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The CD44(high) tumorigenic subsets in lung cancer biospecimens are enriched for low miR-34a expression.

Basak SK, Veena MS, Oh S, Lai C, Vangala S, Elashoff D, Fishbein MC, Sharma S, Rao NP, Rao D, Phan R, Srivatsan ES, Batra RK - PLoS ONE (2013)

Bottom Line: Progression can be associated with emergence of cells that exhibit high phenotypic plasticity (including "de-differentiation" to primitive developmental states), and aggressive behavioral properties (including high tumorigenic potentials).The colony forming efficiency of CD44(hi) cells, characteristic property of CSC, can be inhibited by mir-34a replacement in these samples.In addition the highly tumorigenic CD44(hi) cells are enriched for cells in the G2 phase of cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Stem Cell Institute, Veterans Affairs Greater Los Angeles Healthcare System (VAGLAHS), Los Angeles, California, United States of America ; Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America.

ABSTRACT
Cellular heterogeneity is an integral part of cancer development and progression. Progression can be associated with emergence of cells that exhibit high phenotypic plasticity (including "de-differentiation" to primitive developmental states), and aggressive behavioral properties (including high tumorigenic potentials). We observed that many biomarkers that are used to identify Cancer Stem Cells (CSC) can label cell subsets in an advanced clinical stage of lung cancer (malignant pleural effusions, or MPE). Thus, CSC-biomarkers may be useful for live sorting functionally distinct cell subsets from individual tumors, which may enable investigators to hone in on the molecular basis for functional heterogeneity. We demonstrate that the CD44(hi) (CD44-high) cancer cell subsets display higher clonal, colony forming potential than CD44(lo) cells (n=3) and are also tumorigenic (n=2/2) when transplanted in mouse xenograft model. The CD44(hi) subsets express different levels of embryonal (de-differentiation) markers or chromatin regulators. In archived lung cancer tissues, ALDH markers co-localize more with CD44 in squamous cell carcinoma (n=5/7) than Adeno Carcinoma (n=1/12). MPE cancer cells and a lung cancer cell line (NCI-H-2122) exhibit chromosomal abnormalities and 1p36 deletion (n=3/3). Since miR-34a maps to the 1p36 deletion site, low miR-34a expression levels were detected in these cells. The colony forming efficiency of CD44(hi) cells, characteristic property of CSC, can be inhibited by mir-34a replacement in these samples. In addition the highly tumorigenic CD44(hi) cells are enriched for cells in the G2 phase of cell cycle.

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Expression of miR-34a in CD44hi and CD44lo cells evaluated by RT-qPCR and exogenous delivery of miR-34a into CD44hi cells inhibits colony formation and anti- miR-34a into CD44lo cells increases colony formation.(A) miR-34a expression in unsorted, CD44hi and CD44lo cells of two primary samples (M-1 and M-2), established NSCLC cell line NCI-H-2122 and normal human fibroblast cell line GM 05399. The miR-34a expression has been normalized with RNU48. (B) Sorted CD44hi cells (samples M-1 and M-2) when transduced with miR-34a show decreased colony forming efficiency in comparison to miR-control transduced CD44hi tumor cells; (Sample M-1: miR control = 131.6 (SD = 30.5),+miR-34a = 7 (SD = 2.6) and P = 0.002; Sample M-2: miR control = 75 (SD = 19.6),+miR-34a = 23 (SD = 5.5) and P = 0.01); The mean effect with miR-34a versus miR-control on CD44hi cells is −88.3 (95% CI: −288.12, 111.45; P = 0.112) (C) Sorted CD44hi tumor cells transduced with miR-34a exhibit small colony size (bottom panel) than miR-control transduced CD44hi tumor cells (upper panel); (D) The CD44lo cells from Sample M-1 and M-2 transduced with anti-miR34a show increased colony forming efficiency than tumor cells transduced with miR-control; (Sample M-1: miR control = 21 (SD = 4.5),+anti- miR-34a = 33 (SD = 5.2) and P = 0.04; Sample M-2: miR control = 24.6 (SD = 4.1),+anti-miR-34a = 39.3 (SD = 5.1) and P = 0.018); The mean effect with anti-miR-34a versus miR-control on CD44lo cells is −13.3 (95% CI: −20.43, 47.10; P = 0.125). (E) Sorted CD44lo cells transduced with anti-miR-34a exhibit bigger colony size (bottom panel) than miR-control transduced tumor cells (upper panel).
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pone-0073195-g006: Expression of miR-34a in CD44hi and CD44lo cells evaluated by RT-qPCR and exogenous delivery of miR-34a into CD44hi cells inhibits colony formation and anti- miR-34a into CD44lo cells increases colony formation.(A) miR-34a expression in unsorted, CD44hi and CD44lo cells of two primary samples (M-1 and M-2), established NSCLC cell line NCI-H-2122 and normal human fibroblast cell line GM 05399. The miR-34a expression has been normalized with RNU48. (B) Sorted CD44hi cells (samples M-1 and M-2) when transduced with miR-34a show decreased colony forming efficiency in comparison to miR-control transduced CD44hi tumor cells; (Sample M-1: miR control = 131.6 (SD = 30.5),+miR-34a = 7 (SD = 2.6) and P = 0.002; Sample M-2: miR control = 75 (SD = 19.6),+miR-34a = 23 (SD = 5.5) and P = 0.01); The mean effect with miR-34a versus miR-control on CD44hi cells is −88.3 (95% CI: −288.12, 111.45; P = 0.112) (C) Sorted CD44hi tumor cells transduced with miR-34a exhibit small colony size (bottom panel) than miR-control transduced CD44hi tumor cells (upper panel); (D) The CD44lo cells from Sample M-1 and M-2 transduced with anti-miR34a show increased colony forming efficiency than tumor cells transduced with miR-control; (Sample M-1: miR control = 21 (SD = 4.5),+anti- miR-34a = 33 (SD = 5.2) and P = 0.04; Sample M-2: miR control = 24.6 (SD = 4.1),+anti-miR-34a = 39.3 (SD = 5.1) and P = 0.018); The mean effect with anti-miR-34a versus miR-control on CD44lo cells is −13.3 (95% CI: −20.43, 47.10; P = 0.125). (E) Sorted CD44lo cells transduced with anti-miR-34a exhibit bigger colony size (bottom panel) than miR-control transduced tumor cells (upper panel).

Mentions: Since expression of miR-34a may contribute to the different biological properties of CD44hi versus CD44lo cells in individual tumors, we evaluated its expression levels in CD44hi, CD44lo and unsorted total cell populations in fractionated MPE-biospecimens (Figure 6A). The expression of small nucleolar RNA-RNU48 was used as a reference for gene expression in this assay (data not shown), and miR-34a results were normalized with the RNU48 expression. Although the expression of the small nucleolar RNA-RNU48 may itself be dysregulated in cancer [29], our study demonstrated a similar basal expression pattern across the sample sets. On RT-qPCR analyses, however, there was no significant difference in miR-34a expression in the CD44hi and CD44lo subsets of the MPE sample M-2; the expression in this sample was similar to that of the control fibroblasts. In contrast, CD44hi cells have significantly lower level of miR-34a than the CD44lo cells in sample M-1, as well as in the immortalized cell line NCI-H2122. These data suggest that loss of miR34a may contribute to aggressive biological properties and high tumorigenic potentials in some lung cancers.


The CD44(high) tumorigenic subsets in lung cancer biospecimens are enriched for low miR-34a expression.

Basak SK, Veena MS, Oh S, Lai C, Vangala S, Elashoff D, Fishbein MC, Sharma S, Rao NP, Rao D, Phan R, Srivatsan ES, Batra RK - PLoS ONE (2013)

Expression of miR-34a in CD44hi and CD44lo cells evaluated by RT-qPCR and exogenous delivery of miR-34a into CD44hi cells inhibits colony formation and anti- miR-34a into CD44lo cells increases colony formation.(A) miR-34a expression in unsorted, CD44hi and CD44lo cells of two primary samples (M-1 and M-2), established NSCLC cell line NCI-H-2122 and normal human fibroblast cell line GM 05399. The miR-34a expression has been normalized with RNU48. (B) Sorted CD44hi cells (samples M-1 and M-2) when transduced with miR-34a show decreased colony forming efficiency in comparison to miR-control transduced CD44hi tumor cells; (Sample M-1: miR control = 131.6 (SD = 30.5),+miR-34a = 7 (SD = 2.6) and P = 0.002; Sample M-2: miR control = 75 (SD = 19.6),+miR-34a = 23 (SD = 5.5) and P = 0.01); The mean effect with miR-34a versus miR-control on CD44hi cells is −88.3 (95% CI: −288.12, 111.45; P = 0.112) (C) Sorted CD44hi tumor cells transduced with miR-34a exhibit small colony size (bottom panel) than miR-control transduced CD44hi tumor cells (upper panel); (D) The CD44lo cells from Sample M-1 and M-2 transduced with anti-miR34a show increased colony forming efficiency than tumor cells transduced with miR-control; (Sample M-1: miR control = 21 (SD = 4.5),+anti- miR-34a = 33 (SD = 5.2) and P = 0.04; Sample M-2: miR control = 24.6 (SD = 4.1),+anti-miR-34a = 39.3 (SD = 5.1) and P = 0.018); The mean effect with anti-miR-34a versus miR-control on CD44lo cells is −13.3 (95% CI: −20.43, 47.10; P = 0.125). (E) Sorted CD44lo cells transduced with anti-miR-34a exhibit bigger colony size (bottom panel) than miR-control transduced tumor cells (upper panel).
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pone-0073195-g006: Expression of miR-34a in CD44hi and CD44lo cells evaluated by RT-qPCR and exogenous delivery of miR-34a into CD44hi cells inhibits colony formation and anti- miR-34a into CD44lo cells increases colony formation.(A) miR-34a expression in unsorted, CD44hi and CD44lo cells of two primary samples (M-1 and M-2), established NSCLC cell line NCI-H-2122 and normal human fibroblast cell line GM 05399. The miR-34a expression has been normalized with RNU48. (B) Sorted CD44hi cells (samples M-1 and M-2) when transduced with miR-34a show decreased colony forming efficiency in comparison to miR-control transduced CD44hi tumor cells; (Sample M-1: miR control = 131.6 (SD = 30.5),+miR-34a = 7 (SD = 2.6) and P = 0.002; Sample M-2: miR control = 75 (SD = 19.6),+miR-34a = 23 (SD = 5.5) and P = 0.01); The mean effect with miR-34a versus miR-control on CD44hi cells is −88.3 (95% CI: −288.12, 111.45; P = 0.112) (C) Sorted CD44hi tumor cells transduced with miR-34a exhibit small colony size (bottom panel) than miR-control transduced CD44hi tumor cells (upper panel); (D) The CD44lo cells from Sample M-1 and M-2 transduced with anti-miR34a show increased colony forming efficiency than tumor cells transduced with miR-control; (Sample M-1: miR control = 21 (SD = 4.5),+anti- miR-34a = 33 (SD = 5.2) and P = 0.04; Sample M-2: miR control = 24.6 (SD = 4.1),+anti-miR-34a = 39.3 (SD = 5.1) and P = 0.018); The mean effect with anti-miR-34a versus miR-control on CD44lo cells is −13.3 (95% CI: −20.43, 47.10; P = 0.125). (E) Sorted CD44lo cells transduced with anti-miR-34a exhibit bigger colony size (bottom panel) than miR-control transduced tumor cells (upper panel).
Mentions: Since expression of miR-34a may contribute to the different biological properties of CD44hi versus CD44lo cells in individual tumors, we evaluated its expression levels in CD44hi, CD44lo and unsorted total cell populations in fractionated MPE-biospecimens (Figure 6A). The expression of small nucleolar RNA-RNU48 was used as a reference for gene expression in this assay (data not shown), and miR-34a results were normalized with the RNU48 expression. Although the expression of the small nucleolar RNA-RNU48 may itself be dysregulated in cancer [29], our study demonstrated a similar basal expression pattern across the sample sets. On RT-qPCR analyses, however, there was no significant difference in miR-34a expression in the CD44hi and CD44lo subsets of the MPE sample M-2; the expression in this sample was similar to that of the control fibroblasts. In contrast, CD44hi cells have significantly lower level of miR-34a than the CD44lo cells in sample M-1, as well as in the immortalized cell line NCI-H2122. These data suggest that loss of miR34a may contribute to aggressive biological properties and high tumorigenic potentials in some lung cancers.

Bottom Line: Progression can be associated with emergence of cells that exhibit high phenotypic plasticity (including "de-differentiation" to primitive developmental states), and aggressive behavioral properties (including high tumorigenic potentials).The colony forming efficiency of CD44(hi) cells, characteristic property of CSC, can be inhibited by mir-34a replacement in these samples.In addition the highly tumorigenic CD44(hi) cells are enriched for cells in the G2 phase of cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Stem Cell Institute, Veterans Affairs Greater Los Angeles Healthcare System (VAGLAHS), Los Angeles, California, United States of America ; Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America.

ABSTRACT
Cellular heterogeneity is an integral part of cancer development and progression. Progression can be associated with emergence of cells that exhibit high phenotypic plasticity (including "de-differentiation" to primitive developmental states), and aggressive behavioral properties (including high tumorigenic potentials). We observed that many biomarkers that are used to identify Cancer Stem Cells (CSC) can label cell subsets in an advanced clinical stage of lung cancer (malignant pleural effusions, or MPE). Thus, CSC-biomarkers may be useful for live sorting functionally distinct cell subsets from individual tumors, which may enable investigators to hone in on the molecular basis for functional heterogeneity. We demonstrate that the CD44(hi) (CD44-high) cancer cell subsets display higher clonal, colony forming potential than CD44(lo) cells (n=3) and are also tumorigenic (n=2/2) when transplanted in mouse xenograft model. The CD44(hi) subsets express different levels of embryonal (de-differentiation) markers or chromatin regulators. In archived lung cancer tissues, ALDH markers co-localize more with CD44 in squamous cell carcinoma (n=5/7) than Adeno Carcinoma (n=1/12). MPE cancer cells and a lung cancer cell line (NCI-H-2122) exhibit chromosomal abnormalities and 1p36 deletion (n=3/3). Since miR-34a maps to the 1p36 deletion site, low miR-34a expression levels were detected in these cells. The colony forming efficiency of CD44(hi) cells, characteristic property of CSC, can be inhibited by mir-34a replacement in these samples. In addition the highly tumorigenic CD44(hi) cells are enriched for cells in the G2 phase of cell cycle.

Show MeSH
Related in: MedlinePlus