Expression of L-type amino acid transporter 1 (LAT1) in esophageal carcinoma

BACKGROUND AND OBJECTIVES: It has been reported that amino acid transport systems play an important role in cell proliferation. Their activity is increased in malignant cells compared to benign cells. In this study, we investigated whether L-type amino acid transporter 1 (LAT1) is expressed in human non-cancerous esophageal mucosa and esophageal squamous cell carcinoma. We also examined whether LAT1 expression is correlated with histopathological features. METHODS: From January 1999 to December 2001, sections of formalin-fixed, paraffin-embedded tissue from 11 cases of early esophageal carcinoma (T1) and 19 cases of advanced esophageal carcinoma (T2, T3) were entered in the study. Histopathologically, all 30 cases were squamous cell carcinoma. Immunohistochemical staining was performed using rabbit anti-LAT1 IgG, with the standard avidin-streptavidin immuno-peroxidase method. Measurement was performed by means of computer-assisted image analysis. The ratio of cells with LAT1 expression in esophageal squamous cell carcinoma and non-cancerous esophageal mucosa was used for analysis in this study. RESULTS: Non-cancerous esophageal mucosa expressed LAT1 only in the basal layer of the esophageal wall. Esophageal squamous cell carcinoma expressed LAT1 throughout the tumor. LAT1 expression in esophageal squamous cell carcinoma was significantly higher than that in non-cancerous esophageal mucosa. LAT1 expression in esophageal squamous cell carcinoma increased as the depth of invasion progressed (T1 < T2 (P = 0.0477), T2 < T3 (P = 0.0415), T1 < T3 (P = 0.0044)), and as the tumor size increased. Also, high LAT1 expression was significantly associated with well-differentiated carcinoma. CONCLUSION: These results suggest that LAT1 plays a significant role in cell proliferation, differentiation, and invasion in esophageal squamous cell carcinoma.     

Expression of L‐type amino acid transporter 1 (LAT1) as a prognostic and therapeutic indicator in multiple myeloma

L‐type amino‐acid transporter 1 (LAT1) plays a key role in cell growth and survival. To determine the prognostic significance of LAT1 in multiple myeloma (MM), we investigated the expression of LAT1 and its functional subunit, 4Fc heavy chain (CD98), on myeloma cells by immunohistochemistry in 100 newly diagnosed MM patients. High expression (moderate or strong staining intensity) of LAT1 and CD98 was detected in 56% and 45% of patients, respectively. The LAT1 expression score was positively correlated with Ki‐67 index (r = 0.631, P < 0.001), and there was a statistically significant difference in Durie–Salmon stage between patients with high and low LAT1 expression (P = 0.03). In 43 patients treated with melphalan and prednisolone, the overall response rate was significantly higher in the high LAT1 expression group (60.0%) than in the low LAT1 expression group (17.6%) (P = 0.03). Multivariate analysis confirmed that high expression of LAT1 was a significant prognostic factor for predicting poor overall survival independently from the International Staging System (both P = 0.01). Here, we show that the overexpression of LAT1 is significantly associated with high proliferation and poor prognosis in newly diagnosed MM patients. Thus, LAT1 may be a promising pathological marker for identifying high‐risk MM.     

Expression of L-type amino acid transporter 1 (LAT1) and 4F2 heavy chain (4F2hc) in liver tumor lesions of rat models.

BACKGROUND AND OBJECTIVES: It has been said that amino acid transporters play an important role in supplying nutrition to cells and for cell proliferation. In this study, we examined whether LAT1 and 4F2hc are closely related to tumor growth. METHODS: Rat colon cancer cells (RCN-9) were injected into the spleen of 12 male rats (inbred F344/DuCrj). In each rat, liver samples including tumor lesions were immunostained with anti-LAT1 and anti-4F2hc antibodies. The staining area of LAT1 and 4F2hc tumor lesions was calculated by computer analysis. RESULTS: Sixty-eight tumor nodules were observed in 12 livers. Out of the 68 tumor nodules, 36 nodules (52.9%) indicated a positive staining of LAT1 and 32 (47.1%) had a negative staining of LAT1. However, the LAT1 expression was scarcely detected in non-tumor areas. In terms of the 4F2hc expression, there were 56 nodules (82.4%) with 4F2hc positive and 12 (17.6) with 4F2hc negative. In addition, the expression of 4F2hc in non-tumor areas was almost the same as the expression of 4F2hc in tumor lesions. The average tumor size of the group with LAT1 positive and 4F2hc positive (n = 31) was 0.845 +/- 0.232 mm(2), which was significantly larger than that of the group with LAT1 negative and 4F2hc negative group (n = 7) (0.090 +/- 0.028 mm(2)) or the group with LAT1 positive and 4F2hc negative (n = 5) (0.097 +/- 0.025 mm(2)), respectively (P = 0.0017, P = 0.007). CONCLUSION: LAT1 was related to tumor growth. We think that LAT1 can possibly enhance its ability to promote tumor growth in cooperation with 4F2hc.     

Antitumor effects of novel mAbs against cationic amino acid transporter 1 (CAT1) on human CRC with amplified CAT1 gene

Copy number alterations detected by comparative genomic hybridization (CGH) can lead to the identification of novel cancer‐related genes. We analyzed chromosomal aberrations in a set of 100 human primary colorectal cancers (CRCs) using CGH and found a solute carrier (SLC) 7A1 gene, which encodes cationic amino acid transporter 1 (CAT1) with 14 putative transmembrane domains, in a chromosome region (13q12.3) with a high frequency of gene amplifications. SLC7A1/CAT1 is a transporter responsible for the uptake of cationic amino acids (arginine, lysine, and ornithine) essential for cellular growth. Microarray and PCR analyses have revealed that mRNA transcribed from CAT1 is overexpressed in more than 70% of human CRC samples, and RNA interference–mediated knockdown of CAT1 inhibited the cell growth of CRCs. Rats were immunized with rat hepatoma cells expressing CAT1 tagged with green fluorescent protein (GFP), and rat splenocytes were fused with mouse myeloma cells. Five rat monoclonal antibodies (mAbs) (CA1 ~ CA5) reacting with HEK293 cells expressing CAT1‐GFP in a GFP expression–dependent manner were selected from established hybridoma clones. Novel anti‐CAT1 mAbs selectively reacted with human CRC tumor tissues compared with adjacent normal tissues according to immuno‐histochemical staining and bound strongly to numerous human cancer cell lines by flow cytometry. Anti‐CAT1 mAbs exhibited internalization activity, antibody‐dependent cellular cytotoxicity, and migration inhibition activity against CRC cell lines. Furthermore, CA2 inhibited the in vivo growth of human HT29 and SW‐C4 CRC tumors in nude mice. This study suggested CAT1 to be a promising target for mAb therapy against CRCs.     

Tumor suppressor XIAP‐Associated factor 1 (XAF1) cooperates with tumor necrosis factor‐related apoptosis‐inducing ligand to suppress colon cancer growth and trigger tumor regression

BACKGROUND:

XIAP‐associated factor 1 (XAF1) antagonizes the anticaspase activity of XIAP (X‐linked inhibitor of apoptosis) and functions as a tumor suppressor in colon cancer. The tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is known as a potential anticancer agent. In this study, the synergistic effect of XAF1 and TRAIL on colon cancer growth was investigated.

METHODS:

Adeno‐XAF1 virus was generated and purified. Cell apoptosis was detected by flow‐cytometry and terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling assay. Protein expression of the different genes was determined by Western blot analysis. Tumorigenesis and tumor growth were assessed in subcutaneous nude mouse xenograft experiments.

RESULTS:

Stable overexpression of XAF1‐sensitized colon cancer cells to TRAIL‐induced apoptosis significantly increased the activity of caspase 3, 7, 8, and 9; released cytochrome c; and down‐regulated XIAP, survivin, and c‐IAP‐2. The restoration of XAF1 expression mediated by adenovirus (adeno‐XAF1) directly induced apoptosis, and synergized TRAIL‐induced apoptosis in colon cancer cells. Ex vivo transduction of adeno‐XAF1 suppressed colon cancer formation in vivo. Furthermore, adeno‐XAF1 treatment of mice significantly inhibited tumor growth, strongly enhanced TRAIL‐induced apoptosis and antitumor activity in colon cancer xenograft models in vivo, and markedly prolonged the survival. Notably, the combined treatment with adeno‐XAF1 and TRAIL completely eradicated the established tumors without detectable toxicity in normal tissue.

CONCLUSIONS:

The combined restoration of XAF1 expression and TRAIL treatment may be a potent strategy for colon cancer therapy. Cancer 2010. © 2010 American Cancer Society.     

Novel cucurmosin‐based immunotoxin targeting programmed cell death 1‐ligand 1 with high potency against human tumor in vitro and in vivo

Immunotoxins are Ab‐cytotoxin chimeric molecules with mighty cytotoxicity. Programmed cell death 1‐ligand 1 (PD‐L1), is a transmembrane protein expressed mainly in inflammatory tumor tissues and plays a pivotal role in immune escape and tumor progression. Although PD‐L1 immune checkpoint therapy has been successful in some cases, many patients have not benefited enough due to primary/secondary resistance. In order to optimize the therapeutic efficacy of anti‐PD‐L1 mAb, we used durvalumab as the payload and CUS_245C, a type I ribosome‐inactivating protein isolated from Cucurbita moschata, as the toxin moiety, to construct PD‐L1‐specific immunotoxin (named D‐CUS_245C) through the engineered cysteine residue. In vitro, D‐CUS_245C selectively killed PD‐L1⁺ tumor cells. In vivo studies also showed that D‐CUS_245C had obvious antitumor effect on PD‐L1⁺ human xenograft tumors in nude mice. In conclusion, in the combination of the toxin with mAb, this study developed a new immunotoxin targeting PD‐L1, emphasizing a novel and promising treatment strategy and providing a valuable way to optimize cancer immunotherapy.     

HAMLET (human α‐lactalbumin made lethal to tumor cells) triggers autophagic tumor cell death

HAMLET, a complex of partially unfolded α‐lactalbumin and oleic acid, kills a wide range of tumor cells. Here we propose that HAMLET causes macroautophagy in tumor cells and that this contributes to their death. Cell death was accompanied by mitochondrial damage and a reduction in the level of active mTOR and HAMLET triggered extensive cytoplasmic vacuolization and the formation of double‐membrane‐enclosed vesicles typical of macroautophagy. In addition, HAMLET caused a change from uniform (LC3‐I) to granular (LC3‐II) staining in LC3‐GFP‐transfected cells reflecting LC3 translocation during macroautophagy, and this was blocked by the macroautophagy inhibitor 3‐methyladenine. HAMLET also caused accumulation of LC3‐II detected by Western blot when lysosomal degradation was inhibited suggesting that HAMLET caused an increase in autophagic flux. To determine if macroautophagy contributed to cell death, we used RNA interference against Beclin‐1 and Atg5. Suppression of Beclin‐1 and Atg5 improved the survival of HAMLET‐treated tumor cells and inhibited the increase in granular LC3‐GFP staining. The results show that HAMLET triggers macroautophagy in tumor cells and suggest that macroautophagy contributes to HAMLET‐induced tumor cell death. © 2008 Wiley‐Liss, Inc.     

Warburg phenotype in renal cell carcinoma: High expression of glucose‐transporter 1 (GLUT‐1) correlates with low CD8+ T‐cell infiltration in the tumor

Many tumor cells are characterized by a dysregulated glucose metabolism associated with increased glycolysis in the presence of oxygen (“Warburg Effect”). Here, we analyzed for the first time a possible link between glucose metabolism and immune cell infiltration in renal cell carcinoma (RCC). RCC specimens revealed a highly significant increase in the expression of lactate dehydrogenase A (LDHA) and glucose‐transporter 1 (GLUT‐1) compared to the corresponding normal kidney tissue on mRNA level. Accordingly, tumor cell lines of different origin such as RCC, melanoma and hepatocellular carcinoma strongly expressed LDHA and GLUT‐1 compared to their nonmalignant counterparts. In line with this finding, tumor cells secreted high amounts of lactate. High expression of GLUT‐1 and LDH5, a tetramer of 4 LDHA subunits, was confirmed by tissue microarray analysis of 249 RCC specimens. Overall, 55/79 (69.6%) and 46/71 (64.7%) cases of clear cell carcinoma showed a constitutive, but heterogeneous expression of GLUT‐1 and LDH5, respectively. The number of CD3⁺, CD8⁺ and FOXP3⁺ T cells was significantly elevated in RCC lesions compared to normal kidney epithelium, but effector molecules such as granzyme B and perforin were decreased in tumor infiltrating T cells. Of interest, further analysis revealed an inverse correlation between GLUT‐1 expression and the number of CD8⁺ T cells in RCC lesions. Together, our data suggest that an accelerated glucose metabolism in RCC tissue is associated with a low infiltration of CD8⁺ effector T cells. Targeting the glucose metabolism may represent an interesting tool to improve the efficacy of specific immunotherapeutic approaches in RCC.     

A phase 1 study of weekly dosing of trastuzumab emtansine (T‐DM1) in patients with advanced human epidermal growth factor 2–positive breast cancer

BACKGROUND:

We conducted a phase 1, multicenter, open‐label, dose‐escalation study (TDM3569g) to assess the safety, tolerability, and pharmacokinetics of single‐agent trastuzumab emtansine (T‐DM1) administered weekly and once every 3 weeks in patients with HER2‐positive metastatic breast cancer previously treated with trastuzumab. The weekly dose results are described here.

METHODS:

Patients were administered escalating doses of T‐DM1 weekly, starting at 1.2 mg/kg. Additional patients were enrolled at the maximum tolerated dose (MTD) to better characterize tolerability and pharmacokinetics.

RESULTS:

Twenty‐eight patients received weekly T‐DM1, and the MTD was determined to be 2.4 mg/kg. In general, T‐DM1 was well tolerated, requiring few dose modifications or discontinuations because of adverse events (AEs). Grade ≥3 AEs were reported in 19 patients (67.9%); treatment‐related AEs occurred in 25 (89.3%) patients. Exposure to weekly T‐DM1 was dose‐proportional at ≥1.2 mg/kg, and accumulation of T‐DM1 and total trastuzumab was observed. Objective partial tumor responses were reported in 13 (46.4%) patients; the median duration of response was 18.6 months, and the 6‐month clinical benefit rate was 57.1%.

CONCLUSION:

The results suggest that a weekly dose of T‐DM1 2.4 mg/kg has antitumor activity and is well tolerated in patients with HER2‐positive metastatic breast cancer. Cancer 2012. © 2012 American Cancer Society.     

The PD‐1/PD‐L1 axis and human papilloma virus in patients with head and neck cancer after adjuvant chemoradiotherapy: A multicentre study of the German Cancer Consortium Radiation Oncology Group (DKTK‐ROG)

We examined the prognostic role of PD‐1+ and CD8+ tumor infiltrating lymphocytes (TILs), and PD‐L1+ cells in patients with squamous cell carcinoma of the head and neck (SCCHN) treated with surgery and postoperative chemoradiotherapy (CRT). FFPE samples from 161 patients were immunohistochemically stained for PD‐1, CD8 and PD‐L1. The immune marker expression was correlated with clinicopathologic characteristics, and overall survival (OS), local progression‐free survival (LPFS) and distant metastases free‐survival (DMFS), also in the context of HPV16 DNA/p16 status. The median follow‐up was 48 months (range: 4–100). The 2‐year‐OS was 84.1% for the entire cohort. High PD‐1 and PD‐L1 expression were more common in patients with positive HPV16 DNA (p < 0.001 and p = 0.008, respectively) and high infiltration by CD8+ TILs (p < 0.001 for both markers). High PD‐L1 expression correlated with superior OS (p = 0.025), LPFS (p = 0.047) and DMFS (p = 0.048) in multivariable analysis, whereas no significance could be demonstrated for PD‐1. Patients with CD8^high/PD‐L1^high expression had favorable outcome (p < 0.001 for all endpoints) compared to other groups. We validated the superior OS data on CD8^high/PD‐L1^high using the Cancer Genome Atlas TCGA dataset (n = 518; p = 0.032). High PD‐L1 expression was a favorable prognostic marker in HPV16‐negative but not HPV16‐positive patients. In conclusion, HPV‐positive tumors showed higher expression of immune markers. PD‐L1 expression constitutes an independent prognostic marker in SCCHN patients post‐adjuvant CRT. In conjunction with CD8 status, these data provide an important insight on the immune contexture of SCCHN and are directly relevant for future treatment stratification with PD‐1/PD‐L1 immune checkpoint inhibitors to complement CRT.     

High IKKα expression is associated with reduced time to recurrence and cancer specific survival in oestrogen receptor (ER)‐positive breast cancer

The aim of our study was to examine the relationship between tumour IKKα expression and breast cancer recurrence and survival. Immunohistochemistry was employed in a discovery and a validation tissue microarray to assess the association of tumour IKKα expression and clinico‐pathological characteristics. After siRNA‐mediated silencing of IKKα, cell viability and apoptosis were assessed in MCF7 and MDA‐MB‐231 breast cancer cells. In both the discovery and validation cohorts, associations observed between IKKα and clinical outcome measures were potentiated in oestrogen receptor (ER) positive Luminal A tumours. In the discovery cohort, cytoplasmic IKKα was associated with disease‐free survival (p = 0.029) and recurrence‐free survival on tamoxifen (p < 0.001) in Luminal A tumours. Nuclear IKKα and a combination of cytoplasmic and nuclear IKKα (total tumour cell IKKα) were associated with cancer‐specific survival (p = 0.012 and p = 0.007, respectively) and recurrence‐free survival on tamoxifen (p = 0.013 and p < 0.001, respectively) in Luminal A tumours. In the validation cohort, cytoplasmic IKKα was associated with cancer‐specific survival (p = 0.023), disease‐free survival (p = 0.002) and recurrence‐free survival on tamoxifen (p = 0.009) in Luminal A tumours. Parallel experiment with breast cancer cells in vitro demonstrated the non‐canonical NF‐κB pathway was inducible by exposure to lymphotoxin in ER‐positive MCF7 cells and not in ER‐negative MDA‐MB‐231 cells. Reduction in IKKα expression by siRNA transfection increased levels of apoptosis and reduced cell viability in MCF7 but not in MDA‐MB‐231 cells. IKKα is an important determinant of poor outcome in patients with ER‐positive invasive ductal breast cancer and thus may represent a potential therapeutic target.