Figure 6.
EGFL7, a KLF2 downstream target, promotes primary MM survival. (A) Fold change in ITGB3, EGFL7, and KLF2 expression in ITGB3 OE RPMI cells when compared with Mock cells, as determined by RT-PCR (n = 3/group). (B) Fold change in KLF2 expression in Mock, si-KLF2, and KLF2 OE RPMI cells as determined by RT-PCR (n = 3/group). (C) Fold change in EGFL7 expression in KLF2 OE or si-KLF2 RPMI cells when compared with KLF2 Mock or si-KLF2 cells, respectively, as determined by RT-PCR. (D) Representative western blot for indicated proteins from lysates of si-Ctrl, si-KLF2, Mock, and KLF2 OE cells. (E) Viable cell quantification of KLF2 OE and Mock cells after 24 hours in culture (n = 6/group). (F) KLF2 OE, Mock, si-Ctrl, and si-KLF2 cells were cultured in the presence or absence of rec. EGFL7. Cells were counted after 24 hours (n = 6/group). (G) Fold change in KLF2 expression in BTZ-treated MM cells at the indicated concentration for 24 hours when compared with non-BTZ-treated controls, as evaluated by RT-PCR. (H) MM cells (EGFL7 OE/EGFL7 KD, KLF2 OE, KLF2 KD, or KLF2 OE + EGFL7 KD) were cultured with/without 10 µM BTZ. After 24 hours, viable cells were enumerated (n = 3/group). (I) Immunohistochemical staining for EGFL7 (background panel; scale bars, 50 µm) and hematoxylin and eosin (insert; scale bars, 10 µm) in BM sections of patients with MM. For patient details, see supplemental Figure 1. (J) KLF2 expression determined by RT-PCR in total BMMCs, MACS-isolated BM CD138+ and CD138− cells derived from MM patients #1, #2, and #3. KLF2 expression was given as a fold change to the expression found in MACS-isolated CD138− cells from a healthy donor as the comparator. (K) EGFL7 KD or OE was achieved in human primary MACS-isolated CD138+ MM cells using a lentiviral virus–containing GFP. Cell proliferation was monitored by GFP positivity using FACS after 24 hours (n = 3/group). (L) EGFL7 OE, KD, KLF2 OE or KD was achieved in primary PCR in CD138+ MM patient cell samples (#1-#3). Cell proliferation of transduced cells was determined after 24 hours (n = 6/group). (M) Fold change in ITGB3 expression, as determined by RT-PCR. For all RT-PCR results, transcripts normalized to b-ACTIN. Experiments were repeated twice with similar results. (N) Model of the EGFL7-ITGB3-KLF2-EGFL7 axis in MM cells. EGFL7 partially through binding to ITGB3 on MM cells upregulates the transcription factor KLF2. In turn, KLF2 augments EGFL7 expression. This leads to a positive forward amplification loop that promotes MM survival, a mechanism that seems especially active under the pressure of chemotherapeutic drugs such as BTZ. We propose that EGFL7 contributes to BTZ-induced drug resistance. Data are represented as mean ± SEM. * P ≤ .05; ** P ≤ .01; *** P ≤ .001. P values were determined using a Student t test.

EGFL7, a KLF2 downstream target, promotes primary MM survival. (A) Fold change in ITGB3, EGFL7, and KLF2 expression in ITGB3 OE RPMI cells when compared with Mock cells, as determined by RT-PCR (n = 3/group). (B) Fold change in KLF2 expression in Mock, si-KLF2, and KLF2 OE RPMI cells as determined by RT-PCR (n = 3/group). (C) Fold change in EGFL7 expression in KLF2 OE or si-KLF2 RPMI cells when compared with KLF2 Mock or si-KLF2 cells, respectively, as determined by RT-PCR. (D) Representative western blot for indicated proteins from lysates of si-Ctrl, si-KLF2, Mock, and KLF2 OE cells. (E) Viable cell quantification of KLF2 OE and Mock cells after 24 hours in culture (n = 6/group). (F) KLF2 OE, Mock, si-Ctrl, and si-KLF2 cells were cultured in the presence or absence of rec. EGFL7. Cells were counted after 24 hours (n = 6/group). (G) Fold change in KLF2 expression in BTZ-treated MM cells at the indicated concentration for 24 hours when compared with non-BTZ-treated controls, as evaluated by RT-PCR. (H) MM cells (EGFL7 OE/EGFL7 KD, KLF2 OE, KLF2 KD, or KLF2 OE + EGFL7 KD) were cultured with/without 10 µM BTZ. After 24 hours, viable cells were enumerated (n = 3/group). (I) Immunohistochemical staining for EGFL7 (background panel; scale bars, 50 µm) and hematoxylin and eosin (insert; scale bars, 10 µm) in BM sections of patients with MM. For patient details, see supplemental Figure 1. (J) KLF2 expression determined by RT-PCR in total BMMCs, MACS-isolated BM CD138+ and CD138− cells derived from MM patients #1, #2, and #3. KLF2 expression was given as a fold change to the expression found in MACS-isolated CD138− cells from a healthy donor as the comparator. (K) EGFL7 KD or OE was achieved in human primary MACS-isolated CD138+ MM cells using a lentiviral virus–containing GFP. Cell proliferation was monitored by GFP positivity using FACS after 24 hours (n = 3/group). (L) EGFL7 OE, KD, KLF2 OE or KD was achieved in primary PCR in CD138+ MM patient cell samples (#1-#3). Cell proliferation of transduced cells was determined after 24 hours (n = 6/group). (M) Fold change in ITGB3 expression, as determined by RT-PCR. For all RT-PCR results, transcripts normalized to b-ACTIN. Experiments were repeated twice with similar results. (N) Model of the EGFL7-ITGB3-KLF2-EGFL7 axis in MM cells. EGFL7 partially through binding to ITGB3 on MM cells upregulates the transcription factor KLF2. In turn, KLF2 augments EGFL7 expression. This leads to a positive forward amplification loop that promotes MM survival, a mechanism that seems especially active under the pressure of chemotherapeutic drugs such as BTZ. We propose that EGFL7 contributes to BTZ-induced drug resistance. Data are represented as mean ± SEM. * P ≤ .05; ** P ≤ .01; *** P ≤ .001. P values were determined using a Student t test.

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