Triggering of CD44 by Ultra-Low-Molecular-Weight Hyaluronan Induces Cell Death by Autophagy Via Endoplasmic Reticulum Stress in Acute Lymphoblastic Leukemia with MLL Gene Rearrangements

Abstract

1361 Acute lymphoblastic leukemia (ALL) with MLL gene rearrangements (MLL+ALL) is chemoresistant and its prognosis is still dismal. MLL+ALL shows a unique gene profile clearly distinguishable from other types of ALL and AML, and CD44 is known as one of specific molecules expressed very highly in MLL+ALL. CD44, a transmembrane glycoprotein recently attracting an attention as a cancer stem cell marker for several types of cancers, is expressed on a variety of cells, and has the specific binding sites for hyaluronic acid (HA, formally hyaluronan). High-molecular-weight (HMW)-HA which exists as extracellular matrix in tissues is incorporated into cells in a CD44-dependent manner, and then is secreted from cells as low-molecular-weight (LMW) to ultra-low-molecular-weight (ULMW)-HA in normal and pathological conditions. The purpose of this study is to investigate the biological events elicited after ligand stimulation of CD44 by using distinct molecular sizes of HA (2.5mg/ml) in MLL+ALL. When cultured in the presence of ULMW-HA (6.4kDa), but not of LMW-HA (31kDa) or HMW-HA (980kDa), thymidine uptakes of cell lines expressing CD44 at very high levels (KOPB-26 and YACL-95) were gradually inhibited in the course of culture from day3 through day5 (greater than 90% decrease at day5), although those expressing CD44 at very low levels (KOCL58, KOCL69) were largely unaffected. Cell cycle analysis of KOPB-26 and YACL-95 using propidium iodide (PI) staining after ULMW-HA stimulation revealed no increase in G0/G1 and G2/M phases and subdiploid population, suggesting that a marked inhibition of thymidine uptakes is neither due to cell cycle arrest nor induction of apoptosis. Dye exclusion test showed a marked decrease in viability at days 3–5 after ULMW-HA stimulation, and flow cytometric analysis showed a gradual increase in the low FSC/high SSC population doubly labeled by Annexin V and PI. However, cytospine smears revealed a massive cell death lacking features characteristic of apoptosis. In addition, pan-caspase inhibitor Z-VAD did not block cell death and cleaved caspase 3 was not detected on flow cytometric analysis after ULMW-HA stimulation, resulting in the conclusion that ULMW-HA-induced cell death is not elicited by induction of apoptosis. Although levels of high-mobility group protein B1 (HMGB1) usually used as a necrosis marker was progressively elevated in culture media after ULMW-HA stimulation, the necrosis inhibitor Necrostatin-1 did not block the ULMW-HA-induced cell death. Finally, electromicroscopic examination was performed and revealed a marked extension of endoplasmic reticulum (ER) and an increase in autophagsomes, and the autophagy inhibitor 3-methyladenine specifically suppressed cell death, indicating that ULMW-HA strongly induces ER stress leading to cell death by autophagy. Changes in ER stress and autophagy-associated molecules after ULMW-HA stimulation were also demonstrated. In conclusion, triggering of CD44 by ULMW-HA stimulation strongly elicits cell death by autophagy via ER stress in chemoresistant MLL+ALL. CD44-targeted therapy by ULMW-HA possibly becomes a useful strategy not only for MLL+ALL but also for CD44-high hematological and non-hematological tumors particularly for those in which the cancer stem cell population is highly positive for CD44. Disclosures: No relevant conflicts of interest to declare.

Biglycan evokes autophagy in macrophages via a novel CD44/Toll-like receptor 4 signaling axis in ischemia/reperfusion injury

“…30 Moreover, by triggering CD44, ultralow-molecular-weight HA and MIF were involved in the induction of autophagy in acute lymphoblastic leukemia and endothelial cells, respectively. 28,29 Importantly, our study has unveiled CD44 as a signaling receptor engaged by a matrixderived DAMP to activate autophagy in immune cells.…”

“…CD44 was attracting attention as a signaling receptor upstream of the autophagic pathway in nonimmune cells. [28][29][30] In vascular smooth muscle cells, the CD44-integrin complex was shown to mediate osteopontininduced autophagy. 30 Moreover, by triggering CD44, ultralow-molecular-weight HA and MIF were involved in the induction of autophagy in acute lymphoblastic leukemia and endothelial cells, respectively.…”

“…27 Importantly, CD44 is essential for HA-, osteopontin-, and MIF-induced autophagy in nonimmune cells. [28][29][30] Autophagy is a conserved cellular process through which misfolded proteins or damaged organelles are engulfed in double layered membrane vacuoles called autophagosomes and subjected to lysosomal degradation. 31,32 Previous studies showed that autophagy induction in tubular epithelial cells and podocytes has renoprotective effects in various inflammatory kidney diseases.…”

Breaking down chronic inflammatory diseases: the role of biglycan in promoting a switch between inflammation and autophagy

“…2) [21]. These effects are solely biglycan dependent and independent of HA, osteopontin, and MIF-2 [21,[61][62][63]. Importantly, biglycan-triggered macrophage autophagy does not involve CD14 both in vitro and in vivo [21].…”

LL-37 alone and in combination with IL17A enhances proinflammatory cytokine expression in parallel with hyaluronan metabolism in human synovial sarcoma cell line SW982—A step toward understanding the development of inflammatory arthritis

“…HA regulates the expression of inflammatory genes in a manner dependent on HA molecular weight, as evidenced by a growing number of reports [6], and several studies have indicated its close involvement in inflammatory arthritis development [7, 8]. High molecular weight (HMW) HA, produced by hyaluronan synthase 1 (HAS1) and HAS2, exerts anti-angiogenic, immunosuppressive, and anti-inflammatory properties [9, 10], whereas low-molecular-weight (LMW) HA, which is synthesized by HAS3, is usually secreted from cells both under physiological and pathological conditions [11]. This LMW-HA functions oppositely to HMW-HA, exerting proinflammatory, angiogenic, and immunostimulatory roles [12].…”