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Alternative splicing of TIA-1 in human colon cancer regulates VEGF isoform expression, angiogenesis, tumour growth and bevacizumab resistance.

Hamdollah Zadeh MA, Amin EM, Hoareau-Aveilla C, Domingo E, Symonds KE, Ye X, Heesom KJ, Salmon A, D'Silva O, Betteridge KB, Williams AC, Kerr DJ, Salmon AH, Oltean S, Midgley RS, Ladomery MR, Harper SJ, Varey AH, Bates DO - Mol Oncol (2014)

Bottom Line: Whereas flTIA-1 selectively bound VEGF-A165 mRNA and increased translation of VEGF-A165b, sTIA-1 prevented this binding.In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies.These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy.

View Article: PubMed Central - PubMed

Affiliation: Microvascular Research Laboratories, Veterinary Sciences Building, School of Physiology and Pharmacology, University of Bristol, Southwell Street, Bristol BS2 8EJ, UK.

No MeSH data available.


Related in: MedlinePlus

sTIA-1 splice variants are present in human colon cancer. A. Upper panel, RT-PCR of mRNA extracted from paired tumour (T) and normal (N) tissues from 6 patients undergoing bowel resection for colon cancer. In 4 of 6 patients, the splice isoform were present in the tumours, but not in the normal tissue. Lower panel, RT-PCR for splice forms of VEGF. B. Quantitation of band intensity of sTIA-1 relative to flTIA-1 in control and tumour tissue. C. Quantitation of band intensity of VEGF-A165b relative to VEGF-A165 in colon cancers compared with normal tissues. D. RNA was extracted from 25 μm thick scrolls of paraffin embedded sections of 40 colorectal carcinoma (20 Duke's B, 20 Duke's C stage) and matched normal (N) samples, and RT-PCR carried out to detect flTIA-1 and sTIA-1. RT-PCR detected TIA-1 RNA expression in 61/80 samples. E. Number of cases in which flTIA-1 was found in tumour or normal (p = 0.02 Fishers exact test). F. Number of cases in which flTIA-1 was found (black bars) or not found (white bars) in wildtype versus ras mutant tumours. G. Number of cases in which sTIA-1 was more highly expressed than flTIA-1 (compared with normal p = 0.0106). G Number of cases in which sTIA-1 was detectable in Duke's B compared with Duke's C grade (p = 0.018, statistics for E, F and G Fisher's exact test). H. Sections adjacent to those from which RNA had been extracted were stained for blood vessels (CD31). Blood vessels were counted per high power field, blinded for sTIA-1 status. The gel shows the PCR product for the two sections shown. Arrows indicate blood vessels. I. Microvascular density for sections where flTIA-1 was more intense than sTIA-1 and vice versa. * = p < 0.05 unpaired t test.
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fig4: sTIA-1 splice variants are present in human colon cancer. A. Upper panel, RT-PCR of mRNA extracted from paired tumour (T) and normal (N) tissues from 6 patients undergoing bowel resection for colon cancer. In 4 of 6 patients, the splice isoform were present in the tumours, but not in the normal tissue. Lower panel, RT-PCR for splice forms of VEGF. B. Quantitation of band intensity of sTIA-1 relative to flTIA-1 in control and tumour tissue. C. Quantitation of band intensity of VEGF-A165b relative to VEGF-A165 in colon cancers compared with normal tissues. D. RNA was extracted from 25 μm thick scrolls of paraffin embedded sections of 40 colorectal carcinoma (20 Duke's B, 20 Duke's C stage) and matched normal (N) samples, and RT-PCR carried out to detect flTIA-1 and sTIA-1. RT-PCR detected TIA-1 RNA expression in 61/80 samples. E. Number of cases in which flTIA-1 was found in tumour or normal (p = 0.02 Fishers exact test). F. Number of cases in which flTIA-1 was found (black bars) or not found (white bars) in wildtype versus ras mutant tumours. G. Number of cases in which sTIA-1 was more highly expressed than flTIA-1 (compared with normal p = 0.0106). G Number of cases in which sTIA-1 was detectable in Duke's B compared with Duke's C grade (p = 0.018, statistics for E, F and G Fisher's exact test). H. Sections adjacent to those from which RNA had been extracted were stained for blood vessels (CD31). Blood vessels were counted per high power field, blinded for sTIA-1 status. The gel shows the PCR product for the two sections shown. Arrows indicate blood vessels. I. Microvascular density for sections where flTIA-1 was more intense than sTIA-1 and vice versa. * = p < 0.05 unpaired t test.

Mentions: sTIA-1 and VEGF-A165b RNA were detected in freshly isolated human colorectal cancer samples (Figure 4A) confirmed by sequencing. Quantification showed both a significant increase in sTIA-1 expression in cancer (Figure 4B) and a significantly lower VEGF-A165b:VEGF-A165 ratio in the tumours than the controls (Figure 4C). In histological samples, flTIA-1 RNA (Figure 4D) was found only in 13 of 31 tumours (42%) but 22 of 30 normal tissues (73%, p = 0.02, Fisher's exact test) samples (Figure 4E). The proportion of tumours that were K-Ras mutant was significantly higher (p < 0.01) in those that had no flTIA-1, and K-Ras mutant tumours rarely (2/13 cases) had flTIA-1 detected (Figure 4F). Quantification showed that sTIA-1 exceeded flTIA-1 in 20 of 31 tumours (65%) but in only 9 of 30 normal tissues (30%, p = 0.01, Figure 4G). Moreover, sTIA-1 was present in 14 of the 15 Dukes' C (93%), but only 9 of the 16 Dukes' B (56%, p < 0.01, Figure 4H). There was a significantly higher microvascular density (Figure 4I) in samples in which sTIA-1 but not flTIA-1 was found (Figure 4J).


Alternative splicing of TIA-1 in human colon cancer regulates VEGF isoform expression, angiogenesis, tumour growth and bevacizumab resistance.

Hamdollah Zadeh MA, Amin EM, Hoareau-Aveilla C, Domingo E, Symonds KE, Ye X, Heesom KJ, Salmon A, D'Silva O, Betteridge KB, Williams AC, Kerr DJ, Salmon AH, Oltean S, Midgley RS, Ladomery MR, Harper SJ, Varey AH, Bates DO - Mol Oncol (2014)

sTIA-1 splice variants are present in human colon cancer. A. Upper panel, RT-PCR of mRNA extracted from paired tumour (T) and normal (N) tissues from 6 patients undergoing bowel resection for colon cancer. In 4 of 6 patients, the splice isoform were present in the tumours, but not in the normal tissue. Lower panel, RT-PCR for splice forms of VEGF. B. Quantitation of band intensity of sTIA-1 relative to flTIA-1 in control and tumour tissue. C. Quantitation of band intensity of VEGF-A165b relative to VEGF-A165 in colon cancers compared with normal tissues. D. RNA was extracted from 25 μm thick scrolls of paraffin embedded sections of 40 colorectal carcinoma (20 Duke's B, 20 Duke's C stage) and matched normal (N) samples, and RT-PCR carried out to detect flTIA-1 and sTIA-1. RT-PCR detected TIA-1 RNA expression in 61/80 samples. E. Number of cases in which flTIA-1 was found in tumour or normal (p = 0.02 Fishers exact test). F. Number of cases in which flTIA-1 was found (black bars) or not found (white bars) in wildtype versus ras mutant tumours. G. Number of cases in which sTIA-1 was more highly expressed than flTIA-1 (compared with normal p = 0.0106). G Number of cases in which sTIA-1 was detectable in Duke's B compared with Duke's C grade (p = 0.018, statistics for E, F and G Fisher's exact test). H. Sections adjacent to those from which RNA had been extracted were stained for blood vessels (CD31). Blood vessels were counted per high power field, blinded for sTIA-1 status. The gel shows the PCR product for the two sections shown. Arrows indicate blood vessels. I. Microvascular density for sections where flTIA-1 was more intense than sTIA-1 and vice versa. * = p < 0.05 unpaired t test.
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fig4: sTIA-1 splice variants are present in human colon cancer. A. Upper panel, RT-PCR of mRNA extracted from paired tumour (T) and normal (N) tissues from 6 patients undergoing bowel resection for colon cancer. In 4 of 6 patients, the splice isoform were present in the tumours, but not in the normal tissue. Lower panel, RT-PCR for splice forms of VEGF. B. Quantitation of band intensity of sTIA-1 relative to flTIA-1 in control and tumour tissue. C. Quantitation of band intensity of VEGF-A165b relative to VEGF-A165 in colon cancers compared with normal tissues. D. RNA was extracted from 25 μm thick scrolls of paraffin embedded sections of 40 colorectal carcinoma (20 Duke's B, 20 Duke's C stage) and matched normal (N) samples, and RT-PCR carried out to detect flTIA-1 and sTIA-1. RT-PCR detected TIA-1 RNA expression in 61/80 samples. E. Number of cases in which flTIA-1 was found in tumour or normal (p = 0.02 Fishers exact test). F. Number of cases in which flTIA-1 was found (black bars) or not found (white bars) in wildtype versus ras mutant tumours. G. Number of cases in which sTIA-1 was more highly expressed than flTIA-1 (compared with normal p = 0.0106). G Number of cases in which sTIA-1 was detectable in Duke's B compared with Duke's C grade (p = 0.018, statistics for E, F and G Fisher's exact test). H. Sections adjacent to those from which RNA had been extracted were stained for blood vessels (CD31). Blood vessels were counted per high power field, blinded for sTIA-1 status. The gel shows the PCR product for the two sections shown. Arrows indicate blood vessels. I. Microvascular density for sections where flTIA-1 was more intense than sTIA-1 and vice versa. * = p < 0.05 unpaired t test.
Mentions: sTIA-1 and VEGF-A165b RNA were detected in freshly isolated human colorectal cancer samples (Figure 4A) confirmed by sequencing. Quantification showed both a significant increase in sTIA-1 expression in cancer (Figure 4B) and a significantly lower VEGF-A165b:VEGF-A165 ratio in the tumours than the controls (Figure 4C). In histological samples, flTIA-1 RNA (Figure 4D) was found only in 13 of 31 tumours (42%) but 22 of 30 normal tissues (73%, p = 0.02, Fisher's exact test) samples (Figure 4E). The proportion of tumours that were K-Ras mutant was significantly higher (p < 0.01) in those that had no flTIA-1, and K-Ras mutant tumours rarely (2/13 cases) had flTIA-1 detected (Figure 4F). Quantification showed that sTIA-1 exceeded flTIA-1 in 20 of 31 tumours (65%) but in only 9 of 30 normal tissues (30%, p = 0.01, Figure 4G). Moreover, sTIA-1 was present in 14 of the 15 Dukes' C (93%), but only 9 of the 16 Dukes' B (56%, p < 0.01, Figure 4H). There was a significantly higher microvascular density (Figure 4I) in samples in which sTIA-1 but not flTIA-1 was found (Figure 4J).

Bottom Line: Whereas flTIA-1 selectively bound VEGF-A165 mRNA and increased translation of VEGF-A165b, sTIA-1 prevented this binding.In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies.These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy.

View Article: PubMed Central - PubMed

Affiliation: Microvascular Research Laboratories, Veterinary Sciences Building, School of Physiology and Pharmacology, University of Bristol, Southwell Street, Bristol BS2 8EJ, UK.

No MeSH data available.


Related in: MedlinePlus