Lactate dehydrogenase isoenzymes 1 and 5: differential expression by neoplastic and stromal cells in non-small cell lung cancer and other epithelial malignant tumors.

BACKGROUND/AIMS: Lactate dehydrogenase-5 (LDH-5), an isoenzyme composed of 4 M-polypeptide chains, catalyzes the conversion of pyruvate to lactate with an unparalleled efficiency (anaerobic oxidation), but this function of LDH gradually fades away as the number of H over M chains increases. Thus, LDH-1, another component isoenzyme made up of 4 H-polypeptide chains, favors aerobic oxidation of pyruvate by pyruvate dehydrogenase. METHODS: Using immunohistochemistry in this study, we explored the expression of LDH-1 and LDH-5 in a variety of normal and malignant tissues, including lung, breast, endometrium, urinary bladder and large intestine. RESULTS: LDH-1 was consistently expressed in all living cells, normal and malignant, epithelial and stromal, including endothelium and lymphocytes. In contrast, LDH-5 was expressed preferentially in tumor cells, while normal tissues were devoid of LDH-5 or expressed it only faintly, and the cellular population of the tumor-supporting stroma showed LDH-5 activity in a small percentage of cases. Interestingly, LDH-5-positive stromal cells were associated with hypoxia-inducible factor-1alpha overexpression. CONCLUSION: It is concluded that normal tissues utilize aerobic oxidation as a means of energy production, while tumor cells are turned to anaerobic glycolysis, a phenomenon which is rarely followed by the mesenchymal cells of the tumor-supporting stroma.     

Endogenous markers of hypoxia/anaerobic metabolism and anemia in primary colorectal cancer

Anemia has been implicated in the decreased oxygen tension noted within the tumor environment. In a series of 79 colorectal adenocarcinomas we investigated the role of anemia in activating molecular pathways regulated by hypoxia. Preoperative Hb levels were correlated with the immunohistochemical expression of HIF1alpha and HIF2alpha, LDH5, GLUT1, VEGF, DEC1 and BNIP3, and with angiogenesis and the cancer cell proliferation index. Upregulation of HIF1alpha and HIF2alpha proteins, found in 43% and 44.3% of cases, respectively, was not related to anemia (Hb < 10 g%). This is in agreement with other studies suggesting that HIF activation occurs for various reasons, such as poor or irregular vascularity, or oncogene activation. Nevertheless, low Hb levels (<10 g%) were linked to activated anaerobic metabolism (LDH5 overexpression) in a subset of tumors not expressing HIF1alpha (P < 0.01). Overexpression of HIFs, whether linked to anemia or not, was associated with a number of factors related to tumor aggressiveness (assessed as local invasion and nodal metastasis), anaerobic metabolism and intratumoral acidosis (LDH5, GLUT1; increased glucose metabolism to lactate), activation of genes related to necrosis (BNIP3) and angiogenesis (VEGF). Expression of BNIP3 emerged as the strongest independent factor related to transmural invasion and metastasis to lymph nodes. Identification of specific patterns of the hypoxia molecular cascade activated in cancer cells might help in developing specific therapeutic policies.     

LDH5 overexpression is associated with poor survival in patients with solid tumors: a meta-analysis

Lactate dehydrogenase 5 (LDH5) is believed to be particularly important and a reliable marker of malignancy. However, it is still controversial whether LDH5 expression can be regarded as a prognostic factor for cancer patients. We reviewed the literature by performing an electronic database search via PubMed to identify eligible studies that assessed the impact of LDH5 as a cancer prognostic marker and its association with HIF-1α. Heterogeneity and publication bias were also assessed. A total of 12 literatures which included 1,892 cancer patients were combined in the final analysis. Meta-analysis revealed that LDH5 overexpression was associated with an unfavorable overall survival (12 studies, 1,597 patients; HR 1.59, 95 % CI 1.17–2.16) and disease/recurrence/progression-free survival (7 studies; 1,086 patients; HR 1.46, 95 % CI 1.04–2.04) among solid tumor patients. Meta-analysis revealed an association between the expression of LDH5 and hypoxia-inducible factors 1 (OR 2.72, 95 % CI 1.66–4.45). Publication bias could not be excluded when investigating the association of LDH5 expression and overall survival. However, when we accounted for publication bias using the trim and fill method, the results remained significant (HR 1.435, 95 % CI 1.071–1.923, P?<?0.05) suggesting the stability of our results. Therefore, our study suggested that LDH5 overexpression had a poor prognosis value in cancer patients. The results of this meta-analysis suggest that high LDH5 expression is associated with HIF-1α and poor overall survival in cancer patients.     

Oxygen and glucose consumption in gastrointestinal adenocarcinomas: correlation with markers of hypoxia, acidity and anaerobic glycolysis

This study gives an insight into tumor metabolic activity by investigating oxygen and glucose content, together with their metabolic products carbon dioxide and acids-pH, in the arterial and venous blood of a tumor. Nineteen patients with gastrointestinal adenocarcinomas undergoing surgery were studied. Biochemical analysis showed that in a large subgroup of tumors, oxygen consumption was reduced while that of glucose was increased in malignant, as compared to normal tissues; these features were more evident in tumors overexpressing lactate dehydrogenase (LDH-5) and hypoxia inducible factors (HIF1alpha/2alpha). An increase in carbon dioxide production in the tumor environment was linked with overexpression of carbonic anhydrase 9 (CA9). The simultaneous overexpression of CA9 and LDH-5 was related to very low pH levels in the veins draining the tumor, suggesting an intense acidification of the tumor microenvironment in such cases. These in vivo data confirm the importance of HIFs and their downstream regulated genes in tumor metabolism, particularly in glycolysis and carbon dioxide buffering.     

c-erbB-2 related aggressiveness in breast cancer is hypoxia inducible factor-1alpha dependent.

c-erbB-2-positive breast carcinomas are highly aggressive tumors. In vitro data on breast cell lines showed that c-erbB-2 enhanced translational efficiency of hypoxia inducible factor-1alpha (HIF1alpha) production (Laughner et al., Mol Cell Biol 2001;21:3995-4005). We investigated the clinical correlate of this observation to assess whether c-erbB-2 expression was related to HIF1alpha expression, angiogenesis, and prognosis. A series of 180 breast carcinomas of known c-erbB-2 status (90 c-erbB-2-positive and 90 c-erbB-2-negative carcinomas) were stained immunohistochemically for HIF1alpha and CD31 endothelial cell antigen. c-erbB-2 positivity was clearly related to HIF1alpha protein expression and high angiogenesis. However, prognosis was decreased only in cases with simultaneous c-erbB-2 and HIF1alpha expression. If activation of c-erbB-2 in humans results in overexpression of HIF1alpha independently of conditions of hypoxia, as occur in experimental studies, this interaction may represent a main pathway conferring clinical aggressiveness to c-erbB-2-positive breast tumors.     

Hypoxia-inducible factor (HIF1A and HIF2A), angiogenesis,and chemoradiotherapy outcome of squamous cell head-and-neck cancer

PURPOSE: Hypoxia-inducible factors HIF1alpha and HIF2alpha (HIFalphas) regulate the expression of a variety of genes encoding proteins related to angiogenesis and to anaerobic metabolism of cells exposed to hypoxic stress. Their putative role as markers of clinically relevant hypoxia and, therefore, as predictors of response to chemoradiotherapy is herein examined. PATIENTS AND METHODS: Using immunohistochemistry, we assessed the expression of HIFalphas in normal head-neck mucosa and in 75 cancer specimens from patients with locally advanced squamous cell head-and-neck cancer (SCHNC), treated with concurrent carboplatin chemoradiotherapy. RESULTS: Head-and-neck mucosa from normal individuals did not show any HIF1alpha or HIF2alpha reactivity. SCHNC showed a varying expression of HIFalphas ranging through negative reactivity, to weak or focally strong cytoplasmic reactivity, or to strong diffuse cytoplasmic/nuclear reactivity. Fifty-two percent and 33% of cancer samples showed the latter expression pattern for HIF1alpha and HIF2alpha, respectively, and were considered to bear "high" HIF reactivity. Bone/cartilage involvement was more frequent in tumors with high HIF1alpha expression (p = 0.05). HIF1alpha and HIF2alpha overexpression were significantly associated with high microvessel density (p = 0.002 and 0.02, respectively) and with VEGF expression (p = 0.01 and 0.005, respectively). HIF1alpha was related to high thymidine phosphorylase expression (p = 0.03), whereas VEGF/KDR-activated tumor vasculature was significantly more frequent in HIF2alpha-overexpressing tumors (p = 0.02). High HIF1alpha and HIF2alpha were associated with incomplete response to chemoradiation (p = 0.007 and p = 0.02, respectively). In univariate analysis, high HIF1alpha and HIF2alpha expression were significantly associated with poor local relapse-free survival (p = 0.003 and 0.003, respectively) and with poor overall survival (p = 0.05 and 0.001, respectively). In multivariate models, HIF2alpha expression was an independent prognostic factor. In biopsies performed after the delivery of 20 Gy of radiotherapy, upregulation of HIFalphas was noted in some cases. CONCLUSIONS: It is concluded that the overexpression of HIFalphas in SCHNC is related to locally aggressive behavior, to intensification of angiogenesis, and to an important resistance to carboplatin chemoradiotherapy.     

Pyruvate dehydrogenase and pyruvate dehydrogenase kinase expression in non small cell lung cancer and tumor-associated stroma

Pyruvate dehydrogenase (PDH) catalyzes the conversion of pyruvate to acetyl-coenzyme A, which enters into the Krebs cycle, providing adenosine triphosphate (ATP) to the cell. PDH activity is under the control of pyruvate dehydrogenase kinases (PDKs). Under hypoxic conditions, conversion of pyruvate to lactate occurs, a reaction catalyzed by lactate dehydrogenase 5 (LDH5). In cancer cells, however pyruvate is transformed to lactate occurs, regardless of the presence of oxygen (aerobic glycolysis/Warburg effect). Although, hypoxic intratumoral conditions account for HIF1alpha stabilization and induction of anaerobic metabolism, recent data suggest that high pyruvate concentrations also result in HIF1alpha stabilization independently of hypoxia. In the present immunohistochemical study, we provide evidence that the PDH/PDK pathway is repressed in 73% of non small cell lung carcinomas, which may be a key reason for HIF1alpha stabilization and "aerobic glycolysis." However, about half of PDH-HIF pathway, and patients harboring these tumors have an excellent postoperative outcome. A small subgroup of clinically aggressive tumors maintains a coherent PDH and HIF/LDH5 expression. In contrast to cancer cells, fibroblasts in the tumor supporting stroma exhibit an intense PDH but reduced PDK1 expression favoring maximum PDH activity. This means that stroma may use lactic acid produced by tumor cells, preventing the creation of an intolerable intratumoral acidic environment at the same time.     

The hypoxic microenvironment of the skin contributes to Akt-mediated melanocyte transformation

Constitutive activation of Akt characterizes a high percentage of human melanomas and represents a poor prognostic factor of the disease. We show that Akt transforms melanocytes only in a hypoxic environment, which is found in normal skin. The synergy between Akt and hypoxia is HIF1alpha mediated. Inhibition of HIF1alpha decreases Akt transformation capacity in hypoxia and tumor growth in vivo, while overexpression of HIF1alpha allows anchorage-independent growth in normoxia and development of more aggressive tumors. Finally, we show that mTOR activity is necessary to maintain the transformed phenotype by sustaining HIF1alpha activity. Taken together, these findings demonstrate that Akt hyperactivation and HIF1alpha induction by normally occurring hypoxia in the skin significantly contribute to melanoma development.     

Beclin 1 over- and underexpression in colorectal cancer: distinct patterns relate to prognosis and tumour hypoxia

Introduction:

Autophagy enables cells to recycle long-lived proteins or damaged organelles. Beclin 1, the mammalian orthologue of the yeast Apg6/Vps30 gene, functions as a scaffold for the formation of autophagosomes.

Materials and method:

The immunohistochemical patterns of Beclin 1 expression and their prognostic relevance were studied in formalin-fixed tissues from 155 patients with colorectal adenocarcinoma treated with surgery alone.

Results:

Using the weak homogeneous expression of Beclin 1 in normal colonic tissues as a basis for assessing tumours, the following grouping/staining patterns were recognised in colorectal carcinomas: a normal-like pattern in 62 of 155 (40%) cases, an underexpression pattern in 24 of 155 (15.5%) cases, extensive overexpression of Beclin 1 in 33 of 155 (21.3%) tumours and limited overexpression of the protein in 36 of 155 (23.2%) tumours. Extensive overexpression of Beclin 1 was significantly linked with overexpression of HIF1α and LDH5, as well as with high histological grade, vascular invasion and nodal involvement. Furthermore, patients with extensive over- or underexpression of Beclin 1 had a significantly poorer overall survival compared with the other two groups (P<0.0001). Beclin 1 had an independent prognostic relevance in multivariate analysis.

Conclusions:

Beclin 1 has an important role in growth and metastasis of colorectal cancer. Loss of Beclin 1 expression (allelic loss or microRNA regulatory activity, as suggested in the literature) defines poor prognosis presumably by promoting anti-apoptotic pathways, while overexpression of the protein, being linked with tumour hypoxia and acidity, also defines subgroups of tumours with aggressive clinical behaviour.     

Targeted inactivation of fh1 causes proliferative renal cyst development and activation of the hypoxia pathway

Germline mutations in the fumarate hydratase (FH) tumor suppressor gene predispose to leiomyomatosis, renal cysts, and renal cell cancer (HLRCC). HLRCC tumors overexpress HIF1alpha and hypoxia pathway genes. We conditionally inactivated mouse Fh1 in the kidney. Fh1 mutants developed multiple clonal renal cysts that overexpressed Hif1alpha and Hif2alpha. Hif targets, such as Glut1 and Vegf, were upregulated. We found that Fh1-deficient murine embryonic stem cells and renal carcinomas from HLRCC showed similar overexpression of HIF and hypoxia pathway components to the mouse cysts. Our data have shown in vivo that pseudohypoxic drive, resulting from HIF1alpha (and HIF2alpha) overexpression, is a direct consequence of Fh1 inactivation. Our mouse may be useful for testing therapeutic interventions that target angiogenesis and HIF-prolyl hydroxylation.     

PLOD2 as a potential regulator of peritoneal dissemination in gastric cancer

Peritoneal dissemination is the most common metastatic pattern in advanced gastric cancer (GC) and has a very poor prognosis. However, its molecular mechanism has not been elucidated. Our study investigated genes associated with peritoneal dissemination of GC. We performed combined expression analysis of metastatic GC cell lines and identified Procollagen‐lysine, 2‐oxoglutarate 5‐dioxygenase2 (PLOD2) as a potential regulator of peritoneal dissemination. PLOD2 is regulated by hypoxia‐inducible factor‐1 (HIF‐1) and mediates extracellular matrix remodeling, alignment, and mechanical properties. We analyzed PLOD2 expression immunohistochemically in 179 clinical samples, and found high PLOD2 expression to be significantly associated with peritoneal dissemination, leading to poor prognosis. In an in vivo‐collected metastatic cell line, downregulation of PLOD2 by siRNA reduced invasiveness and migration. Hypoxia upregulated PLOD2 mediated by HIF‐1, and promoted invasiveness and migration. After exposure to hypoxia, a cell line transfected with siPLOD2 exhibited significantly suppressed invasiveness and migration, despite high HIF‐1 expression. These findings indicate that PLOD2 is a regulator of, and candidate therapeutic target for peritoneal dissemination of GC. Although peritoneal dissemination of GC has a very poor prognosis, its molecular mechanism has not been elucidated. We identified PLOD2 regulated by HIF‐1 as a potential regulator of peritoneal dissemination of GC. Finally, we showed that PLOD2 promotes cell invasiveness and migration in GC under hypoxia and lead to peritoneal dissemination of GC.     

The metabolic interactions between tumor cells and tumor-associated stroma (TAS) in prostatic cancer

Tumor-associated stroma (TAS) is not simply a supporting element for cancer cells, but plays an important role in tumor growth, invasion and metastasis. Changes on the level of stromal constituents, such as loss of Caveolin-1 and increased thymidine phosphorylase (TP) expression, have been associated with tumor aggressiveness. The mutual cooperation between stromal fibroblasts and cancer cells is another distinguishing feature, which has recently emerged. In this investigation, both the loss of Caveolin-1 and the increased TP expression in the prostatic TAS was associated with high Gleason score (p = 0.0002 and 0.003, respectively); the two proteins were acting both independently and synergistically. In addition, TP was significantly associated with high stromal Ki-67 (MIB1) proliferation index (p = 0.03). Analysis of the metabolic interactions between stromal and epithelial elements showed that, while prostatic cancer cells express principally (> 91%) lactate dehydrogenase-5 (LDH-5) (anaerobic metabolism), the tumor-associated fibroblasts/myofibroblasts (TAFs) express largely (67.8%) LDH-1 (aerobic metabolism)—the terms TAFs and TAS are used interchangeably. These two isoenzyme pathways act complementary; the LDH-5 pathway converts pyruvate to lactate, whereas the LDH-1 enzyme system utilizes the secreted metabolite lactate to produce pyruvate, essential for continuous energy supply to tumor cells. Monocarboxylate transporter-1 (MCT-1)—the main facilitator of lactate uptake in tumor cells, was expressed exclusively in prostate cancer cells and related directly to LDH-5 overexpression. These findings support and extend our previous studies on energy recycling between the aerobic stroma and the anaerobic cancer cells within the framework of Warburg effect.     

LDH-A influences hypoxia-inducible factor 1�� (HIF1 ��) and is critical for growth of HT29 colon carcinoma cells in vivo

Serum lactate dehydrogenase (LDH) is a well-known clinical surrogate parameter. A high activity of LDH is associated with a poor prognosis in different tumor types. Here we demonstrate by a gene silencing approach that LDH-A is critical for in vivo but not in vitro growth of HT29 colon carcinoma cells. We provide evidence that the suppression of the LDH-A gene leads to an increased level of hypoxia inducible factor 1α (HIF1α) but in consequence not to an increase of HIF1 regulated proteins such as carbonic anhydrase IX (CAIX), vascular endothelial growth factor (VEGF), prolyl-hydroxylase 2 (PHD2), and factor-inhibiting HIF (FIH) in cell cultures and tumor lysates. This effect is independent of LDH activity in vivo. We conclude that LDH-A has an influence on the activity of HIF1α and thus on the adaptation of cells to a hypoxic tumor microenvironment in HT29 colon cells. We suggest the use of LDH-M as a potential therapeutic target for anticancer treatment.