D-3-phosphoglycerate dehydrogenase from the silkworm Bombyx mori: Identification, functional characterization, and expression.

D-3-phosphoglycerate dehydrogenase (PHGDH) is a key enzyme involved in the synthesis of l-serine. Despite the high serine content in silk proteins and the crucial role of PHGDH in serine biosynthesis, PHGDH has not been described in silkworms to date. Here, we identified PHGDH in the silkworm Bombyx mori and evaluated its biochemical properties. On the basis of the amino acid sequence and phylogenetic tree, this PHGDH has been categorized as a new type and designated as bmPHGDH. The recombinant bmPHGDH was overexpressed and purified to homogeneity. Kinetic studies revealed that PHGDH uses NADH as a coenzyme to reduce phosphohydroxypyruvate. High expression levels of bmphgdh messenger RNA (mRNA) were observed in the middle part of the silk gland and midgut in a standard strain of silkworm. Moreover, a sericin-deficient silkworm strain displayed reduced expression of bmphgdh mRNA. These findings indicate that bmPHGDH might play a crucial role in the provision of l-serine in the larva of B. mori.

Overcoming erlotinib resistance in EGFR mutation-positive lung adenocarcinomas through repression of phosphoglycerate dehydrogenase.

How to improve the efficacy and reverse the resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as erlotinib, remains a major challenge in the targeted therapy of lung adenocarcinoma with EGFR-activating mutation. Phosphoglycerate dehydrogenase (PHGDH) is the key enzyme of de novo serine biosynthesis over-expressed in various types of cancer including lung cancer. Elevated PHGDH expression is correlated with a worse overall survival in clinical lung adenocarcinoma patients. Here we investigated the role of PHGDH in lung adenocarcinoma with the acquisition of resistance to erlotinib. Methods: The necessary genes required for the acquired erlotinib resistance in lung adenocarcinoma cells were screened out by RNA-Seq analysis. Then the protein and mRNA levels of PHGDH were confirmed by immunoblotting and qRT-PCR in the erlotinib resistant cells. The effects of PHGDH inhibition or overexpression on erlotinib resistance were examined using cell culture and tumor xenograft mouse models respectively. To explore mechanism, the ROS level and DNA damage marker, γH2AX, were tested by DCFH-DA staining and immunofluorescence after PHGDH inhibition. Results: We found that PHGDH level was significantly increased in the lung adenocarcinoma PC9ER4 and HCC827ER9 cells that acquired resistance to erlotinib. Perturbation of PHGDH by siPHGDH transfection or NCT-503, a small molecular PHGDH inhibitor, synergistically augmented the tumoricidal effect and restored sensitivity to erlotinib in cell lines and xenografts. Over-expression of PHGDH caused xenografts resistant to erlotinib. Furthermore, multiple DNA damage repair pathways related genes were changed by PHGDH depletion specifically in erlotinib resistant cells. ROS stress and DNA damage marker γH2AX were enhanced by siPHGDH and NCT-503, which was reversed by NAC. Conclusion: Our study indicated that PHGDH inhibition has potential therapeutic value in lung adenocarcinoma with the acquired resistance to EGFR-TKIs.

Metabolic differences in estrogen receptor-negative breast cancer based on androgen receptor status.

This study investigated the relationship between steroid hormone receptor signaling and cellular metabolism in tumorigenesis by examining the expression of metabolic proteins with respect to androgen receptor (AR) and human epidermal growth factor receptor-2 (HER-2) status in estrogen receptor-negative (ER-) breast cancer. ER- breast cancer cases (n = 334) were selected from a microarray analysis, including those that were AR+ and AR- (n = 127 and 207, respectively) and HER-2+ and HER-2- (n = 140 and 194, respectively). The expression of proteins involved in glycolysis, glutaminolysis, and mitochondrial and intermediary (i.e., serine/glycine) metabolism was determined by immunohistochemistry and correlated with AR and HER-2 status. The expression of several proteins involved in glycolysis, glutaminolysis, and serine/glycine metabolism was higher (p < 0.01) in the AR- than in the AR+ group. In the former, the expression of the glycolytic protein carbonic anhydrase (CA)IX was associated with a shorter disease-free survival period (p = 0.029) and overall survival rate (p = 0.001). In a multivariate Cox analysis, immunoreactivity for CAIX (hazard ratio 15.89, 95 % confidence interval (CI) 1.820-131.6; p = 0.010) was an independent factor in predicting the survival of the AR+ group. In conclusion, differential expression patterns of metabolism-related proteins were noted in ER- breast cancer according to AR status. These findings highlight the link between hormone receptor signaling and metabolic pathways whose dysregulation could underlie breast tumorigenesis.

Expression levels of serine/glycine metabolism-related proteins in triple negative breast cancer tissues.

To evaluate the expression levels of serine/glycine metabolism-related proteins (PHGDH, PSAT, PSPH, SHMT, and GLDC) in six different subtypes of triple negative breast cancer (TNBC) patients and gain insight into their implications. Formalin-fixed, paraffin-embedded tissues from 129 TNBC patients were assembled into tissue microarrays. Immunohistochemical staining was performed for serine/glycine metabolism-related proteins (PHGDH, PSAT, PSPH, SHMT, and GLDC) and surrogate immunohistochemical markers (CK5/6, EGFR, claudin 3, claudin 4, claudin 7, E-cadherin, STAT1, interleukin-8 [IL-8], AR, and GGT-1) for identifying the molecular subtype of TNBC. TNBC subtype classifications included the following: basal-like (CK5/6-positive and/or EGFR-positive), molecular apocrine (AR-positive and/or GGT-1-positive), claudin-low (claudin 3-, claudin 4-, claudin 7-negative and/or E-cadherin-negative), immune-related (IL-8-negative and stromal STAT1-positive), mixed (features from two or more of the four subtypes), and null (no features from any of the four subtypes). Tissues from basal marker-positive patients showed increased expression levels of tumoral PHGDH compared with those from basal marker-negative patients (p = 0.029); lack of stromal SHMT1 expression was significantly correlated with T stage (p = 0.016). Multivariate Cox analysis revealed that a lack of stromal SHMT1 expression was an independent prognostic factor for predicting a shorter disease-free survival period (hazard ratio 4.002, 95 % confidence interval [CI] 1.077-14.83, p = 0.038); furthermore, a lack of tumoral PHGDH expression was predictive of a shorter overall survival rate (hazard ratio 3.053, 95 % CI 1.002-9.305, p = 0.050). In conclusion, the most abundantly expressed serine/glycine metabolism-related protein in basal-like TNBC tissues was tumoral PHGDH, and expression levels of stromal SHMT1 and tumoral PHGDH were inversely correlated with clinical prognostic factors. Also, this study is the first to assess serine/glycine relationships at the protein level in regards to clinical outcomes.