Retroviral-infection increases tumorigenic potential of MDA-MB-231 breast carcinoma cells by expanding an aldehyde dehydrogenase (ALDH1) positive stem-cell like population

Abstract

Retroviral transformation has been associated with pro-proliferative oncogenic signaling in human cells. The current study demonstrates that transduction of human breast carcinoma cells (MDA-MB231) with LXSN and QCXIP retroviral vectors causes significant increases in growth rate, clonogenic fraction, and aldehyde dehydrogenase-1 positive cells (ALDH1+), which is associated with increased steady-state levels of cancer stem cell populations. Furthermore, this retroviral-induced enhancement of cancer cell growth in vitro was also accompanied by a significant increase in xenograft tumor growth rate in vivo. The retroviral induced increases in cancer cell growth rate were partially inhibited by treatment with 100 U/ml polyethylene glycol-conjugated-(PEG)-superoxide dismutase and/or PEG-catalase. These results show that retroviral infection of MDA-MB231 human breast cancer cells is capable of enhancing cell proliferation and cancer stem cell populations as well as suggesting that modulation of reactive oxygen species-induced pro-survival signaling pathways may be involved in these effects.

Keywords: Aldehyde dehydrogenase positive; Antioxidant enzymes; Mammary cancer; Oxidative stress; Stem cells; Viral carcinogenesis.

Fig. 1

Retroviral infection alters the colony appearance and morphology of MDA-MB-231 human mammary adenocarcinoma cells. (A), (D), and (G)—representative images of clonogenic assay plates from untransformed MDA-MB231 cells (A), retrovirally transformed cells MDA-MB231-LXSN (D), and MDA-MB231-QCXIP (G). Cells were seeded at the density of 250 cells/60 mm dish and allowed to form colonies for 14 days where cells were fixed and stained. (B), (E), and (H) higher magnification images of a single colony highlighting the piling up of cells in the colonies of the MDA-MB231-LXSN (E) and MDA-MB231-QXCIP (H) versus the single layer of cells characteristic of the MDA-MB231 colonies (B). (C), (F), and (I)—close-up images showing the distinct morphological differences between the parent MDA-MB231 (C) cells and the transformed lines MDA-MB231-LXSN (F) and MDA-MB231-QCXIP (I).

Fig. 2

Retroviral infection alters the clonogenic capacity, growth rate, and ALDH1 stem cell markers of MDA-MB231 cells. (A) Clonogenic survival fraction of the cell lines analyzed. After 14 days of seeding at a density of 250cells/60 mm dish, cells were fixed, stained, and surviving colonies were then counted. (B) Graph displaying the number of cells per colony of the untransformed cells (MDA-MB231) versus the retrovirally transformed lines (MDA-MB231-LXSN and MDA-MB231-QCXIP). (C) Cell doubling time (in hours) of cell lines. All transformed cell lines maintained a shorter doubling time than the parental cell lines. (D) Bar graph charting the increases in % population of transformed cells displaying ALDEFLUOR activity. 1×10⁶ cells were re-suspended in ALDEFLUOR assay buffer as recommended by the manufacturer. Samples were analyzed by flow cytometer. The number of ALDH positive cells of 100,000 live cells was determined by using the sorting gate established using DEAB the ALDH1 inhibitor containing samples. A minimum of 10,000 cells was analyzed from each cell population. P values were calculated using two-sided Student’s t test.

Fig. 3

Retroviral infection increases the oxidation of CDCFH2 in MDA-MB231 cells indicating a shift in the intracellular redox environment. Left side: transformed cells (MDA-MB231-LXSN and MDA-MB231-QCXIP) significantly increased CDCFH2 oxidation, relative to MDA-MB231. Right side: when the cells were labeled with the oxidation insensitive probe, CDCF, no difference was seen in the florescence of the probe between cell lines indicating that the changes in florescent seen in panel A were caused by changes in probe oxidation.

Fig. 4

Cell proliferation was inhibited by treatment with PEG-catalase/PEG-SOD. Left side: doubling time of treated versus untreated MDA-MB231 LXSN. Cells were treated with 100 U/ml of PEG-catalase and/or PEG-SOD and growth rate was determined by counting cells every 24 hours for a period of 14 days to calculate a doubling time. Right side: population doubling time of MDA-MB231 QCXIP cells when treated with PEG-catalase, PEG-SOD, or both.

Fig. 5

Retroviral transfection of MDA-MB231 enhances xenograft tumor growth rate in nude mice. (A) One million cells were subcutaneously injected into the left (MDA-MB231) and right (MDA-MB231-LXSN) flanks of 6–8 weeks old athymic nu/nu mice. (B) Tumor growth was monitored and measured with a caliper for 5 weeks after injection. N = 5 mice. (C) In a separate experiment, one million cells of MDA-MB231 and MDA-MB231-QCXIP were subcutaneously injected into the left and right flank, respectively of 6–8 weeks old athymic nu/nu mice. Tumor growth was monitored and measured with a caliper for a period of 10 weeks. N = 4 mice.