PLSCRs are type II single pass transmembrane proteins initially identified as Ca²⁺-activated PL scramblases. Three other homologs hPLSCR2, hPLSCR3 and hPLSCR4 that exhibited 59, 47 and 46% sequence homology to hPLSCR1 respectively (Wiedmer et al., 2000). PLSCR1 is 37 kDa protein localizes either to PM or nucleus depending upon its state of palmitoylation (Wiedmer et al., 2003). PLSCR3 is localized exclusively to mitochondria and regulates its structure, function and apoptotic responses (Liu et al., 2003). Both PLSCR2 and PLSCR4 are localized to the nucleus and are involved in cell signaling mechanisms (Wiedmer et al., 2000; Sims et al., 2001). Subsequent finding revealed that PLSCRs are multi-functional proteins exhibiting both scrambling and non-scrambling activities. PLSCR1-knockout mice possessed normal scramblase activity, rather exhibited defective hematopoesis (Zhou et al., 2002). Scott syndrome patients, despite being deficient in platelet-scramblase activity, exhibited normal level of PLSCR1 expression in B lymphocytes (Zhou et al., 1998), suggesting the presence of alternative additional scramblases. Investigation revealed that TMEM16F (Ano6) a Ca²⁺-activated Cl⁻ channel that also exhibits PL scrambling in plasma membrane exhibits a homozygous null mutation in Scott Syndrome patients (Nagata et al., 2016). PLSCR1 enhanced granulocyte production in response to growth factors and interferon (IFNs) (Der et al., 1998; Zhou et al., 2002; Dong et al., 2004). These findings revealed PLSCR1 to be a novel signaling protein regulating cellular differentiation and apoptotic responses. Subsequent findings revealed that PLSCRs are onco-regulatory proteins associated with many cancer types such as leukemia and carcinomas.

The TMEM16 (anoctamin) family has 10 isoforms named ANO1 (TMEM16A) to ANO10 (TMEM16K) that were initially discovered as Ca²⁺-activated Cl⁻ ion channels. Subsequent analysis revealed that five members of anoctamin family: TMEM16C (ANO3), TMEM16D (ANO4), TMEM16F (ANO6), TMEM16G (ANO7) and TMEM16J (ANO9) exhibit Ca²⁺-dependent PL scrambling activity in addition to their role as Cl⁻ channel (Suzuki et al., 2013). However, both PL scrambling and Cl⁻ channel activities are independent of each other. In particular, TMEM16F (ANO6) is the major contributor to the process of PS translocation from the inner to the outer leaflet of the plasma membrane (Pedemonte and Galietta, 2014). Different paralogs of anoctamins are expressed during murine embryogenesis, indicating their role in embryonic development and cellular differentiation (Rock and Harfe, 2008). Growing set of evidences suggest that the members of TMEM16 family are over expressed in cancer cells that is associated with poor prognosis and cancer development (Katoh and Katoh, 2003).

Both in silico and in vivo analysis shows that c-Myc is a transcriptional regulator of PLSCR1 with a putative binding site located between −800 and −400 upstream of 5’ the flanking region of PLSCR1 (Vinnakota and Gummadi, 2016). Both PLSCR1 and PLSCR4 are down regulated at mRNA and protein level by a Snail transcription factor (Snail TF) that binds to the putative promoter regions of PLSCR1 [−1,525 to −1,244] and PLSCR4 [−1,521 to −1,516] respectively (Francis et al., 2014; Vinnakota and Gummadi, 2016). This finding demonstrates key regulatory role of PLSCRs in tumorigenesis.

Differentiation inducing chemicals such as Phorbol-12 myristate-13 acetate (PMA), a stimulator of PKCδ is known to enhance transcription of PLSCR1 and stimulates cellular differentiation (Andrews et al., 2002). All-trans retinoic acid (ATRA), elevates PLSCR1 expression in acute promyelocytic leukemic (APL), NB4 and HL60 cells but not in maturation-resistant NB4-LR1 cells (Zhao et al., 2004). ATRA in combination with MTA is shown to be a more robust inducer of PLSCR1 expression that regulates leukemic cell differentiation. In U937 and HT1080 leukemic cells, sequential activation of PKCδ and JNK stimulate phosphorylation of STAT1 at Ser⁷²⁷ that in turn activates transcription of PLSCR1 (Zhao et al., 2005).

Cancer cells exhibit an acidic cytoplasm and tumor cells exhibit an acidic microenvironment. Low pH (<6) leads to transcriptional down regulation of hPLSCR1 that correlates with decreased sensitivity of HEK293 to apoptosis. As the cytosol turns acidic in many malignant cell types, PLSCR1 might be a key regulatory protein of cancer progression (Francis and Gummadi, 2015). Similarly, TME exhibit a low oxygen condition that up regulates PLSCRs. PLSCR1 and 3 are over expressed during oxygen deprivation that negatively regulates cellular stress (Ostojic et al., 2013; Slone et al., 2015).

Sodium selenite enhances apoptosis in NB4 leukemic cells through IRF3-mediated upregulation of PLSCR1 (Shi et al., 2020). III10, a synthetic flavonoid induces differentiation of U937 leukemic cells by enhancing PLSCR1 synthesis (Qin et al., 2012). Paclitaxel, an established anticancer drug increases PS exposure in RBC that leads to blood coagulation and thrombosis. However, if it leads to over expression of any specific scramblase, remains to be determined (Kim et al., 2018). Synthetic enzyme made from eight DNA strands induces scrambling of ∼ 10⁷ lipids in biological membranes. The synthetic DNA scramblase led to rapid PS exposure in human breast cancer cells (MDA-MB-231) (Ohmann et al., 2018). Stimulation of PS exposure in malignant cells using an exogenous scramblase opens a novel anti-cancer therapeutic approach.

Woogonoside, a natural flavonoid from Panax ginseng induces differentiation of U937 and HL-60 leukemia cells by upregulating PLSCR1 (Chen et al., 2013). Woogonoside stimulates differentiation of primary AML cells through activation of PLSCR1/IP3R1/Ca²⁺ axis (Li M. et al., 2017). Similarly, resveratol, a polyphenol isolated from grape skin significantly reduces the cervical tumor through over expression of PLSCR1 (Zhao et al., 2019). Many natural anticancer drugs exhibit anticancer activity through over expression of PLSCR1. These compounds are potent drug leads or adjuvant therapeutics that act through inhibition of scramblases or enhance PS exposure on plasma membrane of cancer cells.

Cell shrinkage is an essential morphological feature of metastiatic tumor cells that enables their invasion into the tissue interstitial space (diapedesis) through narrow gaps of capillary endothelial cells. Anoctamins are the key regulators of cellular volume during cell shrinkage. Activation of TMEM16 members is triggered by increased cytosolic Ca²⁺ that activates Cl⁻ channel activity. Cl⁻ export is accompanied by loss of water as a mechanism of osmotic balance that results in shrinkage of the metastatic tumor cells (Sontheimer, 2008; Ruiz et al., 2012). As all the anoctamins exhibit Cl⁻ channel activity, their activation causes simultaneous cell shrinkage and (or) PS exposure on the cell surface. Polarized activation of TMEM16A in migrating cells allows their diapedesis (Sontheimer H. 2008). Change of shape in metastatic carcinoma cells is facilitated by TMEM16A-inducd cytoskeletal remodeling. TMEM16A associates with scaffolding proteins ezrin, radixin, moesin and RhoA that connect plasma membrane proteins to the cytoskeleton (Perez-Cornejo et al., 2012). TMEM16A interacts with various cell adhesion proteins such as zyxin, fibulin 1, S100A11, twinfilin and catenin that falilitates migration and attachment of metastatic tumor cells (Wanitchakool et al., 2014). siRNA-mediated down regulation of TMEM16F was shown to decrease ionophore-mediated release of TNF-Receptor 1 (TNFR1) in human umbilical vein endothelial cells (HUVECs) (Veit et al., 2018). Both TMEM16A and TMEM16F are expressed in Ehrlich Lettre ascites (ELA) cells that regulates cell migration. In metastatic ELA cells, TMEM16A and TMEM16F regulate the direction and rate of migration respectively (Jacobsen et al., 2013). TMEM16F-induced PE exposure on surface of migrating cells is a biomarker of migrating HELA cells which promotes cell migration and adhesion (Fan et al., 2012; Kato et al., 2013).

Microvesicles (MVs) are small, PM-derived membrane-enclosed entities with diameter 50–200 nm that are shedded by cells of multicellular organisms to transfer cellular components essential for paracrine signaling (Headland et al., 2015; Fujita et al., 2016). Both PLSCRs and TMEM16 regulate shedding of MVs through Ca²⁺-induced PS exposure. PLSCR1 is a positive regulator of MP release as the shedding is greatly reduced in cells deficient in expression of PLSCR1 (Gonzalez et al., 2009). PLSCR1 modulates release of MPs by regulating Ca²⁺ induced K⁺ efflux, PS exposure and enhanced membrane fluidity (Campbell et al., 2014). Both PLSCR1 and 3 are secreted through MVs of colorectal cancer cells that contain signaling machinery for colorectal tumorogenesis (Mathivanan et al., 2010). MPs from both hematological and non-hematological cancer cells carry transcripts of membrane vesiculation machinery, micro RNAs (miRNAs), surface antigens, proteins and genetic materials that re-templates recipient cells so as to reflect traits of the donor cell (Jaiswal et al., 2012).

The long spliced form of TMEM16F is targeted to plasma membrane that regulates PL scrambling, shedding of microvesicles and immune-signaling in TME. TMEM16F regulates the formation of a subset of MVs via PL scrambling. TMEM16G peptides were detected in patient-derived prostate-specific MVs (prostasomes) indicating their role in vesicle maturation and trafficking (Poliakov et al., 2009). During prostate cancer progression, prostasomes are secreted into stromal tissue, where they can support tumor growth and induce immune suppression (Lundholm et al., 2014). TMEM16G is immunogenic and TMEM16G-targeted T cells exhibit specificity against prostate cancer cells (Cereda et al., 2010). MVs spread the chemotherapy resistance among cancer cells. For instance, docetaxel chemotherapy resistance between cells spreads via MVs (Corcoran et al., 2012).

Scramblase-mediated PS exposure on surface of cancer cells is an essential cancer regulatory event that facilitates shedding of many membrane-attached proteins to the extracellular medium. The sheddase activity of disintegrin-metalloproteinase ADAM 17 and ADAM10 is regulated by PS exposure on cell surface (Bleibaum et al., 2019) (Figure 2). Ectodomains of transmembrane substrates including TGF-alpha, CD137, Amphiregulin (AREG) and tumor necrosis factor receptor (TNFR) are shed into the extracellular medium by TMEM16F. Proteins such as neuronal (N)-cadherin, epithelial (E)-cadherin, vascular-endothelial (VE)-cadherin, EGFR ligands, β-cellulin (BTC), EGF and the low affinity IgE receptor CD23 are secreted into the TME that enhances tumor survival, migration and apoptosis. Expression of constitutively active mutant of TMEM16F that leads to spontaneous PS exposure, led to increased release of AREG and TGF-alpha (Veit et al., 2018). Over expression of TMEM16F increases stimulated shedding of soluble CD137 (sCD137), a member of the TNF receptor family in patients with malignancy. ADAM10 and ADAM17 stimulate shedding of sCD137 and it’s ligand (LCD137) through their interaction with PS exposed on cell surface. A hyperactive ANO6 results in maximal constitutive shedding of CD137 that enhances T cell proliferation in TME (Figure 2). ADAM10 activation could not be induced in lymphocytes of Scott syndrome patients that harbor a missense mutation in TMEM16F. A putative PS-binding motif was identified in the conserved stalk region of ADAM10. Replacement of this motif resulted in strong reduction of sheddase activity (Bleibaum et al., 2019). Targeting of CD137/CD137L signaling pathway in tumor cells by targeting TMEM16F could lead to the development of novel immunotherapeutics against cancer (Seidel et al., 2021). In macrophages TMEM16F is necessary for phagocytosis stimulated by the ATP receptor P2X7 and microglia lacking TMEM16F demonstrate defects in process formation and phagocytosis (Ousingsawat et al., 2015; Batti et al., 2016).

Tumor survival and immune escape requires IFN-I and II triggered JAK-STAT signaling through expression of IFN-stimulated genes. PLSCRs regulate the INF-mediated immune-signaling leading to the immune escape of tumor cells. The transcriptional activator of IFN-induced genes is the cytosolic ISGF3 complex which is formed by dimerization of either two phosphorylated STATs (STAT1, STAT2 and STAT3) or binding of the dimer with a interferon regulatory factor (e.g. IRF9). PLSCR2 bind to STAT3 that attenuates the adaptive immunity against tumors (Yu et al., 2009; Kuchipudi, 2015). PLSCR1 binds to STAT2 to regulate the promoter activity ISGF3-mediated transcriptional activation (Tsai and Lee, 2018). PLSCR2 binds to the N-terminal domain of STAT3 that inhibits the STAT3-dependent promoter occupancy of ISGF3 complex leading to attenuation of it’s promoter activity (Tsai and Lee, 2018). Palmitoylation of PLSCR2 is essential for it’s STAT3 binding and blockade of the transcriptional activity (Tsai et al., 2019). STAT3 signaling has been widely linked to cancer cell survival, immune suppression and sustained inflammation in the TME. Hence, PLSCRs are the immune regulatory proteins that regulate tumor progression and survival through STAT-regulatory mechanism.

Ca²⁺ ionophores induce activation of TMEM16F in T cells that stimulates PS exposure that in turn, triggers large-scale cell surface membrane expansion. Continued stimulation of TMEM16F results in shedding of ectosomes that contain T-cell co-receptor PD1. Knocking down of TMEM16F resulted in inhibition of membrane expansion and triggered rapid endocytosis of PD1 upon increased cytosolic Ca²⁺. Presence of TMEM16F prevents PD1 endocytosis by facilitating Ca²⁺-induced lipid scrambling in plasma membrane and activating massive endocytosis (Hilgemann and Fine, 2011). PD-1 is a negative regulator of the immune system that is hijacked by various cancers to evade anti-tumour immune responses. TMEM16F deficiency results in high level expression of PD-1 on cell surface that results in their immune exhaustion (Hu et al., 2016). PD1 is an immune-check point that could be blocked by antibodies which are potent anti-cancer immune therapeutics (Pardoll, 2012). In HEK293T cells, over expression of TMEM16F led to increased Ca²⁺-mediated PS-exposure that was accompanied by enhanced release of amphiregulin (AREG) and TGF-alpha essential for T-cell activation (Figure 2).

Many phytochemicals exhibit anticancer properties through scrambling of plasma membrane lipids in cancer cells. The scrambled plasma membrane that exhibits PS exposure on cancer cell surface results in their apoptotic clearance. However, in solid tumors, the PS exposed on plasma membrane is a novel target for PS-binding anticancer molecules. While many phytochemicals and their synthetic conjugates are increasingly promoted as anti-cancer therapeutics, tumor cells exhibiting high PS exposure on cancer cell surface is immune targeted using high affinity PS-binding agents. Diverse plant compounds such as flavonoids, alkaloids, quinones, terpenoids, polyphenols and tannins exhibit PS exposure in malignant cells (Table 2).