Liver Label Retaining Cancer Cells Are Relatively Resistant to the Reported Anti-Cancer Stem Cell Drug Metformin

Abstract

Background & aims: Recently, we reported that liver Label Retaining Cancer Cells (LRCC) can initiate tumors with only 10 cells and are relatively resistant to the targeted drug Sorafenib, a standard of practice in advanced hepatocellular carcinoma (HCC). LRCC are the only cancer stem cells (CSC) isolated alive according to a stem cell fundamental function, asymmetric cell division. Metformin has been reported to preferentially target many other types of CSC of different organs, including liver. It's important to know if LRCC, a novel class of CSC, are relatively resistant to metformin, unlike other types of CSC. As metformin inhibits the Sorafenib-Target-Protein (STP) PI3K, and LRCC are newly described CSC, we undertook this study to test the effects of Metformin on Sorafenib-treated HCC and HCC-derived-LRCC.

Methods: We tested various STP levels and phosphorylation status, associated genes' expression, proliferation, viability, toxicity, and apoptosis profiles, before and after treatment with Sorafenib with/without Metformin.

Results: Metformin enhances the effects of Sorafenib on HCC, and significantly decreased viability/proliferation of HCC cells. This insulin-independent effect was associated with inhibition of multiple STPs (PKC, ERK, JNK and AKT). However, Metformin increased the relative proportion of LRCCs. Comparing LRCC vs. non-LRCC, this effect was associated with improved toxicity and apoptosis profiles, down-regulation of cell death genes and up-regulation of cell proliferation and survival genes in LRCC. Concomitantly, Metformin up-regulated pluripotency, Wnt, Notch and SHH pathways genes in LRCC vs. non-LRCC.

Conclusions: Metformin and Sorafenib have enhanced anti-cancer effects. However, in contradistinction to reports on other types of CSC, Metformin is less effective against HCC-derived-CSC LRCC. Our results suggest that combining Metformin with Sorafenib may be able to repress the bulk of tumor cells, but as with other anti-cancer drugs, may leave LRCC behind leading to cancer recurrence. Therefore, liver LRCC, unlike other types of CSC, are relatively resistant to the reported anti-cancer stem cell drug metformin. This is the first report that there is a type of CSC that is not relatively resistant to the CSC-targeting drug. Our findings suggest that a drug targeting LRCC may be critically needed to target CSC and prevent cancer recurrence. These may significantly contribute to the understanding of Metformin's anti-cancer effects and the development of novel drugs targeting the relatively resistant LRCC.

Keywords: HCC; LRCC; MAPK; Metformin; PKC/ERK/JNK/AKT phosphorylation; cancer-stem-cells.; sorafenib; stem-like label-retaining cancer cells.

Figure 1

Metformin enhances cancer therapy with sorafenib. Metformin addition to sorafenib cause significant reduction of viable cells compared to sorafenib treatment alone after 48 hours of treatment.

Figure 2

PKC-alfal/delta, MEK1/2, ERK1/2 and JNK1/2 are inhibited in PLC/PRF/5 cells after treatment with metformin. After treatment with metformin phosphorylation of multiple protein kinases were inhibited in PLC/PRF/5 cells: (A) PKC-alfal, (B) PKC-delta, (C) MEK1/2 (pT292), (D) MEK1/2 (pT386), (E) ERK1/2 and (F) JNK1/2. Metformin was used at 200 uM and sorafenib at 4 uM.

Figure 3

Metformin ineffectively kills LRCC. (A) Isolation of HCC derived label-retaining-cancer-cells (LRCC). The full method is described in Daniel et al. , . Whole cell populations of HCC cells were labeled with Cy5-DNA-nucleotides (pulse phase). Subsequently, Cy5-positive-high cells were sorted and grown for 8 cell cycles (chase phase). Finally, Cy5-positive-high cells (99% pure) and Cy5-negative cells were sorted as LRCC and non-LRCC, respectively. (B)-(C) The relative proportion of LRCC is increased after metformin treatment. HuH-7 is shown in (B). Compared to non-LRCC, LRCC exhibit reduced cell viability (D), cell toxicity profile (E), and apoptosis profile (F).

Figure 4

STP and STG expression in LRCC vs. non-LRCC. (A-B) Compared to non-LRCC, metformin treatment of LRCC resulted in increases of the major highly phosphorylated species of AKT1. (C) STG expression in LRCC before and after treatment with metformin. (D) Compared to non-LRCC, metformin treatment of LRCC resulted in opposite effects by up-regulation of RET, STAT5A and AKT3.

Figure 5

Wnt pathway and stem cells associated genes expression. (A) Wnt pathway genes' expression: in LRCC with/without metformin, and (B) in LRCC vs. non-LRCC after treatment with metformin. (C) Stem cells associated genes' expression in LRCC with/without metformin, and (D) in LRCC vs. non-LRCC after treatment with metformin. (E) Stem cell pluripotency genes in LRCC vs. non-LRCC after treatment with metformin.