**5. The role of LSCs in tumor proliferation and anti-apoptosis**

Various signaling pathways that stimulate proliferation or inhibit apoptosis are known to aberrant activate LSCs.

#### **5.1 Hedgehog pathway**

The Hh pathway is a highly conserved pathway that regulates the proliferation, migration, and differentiation of cells during development [62, 63]. Three distinct ligands, namely Sonic (Shh), Indian (Ihh), and Desert (Dhh) Hedgehog, exist in humans. Upon ligand binding to the receptor pat (Ptch), inhibition of smoothened (Smo) receptor is relieved. Smo then activates members of the Gli family of zinc-finger transcription factors, translocating them to the nucleus to regulate the transcription of Hh target genes including Gli1, Gli2, and Ptch, and regulators of cell proliferation and survival [64–66].

The Hh pathway promotes cell proliferation mainly by regulating cell cycle. Its regulation mechanism is as follows [67]: (1) Cyclin D1 and cyclin D2 act as downstream target genes for the transcription factor GLI1 and are involved in cell cycle G1 to S phase transformation; (2) PTCH regulates the activity of cyclin B, which is part of the mitosis promoting factor (MPF) compound. MPF is required for cell entry from the G2 phase to the M phase; and (3) SMO proteins block cellular dormancy by modulating P21, a cyclin-dependent inhibitory protein.

The Hh signaling pathway regulates apoptosis mainly through the following mechanisms: (1) Regulate the activity of the BCL-2 family. The BCL-2 family is divided into anti-apoptotic proteins (such as BCL-2, BCL-XL, and MCL-1) and

pro-apoptotic proteins (such as BAX, BAD, and BAK). The ratio between the two types of proteins will directly affect the stability of the mitochondrial membrane and is the most important regulator of the mitochondrial apoptosis pathway. Overexpression of BCL-2, an increase in the ratio of BCL-2 to BAD, leading to defects in mitochondrial apoptosis, is one of the important mechanisms for LSC multidrug resistance and poor prognosis of AML [68]. BCL-2 is the target gene downstream of the Hh pathway, and Hh pathway blockers can induce apoptosis by downregulating BCL-2 [69]. Kobune et al. found that cyclopamine induces apoptosis of drug-resistant CD34+ AML cells by downregulating BCL-2 and makes them sensitive to Ara-C [70]. MCL1 has also emerged as a mechanism of resistance to apoptosis and to BCL-2/BCL-XL inhibitors, and therefore, it is considered as a potential therapeutic target [71]. (2) Regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apoptosis. TRAIL-R3 is a blank receptor that lacks a functional death region and is highly expressed in CD34+ CD38<sup>−</sup> LSCs; the downregulation of TRAIL-R3 increases apoptosis [72]. (3) Regulation of FAS protein express in the death receptor pathway. Studies have found that the Hh pathway inhibitor GDC-0449 promotes tumor stem cell apoptosis by upregulating FAS protein [73].
