The protein network shown in figure 3 indicates at least several pathways have correlations. We first describes the metabolism in cancer. Gene expressions playing role in metabolisms were G6PD, PFKL and LDHA which mostly found in carbon metabolism.(31) Other proteins were HRPT1, while protein ACSL4 were not connected to any networking. PFKL known as phosphofructokinase is a rate limiting enzyme in glycolisis which supports glucose metabolism. It has strong connection with LDHA protein in anaerobic metabolism known as warburg effect to maintain tumor proliferations.(32,33) Warburg effect produces energy for the cells to maintain the cell growth and and its fast proprety makes it commonly used by tumor cells.(34) This effect also related with hypoxia pathway, which may occured inside the core of spheroid that mimics tumor property.(35,36)
Beside its role in glucose metabolism, PFKL and G6PD expressions revealed deeper knowledge on cancer metabolism. G6PD or Glucose-6-phosphate Dehydrogenase is a rate limiting enzymes of pentose phosphate pathway. It is a pathway that supports ribose-5-phosphate synthesis required to sintesize nucleotide.(31) Another important finding is the connection with HPRT1, a gene responsible in de salvage pathway to provide nucleotides.(37) Therefore, the tumor may activate pathway that produce 5-ribose phosphate for nucleotide backbone and actively reproduce de salvage by expressing HRPT1. The importance of G6PD is its role to provide NADPH for power reducer. It is neccesary to regenerate fatty acid. Inside the networking there were no connections or any other informations related to fatty acid synthesis. However, ACSL4 which is known to ligate long fatty acid to coenzyme A and to synthesize long fatty acid, is essential in metabolism, modification of membranes structures, and proliferation.(38) Together with G6PD expression, we assumed that NADPH was also used by cells for the synthesis of fatty acid.
Interestingly, SOD1 known as cytosolic superoxides were also expressed. this may indicate that the tumors protect its cells from reactive superoxide generated as metabolism byproduct by expressing the SOD1. The SOD1 has a protective property against anion superoxide produced by several metabolic pathway.(39) Taken together with G6PD expression, the tumor cells may provide higher reducing power such as NADPH protein expression and high ROS productions inside the cells to support the tumor growth.
The network analysis conveyed positive expression of CASP7, CFLAR and XIAP. CASP7 overexpression were known to induced apoptosis. (40,41) However, in cancer cells overexpression of CASP7 usually indicates poor prognostic factos. Other protein expressed was CLFAR, known as programmed cell death. It modulates apoptotic pathway by regulating caspase-8 activations. (42) Lastly, XIAP protein, also knowns as modulator. This protein regulates apoptosis by directly bind to binding pocket of caspase-7 and caspase-3.(43,44) The proteins were known to inhibits proteasomal degradations that promotes autophagy(45).
To support cell growth, the cells are mostly maintaining complex mechanisms. One of the main mechanism is angiogenesis. TEK, ANGPT1, VEGFC and FLT1 conveyed that the cells support angiogenesis related pathways. TEK, also known as Tie2, were expressed on tumors vasculature. While ANGPT1 overexpression is related to the increasing tumorigenicy of breast cancer cell lines. (46) TEK and ANGPT1 expression may indicate that spheroids may describe tumors and endothelial crosstalk communications. VEGFC overexpression is known to correlate with poorer survival as it supports tumor growth.(47) Moreover, FLT1 is known to support not only tumor growth but also metastasis and mostly due to its interactions with macrophages.(48) Interestingly, SERPINF1 expression, known as anti-angiogenic factors, may be released by certain cells inside this spheroid cultures to suppress the tumor growth.(49) This is interesting, since suppresion of this SERPINF1 could induce tumor promoting phenotype of cancer associated fibroblast.(50)
We also found interactions of FOXC2 and CDH2. FOXC2 may be related to TEK and angiogenesis since FOXC2 supports angiogenesis and disease progression (51). Its connection with CDH2 may be related to reprogramming event of epithelial to mesenchymal transitions (EMT), indicating celullar plasticity.(52) CDH2 were related to aggresive phenotypes and protections of microenvironment against therapheutical agent.(53) Moreover, the expression of SNAI3 and KRT14 related to CDH2, may indicate EMT process inside the spheroids.(54) Despite its interactions were closely related to apoptosis, PINX-1 and TINF2 expression may be related to tumor suppresion effect inside the spheroids.(55) Therefore, at certain extend some cells may have tried to suppress the tumor growth.
However, the expression of IGFB3 and CCND3 were found. IGFB3 may be related to hypoxia related signaling, induced by hypoxic microenvironment.(56) Serum IGFBP3 is known to correlate with VEGFC and may be related to metastasis insidence.(57) IGFB3 were also shown to bind with several growth factors.(58) Lastly, CCND3 expression may be related to malignancy and tumor growth capacity, since its known as poor overall survival prognostic.(58)