Mary Matli

Induction of Vascular Endothelial Growth Factor by Insulin-like Growth Factor 1 in Colorectal Carcinoma

Vascular endothelial growth factor (VEGF) is an angiogenic hormone that is produced by and supports the growth of many types of malignancies. The present study shows that insulin-like growth factor 1 (IGF-I), a mitogen that promotes the propagation of cancers through autocrine and paracrine mechanisms, increases the expression of mRNA for VEGF and production of VEGF protein by COLO 205 colon carcinoma cells. IGF-I also induces expression of VEGF mRNA in SW620, LSLiM6, and HCT15 colon carcinoma cells showing that this is a common response to IGF-I. Whereas IGF-I induced VEGF mRNA in each cell line examined (2.3-12-fold), it induced proliferation only in COLO 205 and LSLiM6 cells. Thus, the proliferative response induced by IGF-I and its ability to induce VEGF occur through distinguishable mechanisms. IGF-I increases the cellular content of VEGF mRNA by increasing the rate of transcription (5-fold after 4 h) and also by increasing the half-life of VEGF mRNA (0.6 ± 0.07 h in control cells to 2.0 ± 0.37 h in IGF-I-treated cells). Monoclonal antibody (αIR3) directed against the type 1 IGF receptor significantly attenuated the ability of IGF-I to promote expression of VEGF mRNA. Interestingly, by itself αIR3 acted as a weak agonist and induced a modest increase in the cellular content of VEGF mRNA. αIR3 also promoted tyrosine phosphorylation of the β subunit of the IGF-I receptor, and the magnitude of this response was comparable with that induced by IGF-I. These observations point to a nonlinear relationship between activation of the IGF-I receptor and induction of VEGF mRNA. Thus, in addition to its direct, growth stimulatory effect on transformed cells, IGF-I induces the expression of VEGF which can promote the progression of cancer by regulating the development of new blood vessels.

Coordinated induction of VEGF receptors in mesenchymal cell types during rat hepatic wound healing.

Homology PCR has been used to identify receptor tyrosine kinases (RTKs) expressed during activation of rat hepatic stellate cells, the key fibrogenic mesenchymal element in the liver. Partial cDNAs encoding several RTKs were cloned from stellate cells activated in vivo, including those of Flt-1, Flk-1, c-met, PDGFR, and Tyro10/DDR2. RNAse protection from cells activated in vivo demonstrated biphasic induction of flt-1 and flk-1 mRNAs, receptors for vascular endothelial growth factor (VEGF). Culture-activation of stellate cells was associated with increased [125I]VEGF binding and Flt-1 and Flk-1 receptor protein. Induction of VEGF binding sites correlated with an 2.5-fold increase in DNA synthesis in response to VEGF, but only if cells were activated by growth on collagen I, whereas cells maintained in a quiescent state on a basement membrane-like substratum (EHS matrix) were nonproliferative. In both stellate and endothelial cells VEGF-induced mitogenesis was augmented by co-incubation with basic fibroblast growth factor (bFGF), a cytokine with known synergy with VEGF. These findings suggest that the cellular targets of VEGF in liver may not be confined to sinusoidal endothelial cells, and that VEGF responses reflect combined effects on both hepatic stellate cells and sinusoidal endothelium.

Incomplete inhibition of phosphorylation of 4E-BP1 as a mechanism of primary resistance to ATP-competitive mTOR inhibitors

The mammalian target of rapamycin (mTOR) regulates cell growth by integrating nutrient and growth factor signaling and is strongly implicated in cancer. But mTOR is not an oncogene, and which tumors will be resistant or sensitive to new adenosine triphosphate (ATP) competitive mTOR inhibitors now in clinical trials remains unknown. We screened a panel of over 600 human cancer cell lines to identify markers of resistance and sensitivity to the mTOR inhibitor PP242. RAS and phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutations were the most significant genetic markers for resistance and sensitivity to PP242, respectively; colon origin was the most significant marker for resistance based on tissue type. Among colon cancer cell lines, those with KRAS mutations were most resistant to PP242, whereas those without KRAS mutations most sensitive. Surprisingly, cell lines with co-mutation of PIK3CA and KRAS had intermediate sensitivity. Immunoblot analysis of the signaling targets downstream of mTOR revealed that the degree of cellular growth inhibition induced by PP242 was correlated with inhibition of phosphorylation of the translational repressor eIF4E-binding protein 1 (4E-BP1), but not ribosomal protein S6 (rpS6). In a tumor growth inhibition trial of PP242 in patient-derived colon cancer xenografts, resistance to PP242-induced inhibition of 4E-BP1 phosphorylation and xenograft growth was again observed in KRAS mutant tumors without PIK3CA co-mutation, compared with KRAS wild-type controls. We show that, in the absence of PIK3CA co-mutation, KRAS mutations are associated with resistance to PP242 and that this is specifically linked to changes in the level of phosphorylation of 4E-BP1.

Homeodomain transcription factor NKX2.2 functions in immature cells to control enteroendocrine differentiation and is expressed in gastrointestinal neuroendocrine tumors

The homeodomain transcription factor NKX2.2 is necessary for neuroendocrine (NE) differentiation in the central nervous system and pancreas. NE tumors derived from the gut are defined by their NE phenotype, which is used for diagnosis and contributes to tumorigenicity. We hypothesized that NKX2.2 is important for NE differentiation in normal and neoplastic gut. NKX2.2 and NE marker expression was investigated in the small intestine of embryonic and adult mice using immunofluorescence (IF). To determine the role of NKX2.2 in NE differentiation of the intestine, the phenotype of Nkx2.2 (-/-) mice was examined by IF and real-time (RT)-PCR. NKX2.2 and NE marker expression in human NE tumors of the gut and normal tissues were evaluated by immunohistochemistry and qRT-PCR. NKX2.2 expression was detected in the intervillus/crypt regions of embryonic and adult mouse intestine. Co-expression of Nkx2.2 with neurogenin3 (NEUROG3) and hormones was observed in the adult intestinal crypt compartment, suggesting NKX2.2 functions in NEUROG3-positive endocrine progenitors and newly differentiated endocrine cells. In the intestine of Nkx2.2 (-/-) mice, we found a dramatic reduction in the number of cells producing numerous hormones, such as serotonin, gastrin, cholecystokinin, somatostatin, glucagon-like peptide 1 (GLP-1), and secretin, but an increase in cells producing ghrelin. NKX2.2 was expressed in most (24 of 29) human NE tumors derived from diverse primary sites. We conclude NKX2.2 functions in immature endocrine cells to control NE differentiation in normal intestine and is expressed in most NE tumors of the gut, and is therefore a novel target of diagnosis for patients with gastrointestinal NE tumors.

PTEN expression is consistent in colorectal cancer primaries and metastases and associates with patient survival

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) negatively regulates the phosphoinositide‐3‐kinase (PI3K) signaling pathway. In colorectal cancer (CRC), observed frequencies of loss of PTEN expression, concordant expression in primary tumors and metastases, and the association of PTEN status with outcome vary markedly by detection method. We determined the degree to which PTEN expression is consistent in 70 matched human CRC primaries and liver metastases using a validated immunohistochemistry assay. We found loss of PTEN expression in 12.3% of assessable CRC primaries and 10.3% of assessable liver metastases. PTEN expression (positive or negative) was concordant in 98% of matched colorectal primaries and liver metastases. Next we related PTEN status to mutations in RAS and PI3K pathway genes (KRAS, NRAS, BRAF, and PIK3CA) and to overall survival (OS). PTEN expression was not significantly associated with the presence or absence of mutations in RAS or PI3K pathway genes. The median OS of patients whose tumors did not express PTEN was 9 months, compared to 49 months for patients whose tumors did express PTEN (HR = 6.25, 95% confidence intervals (CI) (1.98, 15.42), P = 0.0017). The association of absent PTEN expression with increased risk of death remained significant in multivariate analysis (HR = 6.31, 95% CI (2.03, 17.93), P = 0.0023). In summary, PTEN expression was consistent in matched CRC primaries and in liver metastases. Therefore, future investigations of PTEN in metastatic CRC can use primary tumor tissue. In patients with liver‐only metastases, loss of PTEN expression predicted poor OS.

RasGRP1 opposes proliferative EGFR-SOS1-Ras signals and restricts intestinal epithelial cell growth

The character of EGFR signals can influence cell fate but mechanistic insights into intestinal EGFR-Ras signalling are limited. Here we show that two distinct Ras nucleotide exchange factors, RasGRP1 and SOS1, lie downstream of EGFR but act in functional opposition. RasGRP1 is expressed in intestinal crypts where it restricts epithelial growth. High RasGRP1 expression in colorectal cancer (CRC) patient samples correlates with a better clinical outcome. Biochemically, we find that RasGRP1 creates a negative feedback loop that limits proliferative EGFR–SOS1–Ras signals in CRC cells. Genetic Rasgrp1 depletion from mice with either an activating mutation in KRas or with aberrant Wnt signalling due to a mutation in Apc resulted in both cases in exacerbated Ras–ERK signalling and cell proliferation. The unexpected opposing cell biological effects of EGFR–RasGRP1 and EGFR–SOS1 signals in the same cell shed light on the intricacy of EGFR-Ras signalling in normal epithelium and carcinoma.

Use of recombinant interferon to prevent recurrent herpesvirus shedding

Iontophoresis with 6-hydroxydopamine was performed in rabbits previously infected with herpes simplex virus, McKrae strain. Viral shedding into the tear film was significantly decreased by the use of recombinant alpha interferon subtype D given as one drop qid. Interferon was noted in the tear film of rabbits 16-18 hours after the last placement of interferon drops into the inferior cul-de-sac.

Interferon effects on herpes simplex virus in rabbit and human cell cultures

The effects of four subtypes of recombinant human alpha interferon (RIFN alpha), (A,B,D, and the hybrid A/D) were tested on six strains of herpes simplex virus (HSV). RIFN alpha -D was the most effective subtype in rabbit kidney cells, which is consistent with our previous in vivo results in the rabbit herpetic keratitis model. In human corneal cells, however, RIFN alpha -D was one of the least effective IFN subtypes tested. Conversely, RIFN alpha-A appeared to be relatively more effective in the human corneal cells than in the rabbit kidney cells, but RIFN alpha -B and RIFN alpha -A/D were the most effective interferon subtypes in human corneal cells. Different strains of HSV had different susceptibilities to the various IFN subtypes tested.

Abstract 1201: PTEN null expression is concordant in colorectal cancer primaries and metastases and associates with poor survival.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a negative regulator of the phosphoinositide-3-kinase (PI3K) survival pathway. In colorectal cancer, the method of detection is relevant in determining the frequency of loss of PTEN and the association of PTEN status with outcome. Earlier studies reported discordant PTEN expression in an average of 1 in 3 matched colorectal primaries and metastases. Ours is the first investigation of PTEN concordance using a standardized immunohistochemistry assay. Objectives: Our primary aim was to determine the level of concordant PTEN expression in matched human colorectal primaries and liver metastases using a robust assay. Secondary aims were to relate PTEN status to oncogenic mutations in the RAS and PI3K pathways, and to overall survival. Experimental Design: We used a standardized immunohistochemistry assay and a reproducible method of interpretation to characterize PTEN expression in paired colorectal primaries and liver metastases collected from 70 patients. Mutational hotspots in KRAS, NRAS, BRAF and PIK3CA were sequenced. Results: 7 patients were excluded due to insufficient primary tumor. PTEN null expression was found in 7 of 62 assessable colorectal primaries (12.3%) and 6 of 58 assessable liver metastases (10.3%). PTEN expression (positive or null) was concordant in 98% of matched primaries and liver metastases. There was no significant association between PTEN status and RAS family or PIK3CA mutations. The median overall survival of patients with PTEN null tumors was 9 months, compared to 49 months with PTEN expressing tumors (HR=6.25, 95% CI (1.98, 15.42), p = 0.0017). The association of increased risk of death with PTEN null expression remained significant in multivariate analysis (HR=6.31, 95% CI (2.03, 17.93), p = 0.0023). Conclusion: PTEN status was highly concordant in matched colorectal primaries and liver metastases. Loss of PTEN expression predicted poor overall survival. | | | | PRIMARIES | | | | |:----------- | ----------------- | ---------------------------- | ---------------- | ------- | ------------------ | -------- | | | | PTEN | | PIK3CA | | RAS/BRAF | | | | (−),(+),equivocal | | WT, Mut | | WT, Mut | | METASTASES | PTEN null (−) | 6, 0, 0 | PIK3CA wild-type | 54, 0 | RAS/BRAF wild-type | 29, 1 | | | PTEN positive (+) | 1, 43, 5 | PIK3CA mutant | 2, 5 | RAS/BRAF mutant | 0, 31 | | | PTEN equivocal | 0, 2, 0 | | | | | | CONCORDANCE | | 98%(86% including equivocal) | | 96.7% | | 98.4% | Concordance of PTEN, PIK3CA, RAS, and BRAF in paired colorectal primaries and liver metastases Citation Format: Chloe E. Atreya, Zaina Sangale, Nafei Xu, Mary R. Matli, Eliso Tikishvili, William Welbourn, Steve Stone, Kevan M. Shokat, Robert S. Warren. PTEN null expression is concordant in colorectal cancer primaries and metastases and associates with poor survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1201. doi:10.1158/1538-7445.AM2013-1201

Use of recombinant interferon in HSV-1 recurrence in the rabbit

The use of recombinant human interferon alpha subtype D (RIFN alpha D) was effective in reducing shedding of herpes simplex virus type-1 induced by iontophoresis of 6-hydroxydopamine and epinephrine. A post stimulation treatment schedule of RIFN alpha D, one drop four times a day was as effective as pretreatment, using the same dose regimen. The levels of interferon (IFN) present in the assay system are not sufficient to produce an antiviral effect. The levels of IFN required to suppress cell growth are substantially higher than the concentrations detected in the assay system.