IP₃ Receptor Biology and Endoplasmic Reticulum Calcium Dynamics in Cancer
癌症中的 IP ₃受体生物学和内质网钙动态

Abstract  抽象的

Intracellular Ca²⁺ signaling regulates a plethora of cellular functions. A central role in these processes is reserved for the inositol 1,4,5-trisphosphate receptor (IP₃R), a ubiquitously expressed Ca²⁺-release channel, mainly located in the endoplasmic reticulum (ER). Three IP₃R isoforms (IP₃R1, IP₃R2 and IP₃R3) exist, encoded respectively by ITPR1, ITPR2 and ITPR3. The proteins encoded by these genes are each about 2700 amino acids long and assemble into large tetrameric channels, which form the target of many regulatory proteins, including several tumor suppressors and oncogenes. Due to the important role of the IP₃Rs in cell function, their dysregulation is linked to multiple pathologies. In this review, we highlight the complex role of the IP₃R in cancer, as it participates in most of the so-called “hallmarks of cancer”. In particular, the IP₃R directly controls cell death and cell survival decisions via regulation of autophagy and apoptosis. Moreover, the IP₃R impacts cellular proliferation, migration and invasion. Typical examples of the role of the IP₃Rs in these various processes are discussed. The relative levels of the IP₃R isoforms expressed and their subcellular localization, e.g. at the ER-mitochondrial interface, is hereby important. Finally, evidence is provided about how the knowledge of the regulation of the IP₃R by tumor suppressors and oncogenes can be exploited to develop novel therapeutic approaches to fight cancer.
细胞内 Ca ²⁺信号传导调节多种细胞功能。在这些过程中，肌醇 1,4,5-三磷酸受体 (IP ₃ R) 发挥着核心作用，它是一种普遍表达的 Ca ²⁺释放通道，主要位于内质网 (ER) 中。存在三种IP ₃ R同种型(IP ₃ R1、IP ₃ R2和IP ₃ R3)，分别由ITPR1 、 ITPR2和ITPR3编码。这些基因编码的蛋白质各自长约 2700 个氨基酸，并组装成大的四聚体通道，形成许多调节蛋白的靶标，包括几种肿瘤抑制因子和癌基因。由于 IP ₃ R 在细胞功能中的重要作用，它们的失调与多种病理有关。在这篇综述中，我们强调了 IP ₃ R 在癌症中的复杂作用，因为它参与了大多数所谓的“癌症标志”。特别是，IP ₃ R通过调节自噬和细胞凋亡直接控制细胞死亡和细胞生存决策。此外，IP ₃ R 影响细胞增殖、迁移和侵袭。讨论了 IP ₃ R 在这些不同过程中的作用的典型示例。因此，表达的IP ₃ R 同工型的相对水平及其亚细胞定位（例如在ER-线粒体界面处）非常重要。最后，提供了关于如何利用肿瘤抑制因子和癌基因调节 IP ₃ R 的知识来开发抗癌新治疗方法的证据。

11.1 Introduction
11.1简介

Ca²⁺ is in all cell types a crucial intracellular messenger, controlling a plethora of cellular functions, including fertilization, differentiation, secretion, muscle contraction, synaptic plasticity and memory formation, gene transcription and metabolism (Berridge et al. 2000). Meanwhile it became clear that Ca²⁺ is also controlling cell proliferation (Humeau et al. 2018; Roderick and Cook 2008) and processes as macroautophagy (further named autophagy) and apoptosis (Dubois et al. 2016; Hu et al. 2019; La Rovere et al. 2016; Smaili et al. 2013) thereby directly affecting cell death and cell survival decisions. To perform this wide variety of functions, Ca²⁺ is encoded as complex spatio-temporal signals varying in frequency, amplitude, kinetics and subcellular localization which depend on the interplay between the members of the so-called “Ca²⁺-signaling toolkit”, a whole set of Ca²⁺-binding proteins, Ca²⁺ transporters and modulators of these proteins (Berridge et al. 2000; Bootman and Bultynck 2020).
Ca ²⁺在所有细胞类型中都是重要的细胞内信使，控制着大量的细胞功能，包括受精、分化、分泌、肌肉收缩、突触可塑性和记忆形成、基因转录和代谢(Berridge et al. 2000 )。同时，很明显Ca ²⁺还控制着细胞增殖（Humeau et al. 2018 ；Roderick and Cook 2008 ）以及巨自噬（进一步称为自噬）和细胞凋亡过程（Dubois et al. 2016 ；Hu et al. 2019 ；La Rovere 等人， 2016 ；Smaili 等人， 2013 ）从而直接影响细胞死亡和细胞存活。决定。为了执行这些广泛的功能，Ca ²⁺被编码为复杂的时空信号，其频率、幅度、动力学和亚细胞定位各不相同，这取决于所谓的“Ca ²⁺信号工具包”成员之间的相互作用、一整套 Ca ²⁺结合蛋白、Ca ²⁺转运蛋白和这些蛋白的调节剂（Berridge 等人2000 ；Bootman 和 Bultynck 2020 ）。

A central tenant hereby is the inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R), a ubiquitously expressed, intracellular Ca²⁺-release channel mainly localized on the endoplasmic reticulum (ER). This ER Ca²⁺ channel is activated by IP₃, which is produced from phosphatidylinositol 1,4-bisphosphate by various phospholipase C isoforms after cellular stimulation by e.g. hormones, growth factors or neurotransmitters (Foskett et al. 2007; Parys and De Smedt 2012; Parys and Vervliet 2020; Prole and Taylor 2019).
此处的中心租户是肌醇1,4,5-三磷酸(IP ₃ )受体(IP ₃ R)，一种普遍表达的细胞内Ca ²⁺释放通道，主要位于内质网(ER)上。该 ER Ca ²⁺通道由 IP ₃激活，IP 3 是在激素、生长因子或神经递质等细胞刺激后通过各种磷脂酶 C 同工型从磷脂酰肌醇 1,4-二磷酸产生的（Foskett 等人， 2007 年；Parys 和 De Smedt 2012 年） ；帕里斯和维利特2020 ；普罗尔和泰勒2019 ）。

Activation of the IP₃R leads to Ca²⁺ release from the ER into the cytoplasm, setting in motion various cellular signaling cascades. Depending on the cellular situation, the released Ca²⁺ can be funneled to other organelles, transported back into the ER or out of the cell. Meanwhile, the decreased Ca²⁺ load in the ER may lead to the dissociation of Ca²⁺ from the ER Ca²⁺ sensors Stim1/Stim2, leading to their oligomerisation and a rearrangement of their cytosolic domains. This enables the interaction of Stim1 and/or Stim2 with the plasma membrane Orai channels resulting in their opening (Lewis 2020; Zhou et al. 2019). The subsequent Ca²⁺ entry in the cell has a dual role, allowing a more prolonged Ca²⁺ signaling in the cell as well as a replenishment of the ER Ca²⁺ stores. As this form of Ca²⁺ entry primarily depends on the Ca²⁺-store content, this process is known as store-operated Ca²⁺ entry (SOCE).
IP ₃ R 的激活导致 Ca ²⁺从 ER 释放到细胞质中，从而启动各种细胞信号级联。根据细胞情况，释放的 Ca ²⁺可以输送到其他细胞器，运回内质网或运出细胞。同时，内质网中Ca ²⁺负载的减少可能导致Ca ²⁺从内质网Ca ²⁺传感器Stim1/Stim2 解离，导致它们的寡聚化和胞质结构域的重排。这使得 Stim1 和/或 Stim2 与质膜 Orai 通道相互作用，导致其开放（Lewis 2020 ；Zhou et al. 2019 ）。随后的 Ca ²⁺进入细胞具有双重作用，允许细胞内更长时间的 Ca ²⁺信号传导以及补充内质网 Ca ²⁺储存。由于这种形式的Ca ²⁺进入主要取决于Ca ²⁺储存内容，因此该过程被称为储存操作的Ca ²⁺进入(SOCE)。

11.2 IP₃R Structure-Function Relationship
11.2 IP _3R结构-功能关系

In higher organisms, there are three IP₃R isoforms (IP₃R1, IP₃R2, and IP₃R3), encoded by three different genes (ITPR1, ITPR2 and ITPR3) and further diversity is provided at the protein level by splicing events. The homology among the three isoforms is rather high (around 75% at the amino acid level) and the IP₃Rs form homo- or heterotetrameric complexes with each a total molecular mass of about 1.2 MDa (Foskett et al. 2007; Parys and De Smedt 2012).
在高等生物中，存在三种 IP ₃ R 同工型（IP ₃ R1、IP ₃ R2 和 IP ₃ R3），由三种不同的基因（ ITPR1 、 ITPR2和ITPR3 ）编码，并且通过剪接事件在蛋白质水平上提供了进一步的多样性。三种异构体之间的同源性相当高（氨基酸水平约为 75%），IP ₃ R 形成同源或异源四聚体复合物，每个复合物的总分子量约为 1.2 MDa（Foskett 等人， 2007 年；Parys 和 De）斯梅特2012 ）。

Each monomer has a length of about 2700 amino acids and can be divided in three large regions: at its N-terminal extremity the cytosolic ligand-binding region (for IP₃R1: amino acids 1–578), subsequently the large regulatory and coupling region (amino acids 579–2275), and finally the C-terminal part (amino acids 2276–2749) consisting of 6 transmembrane helices forming the ion channel proper, followed by a short cytosolic tail (Südhof et al. 1991) (Fig. 11.1).
每个单体的长度约为 2700 个氨基酸，可分为三个大区域：位于其 N 末端的胞质配体结合区域（对于 IP ₃ R1：氨基酸 1-578），随后是大的调节和偶联区域区域（氨基酸 579-2275），最后是 C 端部分（氨基酸 2276-2749），由 6 个跨膜螺旋组成，形成离子通道本身，后面是短的胞质尾（Südhof 等人， 1991 ）（图11.1 ）。

Fig. 11.1  图11.1

Oncogenes and tumor suppressors interact with and influence IP₃R function. Linear representation of a generic IP₃R showing the location of the ligand-binding region, the regulatory and coupling region and the C-terminal part. A scale in amino acids is also provided. The oncogenes (top panel) and tumor suppressors (bottom panel) listed in the Tables are depicted at the proposed site of their interaction with the IP₃R. Some of them have multiple interaction sites on the IP₃R, and are therefore depicted multiple times, the same color always denoting the same protein. From only some of those proteins the exact interaction sites are known at the amino acid level. These are (in alphabetical order): Bcl-2 in the regulatory and coupling region (amino acids 1389–1408 of IP₃R1), Bcl-X_L in the C-terminal part (amino acids 2571–2606 and 2690–2732), Bok (amino acids 1895–1903 of IP₃R1), FBXL2 (amino acids 545–566 of IP₃R3), and the PKB/Akt phosphorylation site (S²⁶⁸¹ of IP₃R1). In general, oncogenes stimulate pro-survival Ca²⁺ oscillations while inhibiting pro-apoptotic Ca²⁺ signaling. The latter can occur by direct inhibition of the IP₃R, by inducing post-translational modification of the IP₃R or by stimulating its degradation. Tumor suppressors in contrast will stimulate pro-apoptotic Ca²⁺ signaling by either sensitizing the IP₃R or by preventing its degradation. Of note, there are special situations in which a tumor suppressor may compete with an oncogene for binding to the IP₃R, e.g. PTEN and FBXL2, or can reverse post-translational modifications thereby negating the pro-survival effects of the oncogenes. IICR: IP₃-induced Ca²⁺ release
癌基因和肿瘤抑制因子与 IP ₃ R 功能相互作用并影响其功能。通用 IP ₃ R 的线性表示，显示配体结合区、调节和偶联区以及 C 末端部分的位置。还提供了氨基酸的比例。表中列出的癌基因（上图）和肿瘤抑制基因（下图）在它们与 IP ₃ R 相互作用的拟议位点处进行了描述。其中一些在 IP ₃ R 上具有多个相互作用位点，因此在多个位置进行了描述有时，相同的颜色总是表示相同的蛋白质。仅从其中一些蛋白质中，在氨基酸水平上已知确切的相互作用位点。它们是（按字母顺序排列）：调节和偶联区域中的 Bcl-2（IP ₃ R1 的氨基酸 1389-1408）、C 末端部分的 Bcl- _XL （氨基酸 2571-2606 和 2690-2732） , Bok (IP ₃ R1 的氨基酸 1895–1903), FBXL2 (IP 的氨基酸 545–566) ₃ R3) 和 PKB/Akt 磷酸化位点 (IP ₃ R1 的 S ²⁶⁸¹ )。一般来说，癌基因会刺激促存活的 Ca ²⁺振荡，同时抑制促凋亡的 Ca ²⁺信号传导。后者可以通过直接抑制 IP ₃ R、诱导 IP ₃ R 翻译后修饰或刺激其降解来发生。相反，肿瘤抑制因子会通过使 IP ₃ R 敏感或防止其降解来刺激促凋亡 Ca ²⁺信号传导。值得注意的是，在某些特殊情况下，肿瘤抑制因子可能会与癌基因竞争与 IP ₃ R 的结合，例如PTEN 和 FBXL2 可以逆转翻译后修饰，从而消除癌基因的促生存作用。IICR：IP ₃诱导的 Ca ²⁺释放

The ligand-binding region can functionally be divided in a suppressor (amino acids 1–225) and an IP₃-binding core (amino acids 226–578). The IP₃-binding core of each of the three IP₃R isoforms forms a cleft with very high affinity for IP₃ (K_d 2 nM), which however is to a variable extent affected by its respective upstream suppressor sequence. The rank order of eventual affinities of the IP₃Rs for IP₃ is therefore IP₃R2 (K_d 14 nM) > IP₃R1 (K_d 50 nM) > IP₃R3 (K_d 163 nM) (Iwai et al. 2007).
配体结合区在功能上可分为抑制子（氨基酸 1-225）和 IP ₃结合核心（氨基酸 226-578）。三种IP ₃ R 同种型中每一种的IP ₃结合核心形成对IP ₃具有非常高亲和力的裂口(K _d 2 nM)，然而，其在不同程度上受到其各自上游抑制序列的影响。因此，IP ₃ R 对 IP ₃的最终亲和力的排序是 IP ₃ R2 (K _d 14 nM) > IP ₃ R1 (K _d 50 nM) > IP ₃ R3 (K _d 163 nM) (Iwai et al. 2007 ）。

IP₃R channel opening needs the binding of IP₃ to each of the four IP₃R monomers (Alzayady et al. 2016). This activation of the channel must be fine-tuned and occurs via a multitude of mechanisms. The regulators can be divided in 3 broad classes, small soluble cytosolic factors (e.g. Ca²⁺, ATP, …) (Foskett et al. 2007; Taylor and Tovey 2010), regulatory proteins (Parys and Vervliet 2020; Prole and Taylor 2016) and post-translational modifications such as phosphorylation/dephosphorylation events (Vanderheyden et al. 2009).
IP ₃ R 通道开放需要将 IP ₃与四个 IP ₃ R 单体中的每一个结合（Alzayady 等人， 2016 ）。通道的激活必须进行微调，并通过多种机制进行。调节因子可分为 3 大类：小可溶性胞质因子（例如Ca ²⁺ 、 ATP 等）（Foskett et al. 2007 ；Taylor and Tovey 2010 ）、调节蛋白（Parys and Vervliet 2020 ；Prole and Taylor 2016 ）和翻译后修饰，例如磷酸化/去磷酸化事件（Vanderheyden 等，2017） 2009 ）。

The original analysis of the crystal structure of the N-terminal ligand-binding region (Bosanac et al. 2002, 2005), only quite recently extended to the full cytosolic part of the IP₃R (Hamada et al. 2017), has been complemented by high-quality cryo-electron microscopy (Taylor et al. 2004). The latter studies have tremendously increased our knowledge of the structure of the IP₃R.
对 N 端配体结合区域晶体结构的最初分析（Bosanac 等人， 2002 年， 2005 年），直到最近才扩展到 IP ₃ R 的完整胞质部分（Hamada 等人， 2017 年），辅以高质量冷冻电子显微镜（Taylor et al. 2004 ）。后面的研究极大地增加了我们对 IP ₃ R 结构的了解。

Presently, the view of the general structure of the IP₃R1 can be summarized as follows. The IP₃R1 has a mushroom-like structure with a height of 19 nm. High-resolution analysis of its closed form (no IP₃, no Ca²⁺ present) allowed the identification of ten domains: β-trefoil domains 1 and 2 (amino acids 1–436), followed by armadillo solenoid folds (ARM1–ARM3, amino acids 437–2192) with an α-helical domain between ARM1 and 2, the intervening lateral domain (amino acids 2193–2272), the transmembrane region with six transmembrane α-helices (amino acids 2273–2600), a linker domain (amino acids 2601–2680) and the C-terminal domain (amino acids 2681–2731) (Serysheva et al. 2017). The activation mechanism responsible for the channel opening in the presence of IP₃ however remains to be elucidated. Recent studies have provided additional information on the structure of IP₃R3 both in the presence and absence of IP₃ and/or Ca²⁺ (Paknejad and Hite 2018) and indicated in the regulatory and coupling region the presence of an auto-inhibitory peptide that can modulate the IP₃-binding site (Azumaya et al. 2020).
目前，IP ₃ R1的总体结构可以概括如下。 IP ₃ R1 具有蘑菇状结构，高度为 19 nm。对其封闭形式（不存在 IP ₃ ，不存在 Ca ²⁺ ）进行高分辨率分析，可以识别出 10 个结构域：β-三叶结构域 1 和 2（氨基酸 1–436），然后是犰狳螺线管折叠（ARM1–ARM3） ，氨基酸 437–2192），在 ARM1 和 2 之间有一个 α-螺旋结构域，中间的侧向结构域（氨基酸2193–2272），具有六个跨膜 α 螺旋（氨基酸 2273–2600）的跨膜区域，一个接头结构域（氨基酸 2601–2680）和 C 端结构域（氨基酸 2681–2731）（Serysheva 等人，2016）。 2017 ）。然而，在 IP ₃存在的情况下导致通道打开的激活机制仍有待阐明。最近的研究提供了有关 IP ₃ R3 在存在和不存在 IP ₃和/或 Ca ²⁺的情况下的结构的更多信息（Paknejad 和 Hite 2018 ），并表明在调节和偶联区域中存在自抑制肽可以调节 IP ₃结合位点（Azumaya 等人， 2020 ）。

Future analysis of the structure of the various IP₃R isoforms under different experimental conditions will allow to better understand the activation and regulation mechanisms of this important class of channels as well as the physiological function of the various isoforms.
未来对不同实验条件下各种IP ₃ R亚型的结构进行分析将有助于更好地了解这一类重要通道的激活和调节机制以及各种亚型的生理功能。

11.3 Cellular and Subcellular Localization of the IP₃R
11.3 IP ₃ R 的蜂窝和亚蜂窝定位

The expression pattern of the various IP₃R isoforms as well as their subcellular localization is strongly tissue-, cell type-, and differentiation stage-dependent (Vermassen et al. 2004). Although most cells express more than one IP₃R isoform, IP₃R1 is the most ubiquitous isoform and can be expressed at very high levels in some tissues as e.g. Purkinje neurons and oocytes. IP₃R2 has a quite restricted expression pattern and is especially present in hepatocytes, glial cells, cardiomyocytes, various types of secretory cells as well as in some hematological cells (Vervloessem et al. 2015). IP₃R3 levels are generally elevated in rapidly proliferating cells, as well as, together with IP₃R2, in secretory cells (Ivanova et al. 2014).
各种IP ₃ R 同工型的表达模式及其亚细胞定位强烈依赖于组织、细胞类型和分化阶段（Vermassen 等人， 2004 ）。尽管大多数细胞表达不止一种IP ₃ R 同工型，但IP ₃ R1 是最普遍存在的同工型，并且可以在一些组织（例如浦肯野神经元和卵母细胞）中以非常高的水平表达。 IP ₃ R2 的表达模式相当有限，尤其存在于肝细胞、神经胶质细胞、心肌细胞、各种类型的分泌细胞以及一些血液细胞中（Vervloessem 等人， 2015 ）。 IP ₃ R3 水平在快速增殖的细胞中通常升高，并且与 IP ₃ R2 一起在分泌细胞中升高（Ivanova 等人， 2014 ）。

The fact that IP₃Rs can be expressed at well-determined subcellular localizations strongly affects their function (Alonso and Garcia-Sancho 2011; Hohendanner et al. 2014; La Rovere et al. 2016; Lock et al. 2019). Examples of very specific subcellular localization of IP₃Rs are at the ER-mitochondrial contact sites (Csordas et al. 1999; Rizzuto et al. 1993; Szabadkai et al. 2006), in the junctional ER close to the plasma membrane (Okeke et al. 2016; Thillaiappan et al. 2017) as well as at contact sites between ER and lysosomes (Atakpa et al. 2018; Kilpatrick et al. 2013; Lopez-Sanjurjo et al. 2013; Morgan et al. 2013).
IP ₃ R 可以在明确的亚细胞定位上表达，这一事实强烈影响其功能（Alonso 和 Garcia-Sancho 2011 ；Hohendanner 等人2014 ；La Rovere 等人2016 ；Lock 等人2019 ）。 IP ₃ R 非常具体的亚细胞定位的例子是在 ER 线粒体接触位点（Csordas 等人， 1999 ；Rizzuto 等人， 1993 ；Szabadkai 等人， 2006 ），在靠近质膜的连接 ER 中（Okeke 等人）等人2016 ；Thillaiappan 等人2017 ）以及联系网站ER 和溶酶体之间的关系（Atakpa 等人， 2018 年；Kilpatrick 等人， 2013 年；Lopez-Sanjurjo 等人， 2013 年；Morgan 等人， 2013 年）。

11.4 Intracellular Ca²⁺ Signaling and Cancer
11.4细胞内Ca ²⁺信号传导与癌症

As IP₃Rs control a plethora of cellular and physiological functions and processes, IP₃R dysregulation (Berridge 2016) and ITPR mutations (Hisatsune and Mikoshiba 2017; Kerkhofs et al. 2018; Terry et al. 2018; Gambardella et al. 2020) have patho(physio) logical consequences. While a number of those defects correlate with one specific disease situation (e.g. the occurrence of spinocerebellar ataxia or anhidrosis), this review will demonstrate that the role of the IP₃R in cancer is more complex and so illustrate the multifaceted aspects of IP₃R-mediated Ca²⁺ signaling.
由于 IP ₃ R 控制大量的细胞和生理功能和过程，IP ₃ R 失调（Berridge 2016 ）和ITPR突变（Hisatsune 和 Mikoshiba 2017 ；Kerkhofs 等人2018 ；Terry 等人2018 ；Gambardella 等人2020 ）具有病理（生理）逻辑后果。虽然其中许多缺陷与一种特定的疾病情况相关（例如脊髓小脑共济失调或无汗症的发生），但本综述将证明 IP ₃ R 在癌症中的作用更为复杂，因此说明了 IP ₃ R 的多方面-介导的Ca ²⁺信号传导。

Hanahan and Weinberg (Hanahan and Weinberg 2011) have put forward the concept that tumor formation depends on the acquisition of the so-called “hallmarks of cancer”. These are the sum of the capabilities of the cells to sustain proliferation, evade growth suppressors, resist to cell death, possess replicative immortality, stimulate angiogenesis, invasion and metastasis, reprogram energy metabolism and evade the immune response. Interestingly, in most of these processes, Ca²⁺ can play a role (Monteith et al. 2017; Prevarskaya et al. 2018; Roberts-Thomson et al. 2019; Roderick and Cook 2008), and in various cancers a changed expression pattern was found for members of the Ca²⁺-signaling toolkit, including the IP₃R, as was recently reviewed (Pierro et al. 2019). Modulation of IP₃R function in cancer cells can be attributed to one or several of the following factors: mutations, change in expression levels, change in subcellular localization or changes in Ca²⁺-release activity (due to post-translational modifications, interaction with regulatory proteins or protein degradation). Especially noteworthy is that various recognized oncogenes and tumor suppressors directly interact with the IP₃Rs, thereby regulating IP₃R activity or stability (Bittremieux et al. 2016). For all these reasons, members of the Ca²⁺-signaling toolkit have been proposed as novel therapeutic targets (Bong and Monteith 2018; Cui et al. 2017).
哈纳汉和温伯格（Hanahan and Weinberg 2011 ）提出了肿瘤形成取决于所谓“癌症标志”的获得的概念。这些是细胞维持增殖、逃避生长抑制剂、抵抗细胞死亡、具有复制永生性、刺激血管生成、侵袭和转移、重新编程能量代谢和逃避免疫反应的能力的总和。有趣的是，在大多数这些过程中，Ca ²⁺都可以发挥作用（Monteith et al. 2017 ；Prevarskaya et al. 2018 ；Roberts-Thomson et al. 2019 ；Roderick and Cook 2008 ），并且在各种癌症中，表达模式发生了变化发现 Ca ²⁺信号工具包的成员，包括 IP ₃ R，正如最近审查的那样（Pierro 等人）等2019 ）。癌细胞中 IP ₃ R 功能的调节可归因于以下一种或多种因素：突变、表达水平的变化、亚细胞定位的变化或 Ca ²⁺释放活性的变化（由于翻译后修饰、相互作用）与调节蛋白或蛋白质降解）。尤其值得注意的是，各种公认的癌基因和抑癌基因直接与IP ₃ R相互作用，从而调节IP ₃ R活性或稳定性(Bittremieux et al. 2016 )。由于所有这些原因，Ca ²⁺信号传导工具包的成员已被提议作为新的治疗靶点（Bong 和 Monteith 2018 ；Cui 等人2017 ）。

In this review, we will start by explaining the role of IP₃R signaling in autophagy and apoptosis as these processes are critical for cancer development. Next, the complex role that IP₃Rs can play during the various phases of cancer development will be discussed. Finally, we will highlight the interrelation that exist between the IP₃R and a number of tumor suppressors and oncogenes and how these interrelations can be exploited for therapeutic purposes.
在这篇综述中，我们将首先解释 IP ₃ R 信号传导在自噬和细胞凋亡中的作用，因为这些过程对于癌症的发展至关重要。接下来，将讨论 IP ₃ R 在癌症发展的各个阶段中发挥的复杂作用。最后，我们将强调 IP ₃ R 与许多肿瘤抑制因子和癌基因之间存在的相互关系，以及如何利用这些相互关系用于治疗目的。

11.5 The IP₃R as Regulator of Autophagy
11.5 IP ₃ R作为自噬调节剂

To support cellular homeostasis and to protect themselves from cellular stress, cells make use of a process called autophagy. In this process, long-lived proteins as well as dysfunctional organelles and intracellular pathogens are engulfed by newly formed double-membrane vesicles named autophagosomes. The latter will eventually fuse with lysosomes, forming autolysosomes in which both the autophagosomes and their content are fully degraded, after which the materials can be recycled. Autophagy forms a multi-step process that can be activated by adenosine monophosphate-activated protein kinase (AMPK), inhibited by the mechanistic target of rapamycin complex 1 (mTORC1) and in which over 30 autophagy-related (ATG) proteins play crucial roles (Ravikumar et al. 2010).
为了支持细胞稳态并保护自身免受细胞应激，细胞利用一种称为自噬的过程。在此过程中，长寿的蛋白质以及功能失调的细胞器和细胞内病原体被新形成的称为自噬体的双膜囊泡吞噬。后者最终会与溶酶体融合，形成自噬溶酶体，其中自噬体及其内容物均被完全降解，之后材料可以回收利用。自噬形成一个多步骤过程，可以被腺苷单磷酸激活蛋白激酶 (AMPK) 激活，并被雷帕霉素复合物 1 (mTORC1) 的机制靶标抑制，其中超过 30 种自噬相关 (ATG) 蛋白发挥着至关重要的作用。拉维库马尔等人， 2010 ）。

As autophagy has essentially a protective function, it can play a dual role in cancer. Initially, by supporting cellular homeostasis and thus antagonizing cellular stress, autophagy will protect cells from tumorigenicity, but once tumorigenesis has started it similarly will protect the tumoral cells from the harsh conditions in the tumor microenvironment (Eskelinen 2011; Liu and Ryan 2012). Due to the oncosuppressive role of several ATG proteins, including Beclin 1 (Table 11.1), these are considered as tumor suppressors (Yue et al. 2003).
由于自噬本质上具有保护功能，因此它可以在癌症中发挥双重作用。最初，通过支持细胞稳态并从而对抗细胞应激，自噬将保护细胞免受致瘤性的影响，但一旦肿瘤发生开始，它同样会保护肿瘤细胞免受肿瘤微环境中恶劣条件的影响（Eskelinen 2011 ；Liu 和 Ryan 2012 ）。由于包括 Beclin 1 在内的几种 ATG 蛋白的肿瘤抑制作用（表11.1 ），这些蛋白被认为是肿瘤抑制因子（Yue 等人， 2003 ）。

Table 11.1 List of tumor suppressors interacting with the IP₃R and their effects
表 11.1 与 IP ₃ R 相互作用的肿瘤抑制因子列表及其作用

Although the first effects of Ca²⁺ on autophagy have been described more than 2 decades ago (Gordon et al. 1993), the exact role(s) of Ca²⁺ signaling in the regulation of autophagy is only now slowly emerging. The main reason for this are the various, apparently contradictory results that have been published about the role of Ca²⁺, as has been reviewed (Decuypere et al. 2011a; Sun et al. 2016). The reasons for these controversies are multiple, and can be due to cell-type dependent context, the occurrence of various Ca²⁺-sensitive steps and the use of non-physiological conditions or of drugs with pleiotropic effects in a number of studies (Bootman et al. 2018).
尽管 Ca ²⁺对自噬的首次影响已在 20 多年前被描述过（Gordon 等人， 1993 ），但 Ca ²⁺信号传导在自噬调节中的确切作用现在才慢慢显现。其主要原因是已发表的有关 Ca ²⁺作用的各种明显相互矛盾的结果，正如已综述的那样（Decuypere 等人， 2011a ；Sun 等人， 2016 ）。这些争议的原因是多方面的，可能是由于细胞类型依赖性背景、各种 Ca ²⁺敏感步骤的发生以及在许多研究中使用非生理条件或具有多效作用的药物（Bootman等2018 ）。

Also the role of the IP₃R itself in autophagy is complex (Parys et al. 2012). On the one hand, since the IP₃R controls mitochondrial [Ca²⁺] and therefore mitochondrial metabolism (vide infra), it is clear that when Ca²⁺ transfer from the ER to the mitochondria is suppressed, ATP production drops, the AMP/ATP ratio increases and AMPK is activated. This increase in AMPK activity will lead to autophagy induction (Cardenas et al. 2010). Therefore, in basal conditions, the IP₃R acts as an inhibitor of autophagy. On the other hand, intracellular Ca²⁺ signals occurring in the cytosol can affect autophagy in a different way, whereby IP₃R-mediated Ca²⁺ release stimulates the autophagy process (Høyer-Hansen et al. 2007; Lam et al. 2008; Liu et al. 2013; Luyten et al. 2017; Wang et al. 2008). At the molecular level, IP₃Rs are directly sensitized by the pro-autophagic protein Beclin 1, driving starvation-induced autophagic flux (Decuypere et al. 2011b). Whether Beclin 1 recruitment by IP₃Rs and subsequent IP₃R sensitization also occurs in other authophagy-inducing conditions remains currently not known.
IP ₃ R 本身在自噬中的作用也很复杂 (Parys et al. 2012 )。一方面，由于 IP ₃ R 控制线粒体 [Ca ²⁺ ]，从而控制线粒体代谢（参见下文），很明显，当 Ca ²⁺从 ER 到线粒体的转移受到抑制时，ATP 产量下降，AMP /ATP 比率增加并且 AMPK 被激活。 AMPK 活性的增加将导致自噬诱导（Cardenas et al. 2010 ）。因此，在基础条件下，IP ₃ R 充当自噬抑制剂。另一方面，细胞质中发生的细胞内 Ca ²⁺信号可以以不同的方式影响自噬，其中 IP ₃ R 介导的 Ca ²⁺释放刺激自噬过程（Høyer-Hansen 等人， 2007 年；Lam 等人， 2008 年）刘等人， 2013 ；在分子水平上，IP ₃ Rs 直接被促自噬蛋白 Beclin 1 敏化，驱动饥饿诱导的自噬流（Decuypere 等人， 2011b ）。目前尚不清楚 IP ₃ R 招募 Beclin 1 以及随后的 IP ₃ R 致敏是否也发生在其他自噬诱导条件下。

Therefore, the IP₃R, via its actions on autophagy, can also affect cancer cells (Kania et al. 2017). Interestingly, in contrast to other cells, cancer cells appear extremely sensitive to conditions in which the Ca²⁺ transfer to the mitochondria is inhibited. Autophagy induction does not guarantee their survival as the cancer cells, in spite of mitochondrial function decline, do not slow down their proliferation rate and thus undergo a necrotic collapse during cytokinesis (Cardenas et al. 2016).
因此，IP ₃ R 通过其对自噬的作用，也可以影响癌细胞 (Kania et al. 2017 )。有趣的是，与其他细胞相比，癌细胞似乎对 Ca ²⁺向线粒体转移受到抑制的条件极其敏感。自噬诱导并不能保证它们的存活，因为尽管线粒体功能下降，癌细胞也不会减慢其增殖速度，从而在胞质分裂期间经历坏死性崩溃（Cardenas et al. 2016 ）。

A specific increase in both IP₃R2 and IP₃R3 expression levels in breast tumor tissues relative to the adjacent non-tumoral tissue was observed in patients (Singh et al. 2017b). These increased IP₃R expression levels correlated with metabolic changes as a higher rate of glycolysis and an increased glutaminolysis. IP₃R inhibition or siRNA-mediated silencing of IP₃R2 and -3 in breast cancer cell models affected exactly those same pathways, showing an association between IP₃R levels and the dysregulated metabolism of cancer cells. Interestingly, and similar to the study by Cardenas et al. mentioned above, the inhibition of IP₃Rs in breast cancer cells induced autophagy but the compromised energetics together with reactive oxygen species (ROS) production led to their demise (Singh et al. 2017a). Conversely, in clear cell renal cell carcinomas the increased hypoxia-inducible factor 2α level leads to IP₃R1 overexpression and autophagy induction (Messai et al. 2014). This autophagy induction protects the cancer cells against lysis mediated by natural killer cells.
在患者中观察到乳腺肿瘤组织中 IP ₃ R2 和 IP ₃ R3 表达水平相对于邻近非肿瘤组织的特异性增加（Singh et al. 2017b ）。这些增加的 IP ₃ R 表达水平与代谢变化相关，如更高的糖酵解速率和增加的谷氨酰胺分解。在乳腺癌细胞模型中，IP ₃ R 抑制或 siRNA 介导的 IP ₃ R2 和 -3 沉默会影响完全相同的途径，这表明 IP ₃ R 水平与癌细胞代谢失调之间存在关联。有趣的是，与卡德纳斯等人的研究相似。如上所述，乳腺癌细胞中 IP ₃ R 的抑制会诱导自噬，但能量学受损以及活性氧 (ROS) 的产生导致癌细胞死亡 (Singh et al. 2017a )。相反，在透明细胞肾细胞癌中，缺氧诱导因子 2α 水平增加导致 IP ₃ R1 过度表达和自噬诱导 (Messai et al. 2014 )。这种自噬诱导可以保护癌细胞免受自然杀伤细胞介导的裂解。

11.6 The IP₃R in Apoptosis
11.6细胞凋亡中的IP ₃ R

An important hallmark of cancer is the avoidance of cell death and cancer cells therefore often have upregulated anti-apoptotic mechanisms. Apoptosis can originate as the consequence of perturbations in the intracellular milieu (intrinsic apoptosis) or due to the presence of extracellular factors (extrinsic apoptosis) (Galluzzi and Vanpouille-Box 2018).
癌症的一个重要标志是避免细胞死亡，因此癌细胞通常具有上调的抗凋亡机制。细胞凋亡可能源于细胞内环境扰动（内在细胞凋亡）或细胞外因素的存在（外在细胞凋亡）（Galluzzi 和 Vanpouille-Box 2018 ）。

In intrinsic apoptosis, the crucial step is the large structural changes occurring at the level of the mitochondria, called mitochondrial outer membrane permeabilization (MOMP), a process that occurs upon Bax/Bak activation and that is mediated by proteinaceous channel complexes formed by Bax/Bak oligomers (Kale et al. 2018; Singh et al. 2019). MOMP results in the release of pro-apoptotic factors as cytochrome c and second mitochondria-derived activator of caspases (SMAC) from the mitochondrial intermembrane space into the cytosol. Here, cytochrome c forms a complex with apoptotic protease-activating factor 1 (APAF1) forming the apoptosome, leading to subsequent activation of caspase 9 and of the executioner caspases 3 and 7 and thus apoptosis (Galluzzi and Vanpouille-Box 2018). SMAC supports these processes by inhibiting X-linked inhibitor of apoptosis protein (XIAP), a factor that blocks caspase activation.
在内源性细胞凋亡中，关键步骤是发生在线粒体水平的大结构变化，称为线粒体外膜透化 (MOMP)，该过程发生在 Bax/Bak 激活时，由 Bax/Bak 形成的蛋白质通道复合物介导。 Bak 低聚物（Kale 等人， 2018 年；Singh 等人， 2019 年）。 MOMP 导致促凋亡因子作为细胞色素 c 和第二种线粒体衍生的半胱天冬酶激活剂 (SMAC) 从线粒体膜间隙释放到细胞质中。在此，细胞色素 c 与凋亡蛋白酶激活因子 1 (APAF1) 形成复合物，形成凋亡体，导致随后激活 caspase 9 以及刽子手 caspase 3 和 7，从而导致细胞凋亡 (Galluzzi 和 Vanpouille-Box 2018 )。 SMAC 通过抑制 X 连锁凋亡蛋白抑制剂 (XIAP)（一种阻止 caspase 激活的因子）来支持这些过程。

It is known that IP₃Rs play a crucial role in the various processes linked to cellular survival or cell death. These properties are linked to the fact that (a fraction of the) IP₃Rs are localized at ER-mitochondrial contact sites and control therefore the transfer of Ca²⁺ from the ER to the mitochondria (Csordas et al. 1999; Giacomello et al. 2010; Rizzuto et al. 1993). If a too high level of Ca²⁺ transfer occurs from the ER to the mitochondria, mitochondrial Ca²⁺ overload follows, which leads to the opening of the mitochondrial permeability transition pore (mPTP) in the inner mitochondrial membrane. The molecular nature of the mPTP is still controversial (Carroll et al. 2019; Urbani et al. 2019) and the link between mitochondrial Ca²⁺ overload and mPTP opening is also not completely understood. The latter can involve Ca²⁺ binding to cardiolipin leading to the disintegration of mitochondrial complex II and a massive production of ROS that triggers mPTP opening (Hwang et al. 2014) and/or a Ca²⁺-mediated conformational change of the F-ATP synthase which was proposed to form an essential component of the mPTP (Giorgio et al. 2017). Independently of the exact mechanism, the opening of the mPTP corresponds to the opening of a large, non-specific channel in the inner mitochondrial membrane, and consequently depolarization of the mitochondria, ATP hydrolysis, matrix swelling due to osmotic forces, and ultimately rupture of the outer mitochondrial membrane and the release of the above-mentioned pro-apoptotic factors.
众所周知，IP ₃ R 在与细胞存活或细胞死亡相关的各种过程中发挥着至关重要的作用。这些特性与以下事实有关：（一部分）IP ₃ R 位于 ER 线粒体接触位点，因此控制 Ca ²⁺从 ER 转移到线粒体（Csordas 等人， 1999 年；Giacomello 等人） 2010 ；里祖托等人。如果从内质网到线粒体的 Ca ²⁺转移水平过高，则会发生线粒体 Ca ²⁺超载，从而导致线粒体内膜中的线粒体通透性转换孔 (mPTP) 打开。 mPTP 的分子性质仍然存在争议（Carroll et al. 2019 ；Urbani et al. 2019 ），线粒体 Ca ²⁺超载与 mPTP 打开之间的联系也尚未完全了解。后者可能涉及 Ca ²⁺与心磷脂结合，导致线粒体复合物 II 解体，并大量产生 ROS，从而触发 mPTP 打开（Hwang 等人， 2014 ）和/或 Ca ²⁺介导的 F- 构象变化。 ATP 合酶被认为是 mPTP 的重要组成部分（Giorgio et al. 2017 ）。与确切的机制无关，mPTP 的打开对应于线粒体内膜中大的非特异性通道的打开，从而导致线粒体去极化、ATP 水解、渗透力导致的基质膨胀，以及最终线粒体的破裂。线粒体外膜和上述促凋亡因子的释放。

Although originally the idea prevailed that IP₃R3 was particularly expressed at the ER-mitochondria contact sites and therefore most important for triggering Ca²⁺-dependent apoptosis (Blackshaw et al. 2000; Mendes et al. 2005), other evidence also pointed to an important role in these processes for IP₃R1 (Feriod et al. 2017; Szabadkai et al. 2006) or IP₃R2 (Akl et al. 2013), suggesting a cell-type rather than a function-specific isoform distribution. Recent work extended these findings by conclusively demonstrating in cell line models that all three IP₃R isoforms can structurally participate in the ER-mitochondria contacts and that all three can flux Ca²⁺ to the mitochondria (Bartok et al. 2019). IP₃R2 appeared in fact the most effective for this process followed by IP₃R3 and IP₃R1. The latter was however the most effective inducer of SOCE leading to a sustained mitochondrial Ca²⁺ uptake.
尽管最初普遍认为 IP ₃ R3 在 ER-线粒体接触位点特别表达，因此对于触发 Ca ²⁺依赖性细胞凋亡最为重要（Blackshaw 等人， 2000 年；Mendes 等人， 2005 年），但其他证据也指出IP ₃ R1（Feriod 等人， 2017 年；Szabadkai 等人， 2006 年）或 IP ₃在这些过程中发挥着重要作用R2（Akl 等人， 2013 ），表明细胞类型而不是功能特异性亚型分布。最近的工作扩展了这些发现，在细胞系模型中最终证明所有三种 IP ₃ R 同工型都可以在结构上参与 ER-线粒体接触，并且所有三种都可以将 Ca ²⁺流动到线粒体（Bartok 等人， 2019 ）。事实上，IP ₃ R2 对于该过程似乎是最有效的，其次是 IP ₃ R3 和 IP ₃ R1。然而，后者是导致线粒体Ca ²⁺持续摄取的最有效的SOCE 诱导剂。

The most important factor determining apoptosis sensitivity will therefore rather be the amplitude of the IP₃R-mediated Ca²⁺ transfer between ER and mitochondria than which IP₃R isoform is present at the ER-mitochondria contact sites. Interestingly, it was shown that cytochrome c, which is released from the mitochondria after MOMP, not only induces apoptosome formation, but also interacts with the IP₃R. This interaction antagonizes Ca²⁺-mediated inhibition of the IP₃R and thus amplifies pro-apoptotic Ca²⁺ signaling (Boehning et al. 2003).
因此，决定细胞凋亡敏感性的最重要因素将是内质网和线粒体之间IP ₃ R介导的Ca ²⁺转移的幅度，而不是内质网-线粒体接触位点处存在的IP ₃ R亚型。有趣的是，MOMP 后从线粒体释放的细胞色素 c 不仅诱导凋亡体形成，而且与 IP ₃ R 相互作用。这种相互作用拮抗 Ca ²⁺介导的 IP ₃ R 抑制，从而放大促凋亡 Ca ²⁺信号传导（Boehning et al. 2003 ）。

Extrinsic apoptosis is initiated by extracellular factors interacting with the so-called death receptors at the plasma membrane as the FAS and TRAIL receptors. Binding of respectively FAS ligand and TRAIL leads to a conformational change that allows for the formation of an intracellular “death-inducing signaling complex” leading eventually to activation of the initiator caspases 8 and 10. These caspases can either directly activate the effector caspases and/or activate them via cleaving the pro-apoptotic protein Bid thus triggering MOMP and apoptosome formation. This shows a convergence of the extrinsic and the intrinsic pathways (Barnhart et al. 2003; Scaffidi et al. 1998). Interestingly, it was shown that in some cells, including Jurkat T cells and hepatocytes, both phospholipase C γ1 and IP₃R1 were needed for Fas-mediated apoptosis (Wozniak et al. 2006). Moreover, Fas-dependent killing of Jurkat T cells by colon cancer cells depends on IP₃R-mediated Ca²⁺ release, which in turn is amplified by cytochrome c release (Steinmann et al. 2008). Apart from the mitochondrial Ca²⁺ overload that leads to a generalized MOMP and apoptosis, it has been proposed that IP₃R-mediated Ca²⁺ release also stimulates apoptosis by supporting lipidation of various proteins including the IP₃R itself (Fredericks et al. 2014), various components of the T cell receptor complex and proteins of the Wnt signaling pathway (Chen and Boehning 2017).
外源性细胞凋亡是由细胞外因子与质膜上所谓的死亡受体（如 FAS 和 TRAIL 受体）相互作用引发的。 FAS 配体和 TRAIL 的结合分别导致构象变化，从而形成细胞内“死亡诱导信号复合物”，最终导致启动子 caspase 8 和 10 的激活。这些 caspase 可以直接激活效应 caspase 和/或者通过裂解促凋亡蛋白 Bid 来激活它们，从而触发 MOMP 和凋亡体形成。这显示了外在途径和内在途径的融合（Barnhart 等人， 2003 年；Scaffidi 等人， 1998 年）。有趣的是，研究表明，在一些细胞中，包括 Jurkat T 细胞和肝细胞，Fas 介导的细胞凋亡需要磷脂酶 C γ1 和 IP ₃ R1 (Wozniak et al. 2006 )。此外，结肠癌细胞对Jurkat T细胞的Fas依赖性杀伤依赖于IP ₃ R介导的Ca ²⁺释放，而Ca 2+ 释放又被细胞色素c释放放大(Steinmann et al. 2008 )。除了导致普遍 MOMP 和细胞凋亡的线粒体 Ca ²⁺超载之外，有人提出 IP ₃ R 介导的 Ca ²⁺释放还通过支持包括 IP ₃ R 本身在内的各种蛋白质的脂化来刺激细胞凋亡（Fredericks 等） . 2014 )、T 细胞受体复合物的各种成分和 Wnt 信号通路的蛋白质 (Chen 和 Boehning 2017 )。

11.7 The Complex Role of the IP₃R in Tumorigenesis: An Integrated View
11.7 IP ₃ R 在肿瘤发生中的复杂作用：综合观点

Tumorigenesis is typically a multi-step process. After oncogenic transformation, a first characteristic of tumor growth is the acquired capacity for quasi-unlimited cellular proliferation. The metastasis of the tumor subsequently depends on the migration capabilities of the cells followed by the invasion of other tissues. As will be discussed below, IP₃Rs actively participate in these key processes of tumorigenesis in various cancer types. The role of the IP₃R has particularly been investigated in breast and colorectal cancer and these cancer types will be discussed in more detail.
肿瘤发生通常是一个多步骤的过程。致癌转化后，肿瘤生长的第一个特征是获得准无限细胞增殖的能力。肿瘤的转移随后取决于细胞的迁移能力以及随后侵袭其他组织的能力。正如下面将要讨论的，IP ₃ Rs 积极参与各种癌症类型中肿瘤发生的这些关键过程。 IP ₃ R 的作用在乳腺癌和结直肠癌中得到了特别研究，这些癌症类型将得到更详细的讨论。

11.7.1 Breast Cancer
11.7.1乳腺癌

The growth stimulation of the MCF-7 breast cancer cell line by 17β-estradiol is coupled to an increased expression of IP₃R3 and a corresponding change in intracellular Ca²⁺ signaling (Szatkowski et al. 2010). siRNA-mediated IP₃R3 down-regulation modified ATP-evoked Ca²⁺ signals from sustained to oscillatory and antagonized the growth-promoting effect of 17β-estradiol, supporting a role for IP₃R3 in proliferation. This latter effect appeared to depend on a direct interaction between ER-localized IP₃R3 and the plasma membrane large conductance Ca²⁺-activated K⁺ (BK_Ca) channels (Mound et al. 2013). This coupling allows IP₃R3 to activate the BK_Ca channels leading to cellular hyperpolarization. Down-regulation in MCF-7 cells of either IP₃R3 or BK_Ca (or of both) led to a decrease in cyclin D1 and in cyclin-dependent kinase 4 levels and to cell cycle arrest in the G0/G1 phase. Furthermore, when comparing the low-migrating MCF-7 and the highly migrating and invasive MDA-MB-231 and MDA-MB-435S cell lines, it appears that specifically a higher expression level of IP₃R3 correlates with a stronger migration capacity that can be reversed by IP₃R3 down-regulation or amplified by IP₃R3 overexpression (Mound et al. 2017). This property could be linked to the fact that the Ca²⁺ signals evoked by IP₃R3 affect the cellular morphology by impacting profilin levels and therefore the actin cytoskeleton (Vautrin-Glabik et al. 2018).
17β-雌二醇对 MCF-7 乳腺癌细胞系的生长刺激与 IP ₃ R3 表达的增加以及细胞内 Ca ²⁺信号传导的相应变化有关（Szatkowski 等人， 2010 ）。 siRNA 介导的 IP ₃ R3 下调将 ATP 诱发的 Ca ²⁺信号从持续变为振荡，并拮抗 17β-雌二醇的生长促进作用，支持 IP ₃ R3 在增殖中的作用。后一种效应似乎取决于内质网定位的 IP ₃ R3 和质膜大电导 Ca ²⁺激活的 K ⁺ (BK _Ca ) 通道之间的直接相互作用 (Mound et al. 2013 )。这种耦合允许 IP ₃ R3 激活 BK _Ca通道，导致细胞超极化。 MCF-7 细胞中 IP ₃ R3 或 BK _Ca （或两者）的下调导致细胞周期蛋白 D1 和细胞周期蛋白依赖性激酶 4 水平降低，并导致细胞周期停滞在 G0/G1 期。此外，当比较低迁移 MCF-7 与高迁移和侵袭性 MDA-MB-231 和 MDA-MB-435S 细胞系时，似乎 IP ₃ R3 的较高表达水平与较强的迁移能力相关，可以通过 IP ₃ R3 下调来逆转或通过 IP ₃ R3 过表达来放大 (Mound et al. 2017 )。这一特性可能与以下事实有关：IP ₃ R3 诱发的 Ca ²⁺信号通过影响 Profilin 水平进而影响肌动蛋白细胞骨架来影响细胞形态（Vautrin-Glabik 等人， 2018 ）。

11.7.2 Colorectal Cancer
11.7.2结直肠癌

A study performed in 116 patients indicated that while in normal colorectal mucosa only IP₃R1 and IP₃R2 are expressed, in colorectal carcinomas all three IP₃R isoforms are expressed (Shibao et al. 2010). Moreover, not unlike the case of breast cancer, the IP₃R3 expression was especially high in actively invading cancer cells. IP₃R3 levels thus correlated with cancer aggressiveness and poorer patient survival (Shibao et al. 2010). Noteworthy is the fact that also in Caco-2 colon adenocarcinoma cells IP₃R3 levels inversely correlated with their sensitivity to apoptosis.
对 116 名患者进行的一项研究表明，在正常结直肠粘膜中仅表达 IP ₃ R1 和 IP ₃ R2，而在结直肠癌中则表达所有三种 IP ₃ R 亚型（Shibao 等， 2010 ）。此外，与乳腺癌的情况不同，IP ₃ R3 表达在积极侵袭的癌细胞中特别高。因此，IP ₃ R3 水平与癌症侵袭性和较差的患者生存率相关（Shibao 等人， 2010 ）。值得注意的是，在 Caco-2 结肠腺癌细胞中，IP ₃ R3 水平也与其细胞凋亡敏感性呈负相关。

The role of IP₃R3 in tumor progression was further highlighted in a colorectal adenocarcinoma cell line DLD1 expressing a single oncogenic K-Ras mutation (G13D) making the protein constitutively active. K-Ras is a small monomeric GTPase and a well-known proto-oncogene. Mutations in the K-Ras isoform occur in 30–40% of colorectal cancers (Arrington et al. 2012). In accordance with the results discussed above (Shibao et al. 2010), IP₃R3 silencing made DLD1 colorectal adenocarcinoma cells more sensitive to apoptosis and decreased their migration capacity (Rezuchova et al. 2019). Moreover, inoculation in nude mice of DLD1 cells with complete IP₃R3 knockout led to smaller tumors than when parental DLD1 cells were inoculated.
IP ₃ R3 在肿瘤进展中的作用在结直肠腺癌细胞系 DLD1 中得到进一步强调，该细胞系表达单个致癌 K-Ras 突变 (G13D)，使该蛋白持续活跃。 K-Ras 是一种小型单体 GTP 酶，也是一种众所周知的原癌基因。 K-Ras 亚型突变发生在 30-40% 的结直肠癌中（Arrington et al. 2012 ）。根据上述结果（Shibao et al. 2010 ），IP ₃ R3沉默使DLD1结直肠腺癌细胞对细胞凋亡更加敏感，并降低其迁移能力（Rezuchova et al. 2019 ）。此外，与接种亲代DLD1细胞相比，在裸鼠中接种完全IP ₃ R3敲除的DLD1细胞导致肿瘤更小。

Furthermore, comparison of colorectal cancer cell lines (expressing K-Ras^G13D) with their isogenic derivatives in which the mutated K-Ras allele was deleted (HKH2 and DKO4 cells), allowed a detailed investigation of the relation between oncogenic Ras and Ca²⁺ handling by the IP₃R (Pierro et al. 2014). In absence of oncogenic Ras, a remodeling occurred towards an increased expression of SERCA2B and of IP₃R3 while IP₃R1 level decreased. This remodeling correlated with an increased ER Ca²⁺ content, a higher level of IP₃-induced Ca²⁺ release (IICR), and subsequently more mitochondrial Ca²⁺ uptake and an increased sensitivity to apoptosis. A follow-up study furthermore indicated that the suppression of the oncogenic K-Ras also led to an increased expression of Stim1 (in the HKH2 cell line) or of Stim2 (in the DKO4 cell line) and a larger SOCE in both cell lines, which can contribute to the larger filling state of the ER (Pierro et al. 2018). Although these results fit in the context that increased IP₃R activity is linked to an increased propensity to apoptosis (vide supra) they appear to be in contradiction with the finding mentioned above that higher IP₃R3 levels correlate with increased cancer aggressiveness. Interestingly, the decreased IP₃R activity when oncogenic K-Ras is expressed, is potentially related to the finding that GTP-bound, phosphorylated K-Ras4B forms a trimolecular complex with the IP₃R and the anti-apoptotic protein Bcl-Xl (Sung et al. 2013). Under these conditions, the observed pro-survival stimulatory effect of Bcl-Xl (White et al. 2005) on the IP₃R is counteracted, leading to a less efficient respiration process, induction of autophagy, and ultimately cell death (Sung et al. 2013).
此外，将结直肠癌细胞系（表达 K-Ras ^G13D ）与其删除了突变 K-Ras 等位基因的同基因衍生物（HKH2 和 DKO4 细胞）进行比较，可以详细研究致癌 Ras 和 Ca ²⁺之间的关系由 IP ₃ R 处理（Pierro 等人， 2014 ）。在不存在致癌 Ras 的情况下，发生重构，SERCA2B 和 IP ₃ R3 的表达增加，而 IP ₃ R1 水平降低。这种重塑与增加的 ER Ca ²⁺含量、更高水平的 IP ₃诱导的 Ca ²⁺释放 (IICR) 以及随后更多的线粒体 Ca ²⁺摄取和对细胞凋亡的敏感性增加相关。后续研究进一步表明，抑制致癌 K-Ras 还导致 Stim1（在 HKH2 细胞系中）或 Stim2（在 DKO4 细胞系中）的表达增加，并且这两种细胞系中的 SOCE 更大。这有助于提高内质网的充盈状态（Pierro et al. 2018 ）。尽管这些结果符合IP ₃ R活性增加与细胞凋亡倾向增加相关的背景（见上文），但它们似乎与上述发现相矛盾，即较高的IP ₃ R3水平与癌症侵袭性增加相关。有趣的是，当致癌 K-Ras 表达时，IP ₃ R 活性降低，这可能与 GTP 结合的磷酸化 K-Ras4B 与 IP ₃ R 和抗凋亡蛋白 Bcl-Xl 形成三分子复合物的发现有关。宋等人， 2013 ）。 在这些条件下，观察到的 Bcl-Xl（White 等人， 2005 ）对 IP ₃ R 的促生存刺激作用被抵消，导致呼吸过程效率较低，诱导自噬，并最终导致细胞死亡（Sung 等人） 2013 ）。

11.7.3 Other Cancers
11.7.3其他癌症

Also in other cancer types, a relation between IP₃R levels and/or activity has been found with oncogenic progression. Interestingly, in several types of cancer a specific role was found for IP₃R3, though its role appears to differ between the various cancers, as will be further explained below.
此外，在其他癌症类型中，已发现 IP ₃ R 水平和/或活性与致癌进展之间存在关系。有趣的是，在几种类型的癌症中，发现了 IP ₃ R3 的特定作用，尽管其作用在各种癌症之间似乎有所不同，如下文将进一步解释。

In contrast to healthy hepatocytes, IP₃R3 is for example expressed in patients with hepatocellular carcinoma and this independently of the underlying liver disease (Guerra et al. 2019). This de novo expression of IP₃R3 appears to be due to a specific demethylation of its promoter. Although further work is needed to unravel the mechanisms, the high expression level of IP₃R3 leads not only to enhanced Ca²⁺ signaling but also to changes in expression of various proteins, including upregulation of the anti-apoptotic, pro-survival transcription factor POUAF1. Such remodeling might explain the lower sensitivity to apoptosis and the poorer patient survival.
与健康肝细胞相反，IP ₃ R3 在肝细胞癌患者中表达，并且这与潜在的肝脏疾病无关（Guerra 等人， 2019 ）。 IP ₃ R3 的这种从头表达似乎是由于其启动子的特异性去甲基化。尽管还需要进一步的工作来阐明其机制，但 IP ₃ R3 的高表达水平不仅会导致 Ca ²⁺信号传导增强，还会导致各种蛋白质表达的变化，包括抗凋亡、促生存转录因子的上调POUAF1。这种重塑可能解释了细胞凋亡敏感性较低和患者生存率较低的原因。

In cholangiocarcinoma, the second most important liver malignancy, IP₃R3 levels are increased above normal level in both patient biopsies and in biliary adenocarcinoma cell lines as MzCha1 and HuCCA1 (Ueasilamongkol et al. 2020). Moreover, in MzCha1 cells, the IP₃R3, instead of being concentrated in the subapical ER, was expressed in a more diffuse way, including in close association with the mitochondria. Knockout of IP₃R3 in the cell models led to suppression of mitochondrial Ca²⁺ signaling, arrest of the cells in S-phase, increased cell death, and impaired proliferation and migration.
在胆管癌（第二重要的肝脏恶性肿瘤）中，患者活检组织和 MzCha1 和 HuCCA1 等胆管腺癌细胞系中的 IP ₃ R3 水平均升高至正常水平以上 (Ueasilamongkol et al. 2020 )。此外，在 MzCha1 细胞中，IP ₃ R3 不是集中在顶端下 ER，而是以更分散的方式表达，包括与线粒体密切相关。细胞模型中 IP ₃ R3 的敲除导致线粒体 Ca ²⁺信号传导抑制、细胞停滞在 S 期、细胞死亡增加以及增殖和迁移受损。

In clear cell renal cell carcinoma tumors, IP₃R3 mRNA levels were generally increased, while the mRNA levels of IP₃R1 and IP₃R2 appeared unaffected or even downregulated (Rezuchova et al. 2019). In the corresponding RCC4 cell line model, the silencing of either IP₃R1 or IP₃R3 affected cytosolic Ca²⁺ levels, though interestingly IP₃R1 silencing abolished apoptosis while IP₃R3 silencing strongly upregulated it.
在透明细胞肾细胞癌肿瘤中，IP ₃ R3 mRNA 水平普遍升高，而 IP ₃ R1 和 IP ₃ R2 mRNA 水平似乎不受影响甚至下调（Rezuchova et al. 2019 ）。在相应的 RCC4 细胞系模型中，IP ₃ R1 或 IP ₃ R3 的沉默会影响细胞质 Ca ²⁺水平，但有趣的是，IP ₃ R1 沉默消除了细胞凋亡，而 IP ₃ R3 沉默则强烈上调细胞凋亡。

In contrast with these various cancer cell types, in the lung adenocarcinoma cell line A549, IP₃R2 appeared the most important for cell migration (Huang et al. 2016). Interestingly, overexpression of the intraluminal ER protein PDIA10 (a.k.a. ERp44) inhibited IICR and decreased migration. It however remains to be elucidated how PDIA10 is affecting in those cells IICR, as it was originally described to uniquely interact with IP₃R1 (Higo et al. 2005).
与这些不同的癌细胞类型相比，在肺腺癌细胞系 A549 中，IP ₃ R2 似乎对细胞迁移最重要（Huang et al. 2016 ）。有趣的是，腔内 ER 蛋白 PDIA10（又名 ERp44）的过度表达会抑制 IICR 并减少迁移。然而，PDIA10 如何影响 IICR 细胞仍有待阐明，因为它最初被描述为与 IP ₃ R1 独特地相互作用（Higo 等人， 2005 ）。

From the data presented here, it thus clearly appears that IP₃Rs and IICR are involved in various stages of tumorigenesis, but that their role is not fixed. Depending on the IP₃R isoform expressed, their intracellular localization, and the general cellular context, including cell type-specific expression of various other proteins, IICR can drive or regulate different intracellular pathways and consequently exert another physiological function.
从此处提供的数据可以清楚地看出，IP ₃ Rs 和 IICR 参与肿瘤发生的各个阶段，但它们的作用并不固定。根据表达的 IP ₃ R 同种型、其细胞内定位和一般细胞环境，包括各种其他蛋白质的细胞类型特异性表达，IICR 可以驱动或调节不同的细胞内途径，从而发挥另一种生理功能。

11.8 IP₃Rs Are Targeted by Oncogenes and Tumor Suppressors: A Therapeutic Opportunity?
11.8 IP ₃ R 是癌基因和肿瘤抑制因子的靶标：治疗机会？

Several oncogenes and tumor suppressors were found to regulate IP₃R activity or stability and so to modulate their propensity for apoptosis (Bittremieux et al. 2016; Rosa et al. 2020). Generally speaking, oncogenes promote cell survival and thus tumor formation by suppressing pro-apoptotic Ca²⁺ signaling leading to mitochondrial Ca²⁺ overload, while tumor suppressors will rather stimulate these processes in order to induce apoptosis. Various regulatory proteins derive (part of) their oncogenic or tumor suppressive ability from interfering with the IP₃R-mediated Ca²⁺ signals. As we have reviewed this topic extensively in recent publications (Bittremieux et al. 2016; Parys and Vervliet 2020; Rosa et al. 2020), we have summarized the major properties of these proteins with respect to IP₃R-mediated Ca²⁺ signaling in Table 11.1 (for the tumor suppressors) and Table 11.2 (for the oncogenes) as well as their approximate binding sites on the IP₃R in Fig. 11.1. It should be emphasized that those proteins are not necessarily expressed in all cell types, and that their expression patterns and their functional importance can thus be cancer specific (Morciano et al. 2018). A special case concerns the interaction of various proteins of the Bcl-2 family with the IP₃R. With multiple binding sites and with both inhibitory and stimulatory effects described, the situation is quite complex. As a full description of the effects of those proteins on IICR fall outside the scope of this review, we refer to a recent review for that matter (Ivanova et al. 2020).
发现几种癌基因和肿瘤抑制因子可以调节 IP ₃ R 活性或稳定性，从而调节其凋亡倾向（Bittremieux 等人， 2016 年；Rosa 等人， 2020 年）。一般来说，癌基因通过抑制促凋亡 Ca ²⁺信号传导导致线粒体 Ca ²⁺超载来促进细胞存活，从而促进肿瘤形成，而肿瘤抑制基因会刺激这些过程以诱导细胞凋亡。各种调节蛋白（部分）通过干扰 IP ₃ R 介导的 Ca ²⁺信号而获得致癌或肿瘤抑制能力。正如我们在最近的出版物中广泛回顾了这个主题（Bittremieux et al. 2016 ；Parys and Vervliet 2020 ；Rosa et al. 2020 ），我们总结了这些蛋白质在 IP ₃ R 介导的 Ca ²⁺信号传导方面的主要特性表11.1 （肿瘤抑制基因）和表11.2 （癌基因）以及它们在 IP 上的大致结合位点图11.1中的_3R 。应该强调的是，这些蛋白质不一定在所有细胞类型中表达，因此它们的表达模式和功能重要性可能是癌症特异性的（Morciano et al. 2018 ）。一个特例涉及 Bcl-2 家族的各种蛋白质与 IP ₃ R 的相互作用。由于具有多个结合位点以及所描述的抑制和刺激作用，情况相当复杂。 由于这些蛋白质对 IICR 影响的完整描述超出了本次综述的范围，因此我们参考了最近的一篇综述（Ivanova 等人， 2020 ）。

Table 11.2 List of oncogenes interacting with the IP₃R and their effects
表 11.2 与 IP ₃ R 相互作用的癌基因列表及其影响

To illustrate the potential of the IP₃R in complex with its regulatory proteins to be used as a target for drug development in cancer treatment we would like to highlight some studies in which interfering with IP₃R/Bcl-2 complexes can provoke the death of cancer cells. These translational applications were driven by studies unraveling the molecular determinants underlying IP₃R/Bcl-2 complex formation. In particular, disruption of IP₃R/Bcl-2 complexes can be achieved using peptides containing the interaction site of Bcl-2 in the regulatory and coupling region of the IP₃R1 (amino acids 1389–1408). The binding of Bcl-2 to this site was demonstrated to inhibit IICR and to have an anti-apoptotic action (Rong et al. 2008). Various cell-permeable forms of this disrupting peptide have been developed, which differ in their stability/efficacy, and these have successfully been used to induce apoptosis via enhanced Ca²⁺ release from the ER in chronic lymphocytic leukemia (Zhong et al. 2011), diffuse large B cell lymphoma (Akl et al. 2013; Bittremieux et al. 2019), multiple myeloma and follicular lymphoma (Lavik et al. 2015) and small cell lung cancer (Greenberg et al. 2015) as well as to enhance the sensitivity of ovarian cancer cells towards cisplatin (Xie et al. 2018). Such peptides can also cooperate with BH3-mimetic drugs currently used in the clinic, including venetoclax (Akl et al. 2015; Greenberg et al. 2015; Lavik et al. 2015; Vervloessem et al. 2017). This clearly demonstrates that insights in the molecular underpinnings of IP₃R regulation by regulatory proteins, including oncogenes and tumor suppressors, can successfully be exploited therapeutically.
为了说明 IP ₃ R 与其调节蛋白复合物作为癌症治疗药物开发靶点的潜力，我们想重点介绍一些研究，其中干扰 IP ₃ R/Bcl-2 复合物可能会导致死亡癌细胞。这些转化应用是由揭示 IP ₃ R/Bcl-2 复合物形成的分子决定因素的研究驱动的。特别是，可以使用在 IP ₃ R1 的调节和偶联区域（氨基酸 1389-1408）中含有 Bcl-2 相互作用位点的肽来破坏 IP ₃ R/Bcl-2 复合物。 Bcl-2与该位点的结合被证明可以抑制IICR并具有抗凋亡作用（Rong等人， 2008 ）。人们已经开发出这种破坏性肽的各种细胞渗透形式，其稳定性/功效各不相同，并且这些形式已成功用于通过增强慢性淋巴细胞白血病的内质网释放 Ca ²⁺来诱导细胞凋亡（Zhong 等人， 2011 ） 、弥漫性大 B 细胞淋巴瘤（Akl 等人， 2013 年；Bittremieux 等人， 2019 年）、多发性骨髓瘤和滤泡性淋巴瘤(Lavik et al. 2015 ) 和小细胞肺癌 (Greenberg et al. 2015 ) 以及增强卵巢癌细胞对顺铂的敏感性 (Xie et al. 2018 )。此类肽还可以与目前临床上使用的BH3模拟药物配合使用，包括venetoclax（Akl等人， 2015年；Greenberg等人， 2015年；Lavik等人， 2015年；Vervloessem等人， 2017年）。 这清楚地表明，通过调节蛋白（包括癌基因和肿瘤抑制因子）对 IP ₃ R 调节的分子基础的了解可以成功地用于治疗。

11.9 Conclusions
11.9结论

The IP₃R is a central player controlling many cellular functions. To that effect, IP₃R activity and stability are controlled by a plethora of mechanisms, often involving interacting proteins. Several of these interacting proteins are known oncogenes and tumor suppressors. As such, it comes as no surprise that altered IP₃R signaling is critically altered and contributes to several hallmarks of cancer. This is in large part due to IP₃R localization at the ER-mitochondrial contact sites allowing it to regulate mitochondrial Ca²⁺ levels and subsequently mitochondrial processes including energy production and apoptosis. Interestingly, depending on the cell type under investigation, the various IP₃R isoforms can have different properties and consequently different effects. This can be due to a multitude of reasons, including the presence or absence of regulatory proteins, their subcellular localization and the amplitude of their subsequent Ca²⁺ signals that together will determine which intracellular pathways will be activated. Given the central role of IP₃Rs in most if not all hallmarks of cancer, the IP₃R may have crucial roles in cancer prevention, neoplasia and cancer progression. These insights can be employed to develop novel anti-cancer strategies, as well as providing interesting opportunities for future research and the development of translational applications.
IP ₃ R 是控制许多蜂窝功能的中央播放器。为此，IP ₃ R 活性和稳定性由多种机制控制，通常涉及相互作用的蛋白质。这些相互作用的蛋白质中的一些是已知的癌基因和肿瘤抑制因子。因此，改变的 IP ₃ R 信号传导发生重大改变并导致癌症的多种特征也就不足为奇了。这在很大程度上是由于 IP ₃ R 定位于 ER 线粒体接触位点，使其能够调节线粒体 Ca ²⁺水平以及随后的线粒体过程，包括能量产生和细胞凋亡。有趣的是，根据所研究的细胞类型，各种 IP ₃ R 同工型可能具有不同的特性，从而产生不同的效果。这可能是由于多种原因造成的，包括调节蛋白的存在或不存在、它们的亚细胞定位以及随后的 Ca ²⁺信号的幅度，这些因素共同决定哪些细胞内途径将被激活。鉴于 IP ₃ R 在大多数（如果不是全部）癌症特征中发挥核心作用，IP ₃ R 可能在癌症预防、肿瘤形成和癌症进展中发挥关键作用。这些见解可用于开发新颖的抗癌策略，并为未来的研究和转化应用的开发提供有趣的机会。

11.10 Summary Points
11.10要点总结

• IP₃R receptors control cell death and cell survival decisions critically involved in cancer development
  IP ₃ R 受体控制与癌症发展密切相关的细胞死亡和细胞生存决策

• IP₃R receptors control cellular proliferation, migration and invasion
  IP ₃ R 受体控制细胞增殖、迁移和侵袭

• IP₃R are targets of oncogenes and tumor suppressors
  IP ₃ R 是癌基因和肿瘤抑制因子的靶标

• Expression of specific IP₃R isoforms may differentially impact cancer progression
  特定 IP ₃ R 同工型的表达可能对癌症进展产生不同影响

• Targeting IP₃R-mediated signaling could provide novel therapeutic avenues to fight cancer
  靶向 IP ₃ R 介导的信号传导可以提供对抗癌症的新治疗途径

11.11 Future Issues
11.11未来的问题

• Obtaining detailed structural information about the open and closed states of the various IP₃R isoforms will provide important information about the mode of action of regulatory oncogenes and tumor suppressors
  获得有关各种 IP ₃ R 同种型的开放和关闭状态的详细结构信息将提供有关调节癌基因和肿瘤抑制因子的作用模式的重要信息

• It is anticipated that not all regulatory proteins acting on the IP₃R in cancer have been identified. Moreover, such proteins likely are highly cancer specific, underlying unique opportunities to target such complexes in different cancers. Further research on this topic is urgently needed
  预计并非所有作用于癌症中 IP ₃ R 的调节蛋白均已被鉴定。此外，此类蛋白质可能具有高度的癌症特异性，为在不同癌症中靶向此类复合物提供了独特的机会。迫切需要对该主题进行进一步研究

• The possibility for structural and/or functional interactions between various oncogenes and/or tumor suppressors in the regulation of the IP₃R is presently very insufficiently appreciated. It is however crucial to understand how these proteins affect each other in the regulation of the IP₃R
  目前对IP ₃ R调节中各种癌基因和/或肿瘤抑制因子之间结构和/或功能相互作用的可能性认识还很不够。然而，了解这些蛋白质在 IP ₃ R 调节中如何相互影响至关重要

• Development of (isoform) specific IP₃R inhibitors will further increase the understanding of their exact role in cancer
  （亚型）特异性 IP ₃ R 抑制剂的开发将进一步加深对其在癌症中确切作用的了解

• Targeting the cell-type specific interaction of tumor suppressors and oncogenes with the IP₃R as therapeutic avenue must be further explored and stable peptides and small molecules developed to that effect
  必须进一步探索以肿瘤抑制因子和癌基因与 IP ₃ R 的细胞类型特异性相互作用作为治疗途径，并为此开发稳定的肽和小分子