datasets were used to assess the correlation of genes and their transcript isoforms with drug sensitivity. We identified transcripts associated with typical drugs targeting cell cycle by determining correlation coefficients. Cytotoxicity assays were used to confirm the effect of ENST00000257904 against cyclin dependent kinase 4/6 (CDK4/6) inhibitors. Finally, alternative splicing transcripts associated with mitotic (M) phase arrest were analyzed using an RNA synthesis inhibition assay and transcriptome analysis. RESULTS: We established high-resolution transcriptome datasets of synchronized cell cycle samples from colon cancer HCT116 and breast cancer MDA-MB-231 cells. The results of the cell cycle assessment showed that 43,326, 41,578 and 29,244 transcripts were found to be periodically expressed in HeLa, HCT116 and MDA-MB-231 cells, respectively, among which 1280 transcripts showed this expression pattern in all three cancer cell lines. Drug sensitivity assessments showed that a large number of these transcripts displayed a higher correlation with drug sensitivity than their corresponding genes. Cell cycle-related drug screening showed that the level of the CDK4 transcript ENST00000547281 was more significantly associated with the resistance of cells to CDK4/6 inhibitors than the level of the CDK4 reference transcript ENST00000257904. The transcriptional inhibition assay following M phase arrest further confirmed the M-phase-specific expression of the splicing transcripts. Combined with the cell cycle-related drug screening, the results also showed that a set of periodic transcripts, for example, ENST00000314392 (a dolichyl-phosphate mannosyltransferase polypeptide 2 isoform transcript), was more associated with drug sensitivity than the levels of their corresponding gene transcripts. CONCLUSIONS: In summary, we identified a panel of cell cycle-related periodic transcripts and found that the levels of transcripts of drug target genes showed different values for predicting drug sensitivity, providing novel insights into alternative splicing-related drug development and evaluation.

EGFR targeting enhances the efficiency of chemotherapy through inhibiting IRE1α-XBP1s pathway in colorectal cancer cells.

Targeting EGFR combined with chemotherapy is one of the most valuable therapeutic strategies in colorectal cancer. However, resistance remains a major obstacle to improve efficacy. IRE1α-XBP1s signaling pathway is activated in many malignant tumors, and plays important roles in chemoresistance. Therefore, IRE1α-XBP1s might be a potential target to overcome the chemoresistance in colorectal cancer. In this study, we detected the activation of IRE1α-XBP1s signaling in patient cancer tissues and colorectal cancer cell lines. The phosphorylation level of IRE1α and the spliced XBP1s were aberrantly elevated in colorectal cancer, and IRE1α-XBP1s signaling activation was correlated with high EGFR expression. By overexpression of EGFR protein or activation by EGF treatment, we found that EGFR activation could enhance the phosphorylation of IRE1α and spliced XBP1s expression. On the contrary, inhibition of EGFR decreased the IRE1α-XBP1s signaling. Further, we examined the downstream signaling pathways regulated by EGFR. Inhibition of ERK activity could reverse the EGFR induced IRE1α-XBP1s activation. Co-IP confirmed the physical interaction of ERK and IRE1α. Cell growth and colony formation assay showed that the inhibition of IRE1α activity could suppress EGFR driven colorectal cancer cell proliferation. Furthermore, we found that oxaliplatin could activate IRE1α-XBP1s signaling, and combination with cetuximab partially reversed the activation. Inhibition of EGFR signaling could enhance the efficacy of oxaliplatin in vitro and in vivo. Our results showed that IRE1α RNase activity is aberrantly elevated in colorectal cancer, and EGFR signaling could activate IRE1α/XBP1s possibly through EGFR-MEK-ERK pathway. IRE1α-XBP1s pathway might involve in EGFR driven tumor cell proliferation. Cetuximab could partially recover oxaliplatin-induced IRE1α-XBP1s activation, and therefore enhance the anti-tumor efficacy of oxaliplatin. Our findings declare a new mechanism that targeting EGFR could inhibit chemotherapy-induced IRE1α-XBP1s activation and therefore enhance the efficacy.

A tritherapy combination of a fusion protein vaccine with immune-modulating doses of sequential chemotherapies in an optimized regimen completely eradicates large tumors in mice.

Tumor-induced immunosuppression plays a critical role in both impeding tumor-specific immune responses and limiting the effects of cancer immunotherapy. Analyses of regulatory cells recruited during the growth of the E7-expressing tumor, TC-1, revealed a high percentage of regulatory T cells (Tregs) as well as myeloid-derived suppressor cells (MDSCs) in spleens and tumors. In this study, we proposed that treatment with immune-modulating doses of cyclophosphamide (CTX) and all-trans retinoic acid (ATRA) would result in a beneficial tumor microenvironment with the suppression of Tregs and MDSCs and, thus, enhance the effect of a human papillomavirus protein vaccine. Our results showed that CTX preconditioning and persistent ATRA treatment along with the vaccine achieved long-term survival and induced long-term memory responses. However, the effect of the antitumor response sharply declined when the tritherapy was initiated after the optimal therapeutic time. The more intensive regimen could rescue the effect of the tritherapy accompanied by the decreased percentage of Tregs and MDSCs in spleens and tumors. Besides, a favorable host environment was created by the reduced secretion of interleukin-10 and 6 and vascular endothelial growth factor (VEGF) in the tumor niche and decreased the expression of phosphorylation-signal transducer and activator of transcription 3 of TC-1 tumors. Our data shed light on the immune-modulating doses of sequential chemoimmunotherapeutic strategy targeting not only the tumor but also its microenvironment, which suggests a potential clinical benefit for the immunotherapy of HPV-associated malignancies.