Julia V. Burnier

Type IV collagen-initiated signals provide survival and growth cues required for liver metastasis

The liver is a major site of metastasis for human malignancies, yet the factors that regulate tumor cell survival and growth in this organ remain elusive. Previously, we reported that M-27IGF−IR murine lung carcinoma cells with ectopic insulin-like growth factor-1 (IGF-I) receptor overexpression acquired a site-specific, liver-metastasizing potential. Gene expression profiling and subsequent RNA and protein analyses revealed that this was associated with major changes to the expression of extracellular matrix (ECM) protein-encoding genes including type III, IV and XVIII collagen genes, and these changes were also observed in the respective tumors in vivo. Because type IV collagen was the most prominently altered ECM protein in this model, we further analyzed its functional relevance to liver metastasis. M-27 cells stably overexpressing type IV collagen α1 and α2 chains were generated and their growth and metastatic properties investigated. We found that these cells acquired a site-selective growth advantage in the liver and this was associated with cell rescue from anoikis in a collagen IV/α2 integrin/FAK-dependent manner and increased responsiveness to IGF-I. Conversely, collagen IV or focal adhesion kinase (FAK) silencing by small-interfering RNA in highly metastatic tumor cells enhanced anoikis and decreased liver metastases formation. Moreover, analysis of human surgical specimens revealed uniformly high collagen IV expression in 65/65 hepatic metastases analyzed, regardless of tissue of origin, whereas it was variable and generally low in 50/50 primary colorectal carcinoma specimens examined. The results suggest that collagen IV-conveyed signals are essential cues for liver metastasis in diverse tumor types and identify mediators of collagen IV signaling as potential therapeutic targets in the management of hepatic metastases.

Autologous Bone Marrow Stromal Cells Genetically Engineered to Secrete an IGF-I Receptor Decoy Prevent the Growth of Liver Metastases

Liver metastases respond poorly to current therapy and remain a frequent cause of cancer-related mortality. We reported previously that tumor cells expressing a soluble form of the insulin-like growth factor-I receptor (sIGFIR) lost the ability to metastasize to the liver. Here, we sought to develop a novel therapeutic approach for prevention of hepatic metastasis based on sustained in vivo delivery of the soluble receptor by genetically engineered autologous bone marrow stromal cells. We found that when implanted into mice, these cells secreted high plasma levels of sIGFIR and inhibited experimental hepatic metastases of colon and lung carcinoma cells. In hepatic micrometastases, a reduction in intralesional angiogenesis and increased tumor cell apoptosis were observed. The results show that the soluble receptor acted as a decoy to abort insulin-like growth factor-I receptor (IGF-IR) functions during the early stages of metastasis and identify sustained sIGFIR delivery by cell-based vehicles as a potential approach for prevention of hepatic metastasis.

Somatic BRCA1/2 Recovery as a Resistance Mechanism After Exceptional Response to Poly (ADP-ribose) Polymerase Inhibition

Purpose Durable and long-term responses to the poly (ADP-ribose) polymerase inhibitor olaparib are observed in patients without BRCA1/2 mutations. However, beyond BRCA1/2 mutations, there are no approved biomarkers for olaparib in high-grade serous ovarian cancer (HGSOC). To determine mechanisms of durable response and resistance to olaparib therapy, we performed an analysis of HGSOC tumors from three patients without germline BRCA1/2 mutations who experienced exceptional responses to olaparib. Patients and Methods We performed integrated exome, low-pass genome, and RNA sequence analysis of tumors at diagnosis and upon relapse from patients with platinum-sensitive HGSOC recurrence who were treated > 5 years with olaparib therapy as a single agent. Results We observed somatic disruption of BRCA1/2 in all three patients at diagnosis, followed by subsequent BRCA recovery upon progression by copy number gain and/or upregulation of the remaining functional allele in two patients. The third patient with ongoing response (> 7 years) had a tumor at diagnosis with biallelic somatic deletion and loss-of-function mutation, thereby lacking a functional allele for recovery of BRCA1 activity and indicating a potential cure. Conclusion Olaparib has durable benefit for patients with ovarian cancer beyond germline BRCA1/2 carriers. These data suggest that biallelic loss of BRCA1/2 in cancer cells may be a potential marker of long-term response to poly (ADP-ribose) polymerase inhibition and that restoration of homologous repair function may be a mechanism of disease resistance.

Incidence of epithelial lesions of the conjunctiva in a review of 12,102 specimens in Canada (Quebec)

PURPOSE The purpose of this study was to assess the relative frequency of epithelial lesions of the conjunctiva in Canada. METHODS A retrospective study of 12,102 consecutive cases received during 16 years (1993-2009) at the Henry C. Witelson Ocular Pathology Laboratory in Montreal, Canada, was performed. Demographic data was retrieved from histopathological request forms and specimens were categorized and analyzed by mean percentage. The relative frequency of epithelial lesions of the conjunctiva from a single center in Canada, representing the province of Quebec was reviewed. RESULTS Of the 12,102 specimens reviewed, 273 were conjunctival lesions (2.25%), including 86 epithelial tumors (0.71%) of the conjunctiva that comprised the studied sample. The average age of these patients was 59.9 ± 17.6 years, and gender distribution was 66 (69%) males and 30 (31%) females. Fifteen lesions (17.4%) were classified as squamous cell papillomas (mean age, 57.3 ± 16.7 years). Within the ocular surface squamous neoplasia (OSSN) spectrum, there were 10 (11.6%) actinic keratosis (63.8 ± 17.6 years), 27 (31.3%) cases of conjunctival intraepithelial neoplasia (CIN) with variable degrees of atypia (mild to moderate) (63.9 ± 15.3 years), 15 (17.4%) carcinomas in situ (66.7 ± 18.0 years), and 17 (19.7%) squamous cell carcinomas (SCC) (56.2 ± 19.4 years). Two other rare cases of malignant tumors included one basal cell carcinoma and one mucoepidermoid carcinoma. CONCLUSIONS The distribution of our sample is similar to the one reported by the American Forces Institute of Pathology (AFIP) in 1994. When we compare our sample to others coming from countries with high levels of sunlight exposure, we found a lower incidence of ocular surface squamous neoplasia, including squamous cell carcinomas in Canada.

The IGF-I Receptor Can Alter the Matrix Metalloproteinase Repertoire of Tumor Cells through Transcriptional Regulation of PKC-α

The IGF-I receptor (IGF-IR) was identified as a tumor progression factor, but its role in invasion and metastasis has been the subject of some controversy. Previously we reported that in murine lung carcinoma M-27 cells, overexpression of IGF-IR increased the synthesis and activation of matrix metalloproteinase (MMP)-2 via Akt/phosphatidylinositol 3-kinase signaling. In contrast, we show here that in these and other cells, IGF-IR overexpression reduced the constitutive and phorbol 12-myristate 13-acetate (PMA)-inducible expression of three protein kinase C (PKC)-regulated metalloproteinases, MMP-3, MMP-9, and MMP-13, in cultured cells as well as in vivo in sc tumors. To elucidate the underlying mechanism, we analyzed the effect of IGF-IR on PKC expression and activity using wild-type and IGF-IR-overexpressing (M-27(IGFIR)) tumor cells. Our results show that overexpression and activation of IGF-IR reduced PKC-alpha expression, PKC activity, and downstream ERK1/2 signaling, and these effects were reversed in cells expressing kinase (Y(1131,1135,1136)F) or C-terminal (Y(1250/51)F) domain mutants of IGF-IR. This reduction was due to transcriptional down-regulation of PKC-alpha as evidenced by reduced PKC-alpha mRNA expression in a phosphatidylinositol 3-kinase-dependent manner and a blockade of PKC-alpha promoter activation as revealed by a reporter gene assay. Finally, reconstitution of PKC-alpha levels could restore MMP-9 expression levels in these cells. Collectively, these results show that IGF-IR can inhibit PKC-alpha gene transcription and thereby block the synthesis of PMA-regulated MMPs, suggesting that within the same cells, IGF-IR can act as both a positive and negative regulator of MMP expression and function.

Vascular endothelial growth factor expression and inhibition in uveal melanoma cell lines

Background: Uveal melanoma (UM) is a disease that affects approximately five people per million in the United States. This disease metastasises predominantly to the liver, and treatment options following the clinical detection of these sequelae are limited. Vascular endothelial growth factor-A (VEGF-A) is the primary activator of tumour angiogenesis and functions by binding to VEGF-Receptor 2 (VEGF-R2) and is often required for tumour growth beyond 2–3 mm. The purpose of this study was to investigate the expression of VEGF-A and the primary VEGF-R2 in three UM cell lines. Furthermore, we investigated the effects of VEGF-A inhibition on receptor activation and production of other cytokines. Finally, the effects of VEGF-A inhibition on the proliferation, migration, and invasion in the cell lines were ascertained. Materials: Three UM cell lines (92.1, OCM-1, and UW-1) were incubated with and without the addition of 100 μg/mL of bevacizumab. VEGF-A expression under both conditions was determined by sandwich enzyme-linked immunosorbent assay (ELISA), and phosphorylated VEGF-R2 expression was determined using western blot. The effects of VEGF-A inhibition on 20 cytokines (IL-1a, IL-2, IL-5, IL-8, IL-12p70, GM-CSF, IFNy, CCL3, MMP-9, TNF-a, IL-1b, IL-4, IL-6, IL-10, IL-13, GRO, MCP-1, MIP-1b, and RANTES) were determined using a multiplex sandwich ELISA. Proliferation rates before and after treatment were evaluated via sulforhodamine B assay, and migration and invasion assays implementing the Boyden chamber technique, the latter with artificial extracellular matrix, were used to assess their respective abilities. The Student’s t-test was used to compare changes in cytokine expression following VEGF-A inhibition. Analysis of variance was used to compare changes in the functional abilities of three uveal melanoma cell lines following VEGF-A inhibition. A P-value < 0.05 was considered statistically significant. Results: All three cell lines produced copious amounts of VEGF-A in culture (92.1, 11785.5 ± 231.8 pg/μL; OCM-1, 4608.0 ± 324.0 pg/μL; UW-1, 8309.3 ± 634.5 pg/μL), which was reduced to undetectable levels following the administration of bevacizumab (P< 0.05). Similarly, detectable phosphorylated VEGF-R2 was present in all cells, which was reduced significantly in all cell lines following bevacizumab treatment (107525.2 ± 8602.0 versus 1024.5 ± 98.2, 46587.3 ± 4192.9 versus 12821.1 ± 1666.7, and 60394.3 ± 4026.4 versus 6908.2 ± 607.2; 92.1, OCM-1, and UW-1, respectively; P< 0.05). Of the cytokines investigated, only MMP-9 and CCL3 were ubiquitously altered across all three cell lines following bevacizumab treatment; they were upregulated (CCL3: 1072.50 ± 18.77 pg/mL versus 1281.00 ± 72.34 pg/mL; 22.5 ± 7.85 pg/mL versus 62.00 ± 9.16 pg/mL; 20.33 ± 6.35 pg/mL versus 35.00 ± 6.22 pg/mL; control versus bevacizumab; MMP-9: 25.50 ± 5.47 pg/mL versus 88.25 ± 13.38 pg/mL; 19.75 ± 4.14 pg/mL versus 45.25 ± 8.36 pg/mL; 3.25 ± 1.09 pg/mL versus 19.25 ± 3.77 pg/mL; control versus bevacizumab; 92.1, OCM-1, and UW-1, respectively; P< 0.05). Bevacizumab significantly reduced the proliferation of one cell line (92.1: 0.405 ± 0.012 versus 0.509 ± 0.033; bevacizumab versus control; values OD; P< 0.05), the migration of two cell lines (92.1: 0.071 ± 0.003 versus 0.115 ± 0.003; OCM-1: 0.049 ± 0.005 versus 0.117 ± 0.014; bevacizumab versus control; values OD; P< 0.05), and did not significantly affect invasion. Conclusion: Despite the significant reduction in phosphorylated VEGF-R2 levels, bevacizumab did not have a dramatic impact on the functional abilities of the three UM cell lines studied. Our results indicate that compensatory mechanisms, such as the upregulation of MMP-9 and CCL-3, following bevacizumab administration may mitigate its effects on these abilities.

The inhibitor of kappa B kinase-epsilon regulates MMP-3 expression levels and can promote lung metastasis.

The factors that determine the ability of metastatic tumor cells to expand and grow in specific secondary site(s) are not yet fully understood. Matrix metalloproteinases (MMP) were identified as potential regulators of the site-specificity of metastasis. We found that lung carcinoma cells ectopically expressing high levels of the receptor for the type I insulin like growth factor receptor (M27R cells) had a significant reduction in MMP-3 expression levels and this coincided with reduced metastasis to the lung. We used these cells to further investigate signaling pathways regulating MMP-3 expression and the role that MMP-3 plays in lung metastasis. We show that ectopic IκB kinase ɛ (IKKɛ) expression in these cells partly restored MMP-3 expression levels and also sensitized MMP-3 transcription to induction by phorbol 12-myristate 13-acetate (PMA). This increase in MMP-3 production was due to increased activation of several signal transduction mediators, including protein kinase C alpha, ERK2, Akt and the transcription factor p65. Furthermore, reconstitution of MMP-3 expression in M27R cells restored their ability to colonize the lung whereas silencing of MMP-3 in M27 cells reduced metastases. Collectively, our results implicate IKKɛ as a central regulator of PMA-induced cell signaling and MMP-3 expression and identify MMP-3 as an enabler of tumor cell expansion in the lung.Oncogenesis (2014) 3, e116; doi:10.1038/oncsis.2014.28; published online 18 August 2014

Experimental and clinical metastasis

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Management of glaucoma in an eye with diffuse iris melanocytoma

A 68-year-old Caucasian male was referred for evaluation and follow-up of unilateral glaucoma resistant to control with topical medication. Corrected visual acuity was 20/20 in the right and 20/25 in the left eye. Biomicroscopy was normal and the intraocular pressure (IOP) was 14 mm Hg in the right eye. In the left eye, ciliary injection, a diffuse iris hyperchromia (figure 1A), discrete corneal oedema with the presence of sparse pigment deposits on the endothelium (figure 1B), and mild anterior chamber reaction (+/4+) were observed. The angle was obstructed inferiorly by the pigment deposition, while the parts of the angle that were visible showed increased pigmentation of the trabecular meshwork during gonioscopy. The iris surface was smooth and the crypts were absent. The pupil was round but fixed in medium mydriasis with a posterior synechia seen at 1 h. In addition, the lens had nuclear sclerosis and the IOP was measured at 58 mm Hg. Funduscopy disclosed increased cup-to-disc ratio in the left (0.7) compared with the right eye (0.4), but was otherwise unremarkable. Figure 1 Patient presents with left hyperchromic heterochromia (A). Observed in (B) is a smooth surface and an absence of crypts. Pigment deposits could be seen over the lens capsule and the endothelium side of the cornea. Diffuse hyperreflectivity of the iris in the left is shown in (C). Normal UBM in the right eye is also shown for comparison (D). Normal reflectivity of the ciliary processes in the left eye can be seen in an UBM (E). The patients relevant medical history included controlled hypertension and a basal cell carcinoma in the left arm resected 2 years before. There was no evidence of congenital ocular or oculodermal melanocytosis in the patient or his family. Presently, differential diagnoses include diffuse iris melanoma and diffuse iris melanocytoma. Ultrasound biomicroscopy was performed showing a …

Eyelid Mycetoma Masquerading as Sebaceous Carcinoma.

A 56-year-old Asian woman presented with an upper eyelid mass. The lesion was exposed after eversion of the eyelid revealing a thickened tarsus with yellowish areas. Working diagnosis was sebaceous carcinoma. Biopsy was performed. Histopathological studies showed a mycotic eumycetoma with Splendore-Hoeppli phenomena and - microbiologic cultures grew Scedosporium apiospermum. The patient was started on voriconazole 200 mg po bid with adequate serum levels. A complete response was observed after 18 weeks of voriconazole therapy. To the best of our knowledge, this is the first published case of S. apiospermum eumycotic mycetoma of the eyelid. It is important to consider mycotic infection in the differential diagnosis of eyelid tumors even in healthy patients.