Study of 320-Slice Dynamic Volume CT Perfusion in Different Pathologic Types of Kidney Tumor: Preliminary Results

Objective

To investigate microcirculatory differences between pathologic types of kidney tumor using 320-slice dynamic volume CT perfusion.

Methods

Perfusion imaging with 320-slice dynamic volume CT was prospectively performed in 85 patients with pathologically proven clear cell renal cell carcinoma (RCC) (n = 66), papillary RCC (n = 7), chromophobe RCC (n = 5), angiomyolipoma (AML) with minimal fat (n = 7), or RCC (n = 78). Equivalent blood volume (Equiv BV), permeability surface-area product (PS; clearance/unit volume = permeability), and blood flow (BF) of tumor and normal renal cortex were measured and analyzed. Effective radiation dose was calculated.

Results

There was a significant difference in all three parameters between tumor and normal renal cortex (P<0.001). Equiv BV was significantly different between RCC and AML with minimal fat (P = 0.038) and between clear cell RCC and AML with minimal fat (P<0.001). Mean Equiv BV and BF were significantly higher in clear cell RCC than in papillary RCC (P<0.001 for both) and mean Equiv BV was higher in clear cell RCC than in chromophobe RCC (P<0.001). The effective radiation dose of the CT perfusion protocol was 18.5 mSv.

Conclusion

Perfusion imaging using 320-slice dynamic volume CT can be used to evaluate hemodynamic features of the whole kidney and kidney tumors, which may be useful in the differential diagnosis of these four pathologic types of kidney tumor.     

Up-regulation of microRNA-21 correlates with lower kidney cancer survival

Background

MicroRNA-21 is up-regulated in a variety of cancers like, breast, colorectal, lung, head and neck etc. However, the regulation of miR-21 in renal cell carcinoma (RCC) has not yet been studied systematically.

Methods and Results

We measured miR-21 levels in 54 pairs of kidney cancers and their normal matched tissues by real-time PCR. The expression level of miR-21 was correlated with 5 year survival and the pathological stage. Functional studies were done after inhibiting miR-21 in RCC cell lines. We studied in vitro and in vivo effects of the chemo preventive agent genistein on miR-21 expression. In 48 cases (90%), miR-21 was increased. All patients with low miR-21 expression survived 5 years, while with high miR-21 expression, only 50% survived. Higher expression of miR-21 is associated with an increase in the stage of renal cancer. Functional studies after inhibiting miRNA-21 in RCC cell lines show cell cycle arrest, induction of apoptosis and reduced invasive and migratory capabilities. Western blot analysis showed an increase in the expression of p21 and p38 MAP kinase genes and a reduction in cyclin E2. Genistein inhibited the expression of miR-21 in A-498 cells and in the tumors formed after injecting genistein treated A-498 cells in nude mice besides inhibiting tumor formation.

Conclusions

The current study shows a clear correlation between miR-21 expression and clinical characteristics of renal cancer. Thus we believe that miR-21 can be used as a tumor marker and its inhibition may prove to be useful in controlling cancers with up-regulated miR-21.     

Down-regulation of PKCζ in renal cell carcinoma and its clinicopathological implications

Background

Metastatic renal cell carcinoma (RCC) is highly resistant to systemic chemotherapy. Unfortunately, nearly all patients die of the metastatic and chemoresistant RCC. Recent studies have shown the atypical PKCζ is an important regulator of tumorigenesis. However, the correlation between PKCζ expression and the clinical outcome in RCC patients is unclear. We examined the level of PKCζ expression in human RCC.

Methods

PKCζ mRNA and protein expressions were examined by real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC) respectively in RCC tissues of 144 patients. Cellular cytotoxicity and proliferation were assessed by MTT.

Results

PKCζ expression was significantly higher in normal than in cancerous tissues (P < 0.0001) by real-time PCR and IHC. Similarly, PKCζ expression was down-regulated in four renal cancer cell lines compared to immortalized benign renal tubular cells. Interestingly, an increase of PKCζ expression was associated with the elevated tumor grade (P = 0.04), but no such association was found in TNM stage (P = 0.13). Tumors with higher PKCζ expression were associated with tumor size (P = 0.048). Expression of higher PKCζ found a poor survival in patients with high tumor grade. Down-regulation of PKCζ showed the significant chemoresistance in RCC cell lines. Inactivation of PKCζ expression enhanced cellular resistance to cisplatin and paclitaxel, and proliferation in HK-2 cells by specific PKCζ siRNA and inhibitor.

Conclusions

PKCζ expression was associated with tumorigenesis and chemoresistance in RCC.     

Gas6 and the Receptor Tyrosine Kinase Axl in Clear Cell Renal Cell Carcinoma

Background

The molecular biology of renal cell carcinoma (RCC) is complex and not fully understood. We have recently found that the expression of the receptor tyrosine kinase Axl in the RCC tumors independently correlates with survival of the patients.

Principal Findings

Here, we have investigated the role of Axl and its ligand Gas6, the vitamin-K dependent protein product of the growth arrest-specific gene 6, in clear cell RCC (ccRCC) derived cells. The Axl protein was highly expressed in ccRCC cells deficient in functional von Hippel-Lindau (VHL) protein, a tumor suppressor gene often inactivated in ccRCC. VHL reconstituted cells expressed decreased levels of Axl protein, but not Axl mRNA, suggesting VHL to regulate Axl expression. Gas6-mediated activation of Axl in ccRCC cells resulted in Axl phosphorylation, receptor down-regulation, decreased cell-viability and migratory capacity. No effects of the Gas6/Axl system could be detected on invasion. Moreover, in ccRCC tumor tissues, Axl was phosphorylated and Gas6 γ-carboxylated, suggesting these molecules to be active in vivo.

Significance

These results provide novel information regarding the complex function of the Gas6/Axl system in ccRCC.     

Solution Structure of Tensin2 SH2 Domain and Its Phosphotyrosine-Independent Interaction with DLC-1

Background

Src homology 2 (SH2) domain is a conserved module involved in various biological processes. Tensin family member was reported to be involved in tumor suppression by interacting with DLC-1 (deleted-in-liver-cancer-1) via its SH2 domain. We explore here the important questions that what the structure of tensin2 SH2 domain is, and how it binds to DLC-1, which might reveal a novel binding mode.

Principal Findings

Tensin2 SH2 domain adopts a conserved SH2 fold that mainly consists of five β-strands flanked by two α-helices. Most SH2 domains recognize phosphorylated ligands specifically. However, tensin2 SH2 domain was identified to interact with nonphosphorylated ligand (DLC-1) as well as phosphorylated ligand.

Conclusions

We determined the solution structure of tensin2 SH2 domain using NMR spectroscopy, and revealed the interactions between tensin2 SH2 domain and its ligands in a phosphotyrosine-independent manner.     

KIAA0101 is overexpressed, and promotes growth and invasion in adrenal cancer

Background

KIAA0101 is a proliferating cell nuclear antigen-associated factor that is overexpressed in some human malignancies. Adrenocortical neoplasm is one of the most common human neoplasms for which the molecular causes are poorly understood. Moreover, it is difficult to distinguish between localized benign and malignant adrenocortical tumors. For these reasons, we studied the expression, function and possible mechanism of dysregulation of KIAA0101 in human adrenocortical neoplasm.

Methodology/Principal Findings

KIAA0101 mRNA and protein expression levels were determined in 112 adrenocortical tissue samples (21 normal adrenal cortex, 80 benign adrenocortical tumors, and 11 adrenocortical carcinoma (ACC). SiRNA knockdown was used to determine the functional role of KIAA0101 on cell proliferation, cell cycle, apoptosis, soft agar anchorage independent growth and invasion in the ACC cell line, NCI-H295R. In addition, we explored the mechanism of KIAA0101 dysregulation by examining the mutational status. KIAA0101 mRNA (9.7 fold) and protein expression were significantly higher in ACC (p<0.0001). KIAA0101 had sparse protein expression in only a few normal adrenal cortex samples, which was confined to adrenocortical progenitor cells. KIAA0101 expression levels were 84% accurate for distinguishing between ACC and normal and benign adrenocortical tumor samples. Knockdown of KIAA0101 gene expression significantly decreased anchorage independent growth by 80% and invasion by 60% (p = 0.001; p = 0.006). We found no mutations in KIAA0101 in ACC.

Conclusions/Significance

KIAA0101 is overexpressed in ACC. Our data supports that KIAA0101 is a marker of cellular proliferation, promotes growth and invasion, and is a good diagnostic marker for distinguishing benign from malignant adrenocortical neoplasm.     

A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy.

Methods

Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC). Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR.

Results

In vitro binding experiments of ¹²⁵I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney.

Conclusions

These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.     

Tumor site immune markers associated with risk for subsequent basal cell carcinomas

Background

Basal cell carcinoma (BCC) tumors are the most common skin cancer and are highly immunogenic.

Objective

The goal of this study was to assess how immune-cell related gene expression in an initial BCC tumor biopsy was related to the appearance of subsequent BCC tumors.

Materials and Methods

Levels of mRNA for CD3ε (a T-cell receptor marker), CD25 (the alpha chain of the interleukin (IL)-2 receptor expressed on activated T-cells and B-cells), CD68 (a marker for monocytes/macrophages), the cell surface glycoprotein intercellular adhesion molecule-1 (ICAM-1), the cytokine interferon-γ (IFN-γ) and the anti-inflammatory cytokine IL-10 were measured in BCC tumor biopsies from 138 patients using real-time PCR.

Results

The median follow-up was 26.6 months, and 61% of subjects were free of new BCCs two years post-initial biopsy. Patients with low CD3ε CD25, CD68, and ICAM-1 mRNA levels had significantly shorter times before new tumors were detected (p = 0.03, p = 0.02, p = 0.003, and p = 0.08, respectively). Furthermore, older age diminished the association of mRNA levels with the appearance of subsequent tumors.

Conclusions

Our results show that levels of CD3ε, CD25, CD68, and ICAM-1 mRNA in BCC biopsies may predict risk for new BCC tumors.     

The Axl-Regulating Tumor Suppressor miR-34a Is Increased in ccRCC but Does Not Correlate with Axl mRNA or Axl Protein Levels

Background

High expression of the receptor tyrosine kinase Axl is associated with poor prognosis in patients with Renal Cell Carcinoma (RCC), the most common malignancy of the kidney. The miR-34a has been shown to directly regulate Axl in cancer cells. The miR-34a is a mediator of p53-dependent tumor suppression, and low expression of miR-34a has been associated with worse prognosis in several cancers. Our aim was to elucidate whether miR-34a or the other members of the miR-34 family (miR-34b/c) regulate Axl in RCC.

Methodology and Results

Using western blot, flow cytometry, and RT-qPCR, we showed that Axl mRNA and protein are downregulated in 786-O cells by miR-34a and miR-34c but not by miR-34b. A luciferase reporter assay demonstrated direct interaction between the Axl 3’ UTR and miR-34a and miR-34c. The levels of miR-34a/b/c were measured in tumor tissue in a cohort of 198 RCC patients, and the levels of miR-34a were found to be upregulated in clear cell RCC (ccRCC) tumors, but not associated with patient outcome. Neither of the miR-34 family members correlated with Axl mRNA, soluble Axl protein in serum, nor with immunohistochemistry of Axl in tumor tissue. In addition, we measured mRNA levels of a known miR-34a target, HNF4A, and found the HNF4A levels to be decreased in ccRCC tumors, but unexpectedly correlated positively rather than negatively with miR-34a.

Conclusions

Although miR-34a and miR-34c can regulate Axl expression in vitro, our data indicates that the miR-34 family members are not the primary regulators of Axl expression in RCC.     

Customized Treatment in Non-Small-Cell Lung Cancer Based on EGFR Mutations and BRCA1 mRNA Expression

Background

Median survival is 10 months and 2-year survival is 20% in metastatic non-small-cell lung cancer (NSCLC) treated with platinum-based chemotherapy. A small fraction of non-squamous cell lung cancers harbor EGFR mutations, with improved outcome to gefitinib and erlotinib. Experimental evidence suggests that BRCA1 overexpression enhances sensitivity to docetaxel and resistance to cisplatin. RAP80 and Abraxas are interacting proteins that form complexes with BRCA1 and could modulate the effect of BRCA1. In order to further examine the effect of EGFR mutations and BRCA1 mRNA levels on outcome in advanced NSCLC, we performed a prospective non-randomized phase II clinical trial, testing the hypothesis that customized therapy would confer improved outcome over non-customized therapy. In an exploratory analysis, we also examined the effect of RAP80 and Abraxas mRNA levels.

Methodology/Principal Findings

We treated 123 metastatic non-squamous cell lung carcinoma patients using a customized approach. RNA and DNA were isolated from microdissected specimens from paraffin-embedded tumor tissue. Patients with EGFR mutations received erlotinib, and those without EGFR mutations received chemotherapy with or without cisplatin based on their BRCA1 mRNA levels: low, cisplatin plus gemcitabine; intermediate, cisplatin plus docetaxel; high, docetaxel alone. An exploratory analysis examined RAP80 and Abraxas expression. Median survival exceeded 28 months for 12 patients with EGFR mutations, and was 11 months for 38 patients with low BRCA1, 9 months for 40 patients with intermediate BRCA1, and 11 months for 33 patients with high BRCA1. Two-year survival was 73.3%, 41.2%, 15.6% and 0%, respectively. Median survival was influenced by RAP80 expression in the three BRCA1 groups. For example, for patients with both low BRCA1 and low RAP80, median survival exceeded 26 months. RAP80 was a significant factor for survival in patients treated according to BRCA1 levels (hazard ratio, 1.3 [95% CI, 1–1.7]; P = 0.05).

Conclusions/Significance

Chemotherapy customized according to BRCA1 expression levels is associated with excellent median and 2-year survival for some subsets of NSCLC patients , and RAP80 could play a crucial modulating effect on this model of customized chemotherapy.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00883480","term_id":"NCT00883480"}}NCT00883480     

ADAM10 Releases a Soluble Form of the GPNMB/Osteoactivin Extracellular Domain with Angiogenic Properties

Background

Glycoprotein non-metastatic melanoma protein B (GPNMB)/Osteoactivin (OA) is a transmembrane protein expressed in approximately 40–75% of breast cancers. GPNMB/OA promotes the migration, invasion and metastasis of breast cancer cells; it is commonly expressed in basal/triple-negative breast tumors and is associated with shorter recurrence-free and overall survival times in patients with breast cancer. Thus, GPNMB/OA represents an attractive target for therapeutic intervention in breast cancer; however, little is known about the functions of GPNMB/OA within the primary tumor microenvironment.

Methodology/Principal Findings

We have employed mouse and human breast cancer cells to investigate the effects of GPNMB/OA on tumor growth and angiogenesis. GPNMB/OA-expressing tumors display elevated endothelial recruitment and reduced apoptosis when compared to vector control-derived tumors. Primary human breast cancers characterized by high vascular density also display elevated levels of GPNMB/OA when compared to those with low vascular density. Using immunoblot and ELISA assays, we demonstrate the GPNMB/OA ectodomain is shed from the surface of breast cancer cells. Transient siRNA-mediated knockdown studies of known sheddases identified ADAM10 as the protease responsible for GPNMB/OA processing. Finally, we demonstrate that the shed extracellular domain (ECD) of GPNMB/OA can promote endothelial migration in vitro.

Conclusions/Significance

GPNMB/OA expression promotes tumor growth, which is associated with enhanced endothelial recruitment. We identify ADAM10 as a sheddase capable of releasing the GPNMB/OA ectodomain from the surface of breast cancer cells, which induces endothelial cell migration. Thus, ectodomain shedding may serve as a novel mechanism by which GPNMB/OA promotes angiogenesis in breast cancer.     

Dll4 Blockade Potentiates the Anti-Tumor Effects of VEGF Inhibition in Renal Cell Carcinoma Patient-Derived Xenografts

Background

The Notch ligand Delta-like 4 (Dll4) is highly expressed in vascular endothelium and has been shown to play a pivotal role in regulating tumor angiogenesis. Blockade of the Dll4-Notch pathway in preclinical cancer models has been associated with non-productive angiogenesis and reduced tumor growth. Given the cross-talk between the vascular endothelial growth factor (VEGF) and Delta-Notch pathways in tumor angiogenesis, we examined the activity of a function-blocking Dll4 antibody, REGN1035, alone and in combination with anti-VEGF therapy in renal cell carcinoma (RCC).

Methods and Results

Severe combined immunodeficiency (SCID) mice bearing patient-derived clear cell RCC xenografts were treated with REGN1035 and in combination with the multi-targeted tyrosine kinase inhibitor sunitinib or the VEGF blocker ziv-aflibercept. Immunohistochemical and immunofluorescent analyses were carried out, as well as magnetic resonance imaging (MRI) examinations pre and 24 hours and 2 weeks post treatment. Single agent treatment with REGN1035 resulted in significant tumor growth inhibition (36–62%) that was equivalent to or exceeded the single agent anti-tumor activity of the VEGF pathway inhibitors sunitinib (38–54%) and ziv-aflibercept (46%). Importantly, combination treatments with REGN1035 plus VEGF inhibitors resulted in enhanced anti-tumor effects (72–80% growth inhibition), including some tumor regression. Magnetic resonance imaging showed a marked decrease in tumor perfusion in all treatment groups. Interestingly, anti-tumor efficacy of the combination of REGN1035 and ziv-aflibercept was also observed in a sunitinib resistant ccRCC model.

Conclusions

Overall, these findings demonstrate the potent anti-tumor activity of Dll4 blockade in RCC patient-derived tumors and a combination benefit for the simultaneous targeting of the Dll4 and VEGF signaling pathways, highlighting the therapeutic potential of this treatment modality in RCC.