Файл: Extracellular and Intracellular Signaling (книга).pdf

immediately post-burn are both capable of inducing uncoupling protein expression in muscle. These hormones presumably also trigger a net negative protein balance (see Sections 5.2.1.2 and 5.2.1.4 for putative mechanisms) with the energy liberated being used to produce heat via less e cient oxidative phosphorylation. The ability to attenuate the hypermetabolic state immediately post-burn by use of beta blockers highlights the role of epinephrine as a key inducer of the hypermetabolic state immediately post-burn. Secondly, increased caloric intake can maintain lean body mass while loss of lean body mass results in delayed healing time. This suggests that at least part of the catabolic state is directly tied to increased caloric demand associated with the healing process. Presumably the signals regulating entry into the catabolic state due to nutritional insu ciency are both decreased plasma AA levels and, consequently, decreased insulin levels (see Section 5.3.1 for details of signals which are presumably lacking). Thirdly, sepsis is quite common after thermal injury and can elevate the metabolic rate by an additional 40% (mechanisms and interventions for sepsis are discussed in Section 5.4.3 above). Fourthly, once patients have recovered from the initial burn injury and/or sepsis stages, resistance exercise training and hormone management have both been shown to improve muscle mass (see Section 5.3.2 for putative mechanisms underlying exercise actions on muscle mass). Because insulin and testosterone levels often drop in response to the burn and recombinant growth hormone (stimulates IGF-1 production), IGF-1, insulin and testosterone (or oxandrolone, which has much less potent androgenic e ects) all can counteract muscle wasting, loss of inhibition of protein degradation by insulin/IGF-1 seems a likely contributor to longer-term muscle loss post-burn (see Sections 5.2.2.1 and 5.2.1.3 for insulin/IGF-1 and testosterone mechanisms, respectively).

5.4.5Cancer Cachexia

Patients with pancreatic or gastric cancer have the highest frequency of weight loss, while patients with non-Hodgkin’s lymphoma, breast cancer, acute nonlymphocytic leukemia and sarcomas have the lowest frequency.39,40 Myosin heavy chain is selectively degraded by the ubiquitin proteasome pathway in the cachectic state, while other core myofibrillar proteins including troponin T, tropomyosin (a- and b-forms) and sarcomeric actin remain unchanged. As with thermal injury and sepsis, glucocorticoids may play a role in the development of cancer cachexia, although adrenalectomy has been shown not to alter the course of cachexia in other animal models, which argues against this. In addition, there is considerable evidence from animal studies that TNFa and IL-6 play a role in muscle loss in cancer cachexia, although its role in the human condition may be more questionable. Further discussion of these is unwarranted as their upregulation likely follows a similar track to sepsis and burns. On the other hand, highly specific to tumors is the production of pro- teolysis-inducing factor (PIF), a 24-kDa molecular mass sulfated glycoprotein, originally isolated from the cachexia-inducing MAC16 tumor. PIF has also been shown to inhibit protein synthesis and stimulate protein degradation

directly in isolated murine myotubes. Moreover, urinary PIF excretion is related to weight loss in patients with prostatic and primary gastrointestinal tumors. Mechanistically, PIF expression leads to increased proteasome expression (20S proteasome subunits, MSS1 and p42, another ATPase subunit of the 19S regulator, as well as an increased chymotrypsin-like enzyme activity of the b5 subunits of the proteasome) and activity is likely via inducing NFkB in a ROS-dependent manner. Indeed, studies in animal models of cancer cachexia, as well as in cancer patients, suggest that the ubiquitin proteasome pathway plays the predominant role in the degradation of myofibrillar proteins, particularly in patients with a weight loss of 410%.

Depressed protein synthesis during cancer cachexia in skeletal muscle is related not only to anorexia, as protein synthesis is also depressed in other animal models of cachexia where anorexia is absent. This suggests an underlying defect in the protein synthetic machinery. For example, gastrocnemius muscles from mice bearing the cachexia-inducing tumor MAC16 show activation of protein kinase R (PKR) when the weight loss is 416% and a corresponding increase in phosphorylation of eIF2a (an inhibitor of eIF2Be), which would suppress mRNA translation. Weight loss in mice bearing the MAC16 tumor is also associated with an increased amount of eIF4E bound to 4E-BP1 in gastrocnemius muscle, due to hypophosphorylation of 4E-BP1, resulting in a progressive decrease in the concentration of the active eIF4GeIF4E complex. This would also contribute to a depression in protein synthesis, as would also a decrease in phosphorylation of mTOR and p70S6K1. There is also an increase in the phosphorylation of eEF2, which would also decrease protein synthesis through a decrease in translation elongation.

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normalization.9 It is the process by which tumor cell growth is normalized by contact with neighboring non-transformed cells.

6.2 Contact Normalization

Contact normalization is a powerful process. To become malignant and metastatic tumor cells must overcome inhibition by contact normalization. Cells transformed by a variety of chemicals,10 viral agents9 and oncogenes including Src11–15 can be normalized by contact with non-transformed cells. This process is clearly exemplified by malignant tumor cells that form normal adult organs when injected into mouse blastocysts.16 Demonstrations that a

population of tumor cells can be normalized by as little as three times as many non-transformed cells illustrate the power of this process.17,18

Contact normalization is an important process in vivo. Genetically trans-

formed cells can assume a normal morphology and reside in many organs including skin,4,5,19 breast2,20 and intestine.3 Moreover, since these ‘‘occult

tumor’’ cells are phenotypically normal, they tend to resist chemotherapy.21–23 As stated above, contact normalization is a powerful process; transformed keratinocytes that comprise up to 4% of epidermal volume can be controlled in human skin for decades.24

Taken together, a number of studies indicate that contact normalization is

mediated through direct contact between transformed and non-transformed cells.9,15,25–29 Direct contact between cells is mediated by intercellular junctions

such as adherens and gap junctions. Though much remains to be elucidated, progress has been made in identifying mediators involved in this process.

While not essential, gap junctions appear to facilitate the process of contact normalization.7,8,15,29 In addition, a number of genes associated with contact

normalization have been identified.29–33

6.3 Cadherins

Cadherins form intercellular junctions that are required to maintain normal cell architecture. Cadherins are tethered by catenins to the actin cytoskeleton. Cadherin junctions are disrupted by tyrosine phosphorylation of b-catenin. In addition to disturbing cell morphology, disruption of cadherin junctions allows b-catenin to enter the nucleus and participate in mitogenic transcriptional signaling events.34,35

Cadherins are tumor suppressors. Aberrant cadherin expression is often found in tumor cells.36,37 Interestingly, loss of cadherin expression was found

necessary, but not su cient, for tumor cell invasion in genetic screens for metastatic behavior of tumor suppressor genes in a Drosophila model.38 As expected, expression of wild-type cadherins can suppress transformed cell growth,39 while expression of dominant negative cadherins can enhance cell invasion.40

in many epithelial malignant tumors. The loss or decrease of E-cadherin expression results in the dedi erentiation, loss of epithelial morphology and increase of cellular motility. In the process of EMT, di erentiated epithelial cells acquire attributes that are similar to embryonic mesenchymal cells.41

Like E-cadherin, N-cadherin is also involved in cell adhesion, di erentiation and invasion.42 As with other ‘‘classic’’ cadherins, protein kinase, exemplified by Src, can phosphorylate b-catenin to break N-cadherin junctions with adjacent cells.43–45 In addition, tumor promoting proteases can disrupt N-cadherin junctions to induce b-catenin nuclear signaling.46 It should be noted that N-cadherin can promote the growth of some epithelial tumor cells.42 However, N-cadherin can suppress the growth of many other tumor cells including osteosarcoma,47 ovarian carcinoma,48 glioblastoma and astrocytoma.49–51

6.4 Gap Junctions

Gap junctions form aqueous channels that connect the cytoplasm of adjacent cells. These channels are formed by integral membrane proteins called connexins.

Connexins have evolved into a family of at least 20 mammalian members, which are commonly named by their predicted molecular weights.52–54

Gap junctions allow adjacent cells to share intracellular signals and function in a coordinated fashion.55–57

Evidence indicates that connexins play an important role in cell growth control.58 Like cadherins, experiments have identified connexins as tumor suppressor genes.59–61 In general, gap junctional communication is blocked between transformed cells.62,63 For example, Cx43 expression is robust in normal glial and mammary epithelial cells, but repressed in some human glioma

and mammary carcinoma cells. Moreover, restoration of Cx43 expression can normalize the growth of human glioma and mammary carcinoma cells.61,64,65

Results from experiments with connexin knockout cells and chemical blockers indicate that gap junctional communication is not required for contact normalization.29 This is consistent with previous reports of contact normalization in the absence of dye transfer between transformed and non-transformed cells.14,66 However, while gap junctions are not absolutely required for contact normalization, many reports suggest that gap junctional communication

augments the ability of normal cells to control the growth of neighboring tumor cells.15,26,64,67,68 This has been demonstrated for cells transformed by a variety of agents.12–15

Cx43 can augment the contact normalization of some transformed cells. We have previously shown that inhibiting gap junctional communication with a Cx43 anti-sense construct curtails the ability of non-transformed cells to normalize Src transformed cells in coculture.15 We have also shown that non-transformed cells normalize gap junctional communication with adjacent Src transformed cells.15,69 Thus, signals passed between transformed and non-transformed cells may help normalize the growth of coupled cells.

For example, we have shown that Cx43 can e ectively equilibrate levels of the high-energy metabolite ATP throughout a population of cells. In this way, gap junctional communication could prevent an individual tumor cell from acquiring a level of metabolic energy required to undergo mitosis.55,56

Src phosphorylates Connexin43, but may require downstream events to block gap junctional communication. Like Cas and b-catenin, Cx43 is a functionally relevant Src substrate. Src phosphorylates Cx43 on critical tyrosine residues, and this event can reduce intercellular communication.70–73 However, modification of tyrosine residues 247 and 265 to glutamate does not a ect channel function.74 It has become apparent that Src requires other factors to block gap junctional communication mediated by Cx43. For example, MAPK acts downstream of Src to phosphorylate Cx43 and actually close the gap junction channel. In addition, other components may be involved since potential SH3 binding domains on Cx43 are required for channel closure in Src transformed cells.75 Src and the focal adhesion adaptor protein Cas both possess SH3 domains.76 In addition to Src, Cas associates with Cx43, and Src

utilizes Cas to block gap junctional communication between transformed cells.77,78

As described above, junctions formed by Cx43 and cadherins are disrupted by oncogenic protein kinases in transformed cells. In addition, tumor promoters such as TPA also disrupt Cx43 and cadherin junctions.79 Interestingly, cadherins are required for Cx43 assembly and function in some cells. Disruption of junctions formed by N-cadherin can block gap junctional communication.80 Moreover, induction of N-cadherin has been shown to increase Cx43 expression while inhibiting tumor cell growth.81

6.5 Contact Normalization and Tumor Suppressors

Comprehensive analysis of gene expression has found that less than about 0.01% of the transcriptome is a ected by contact normalization. The expression of most of these gene products is inhibited in transformed cells and induced during contact normalization. Some of these genes can act as tumor suppressors.29,30

Fhl1 and Sdpr provide examples of tumor suppressors that are induced by contact normalization. Fhl1 consists of four-and-a-half LIM domains.

Fhl1 can move between intercellular junctions,82 focal adhesions and the nucleus,83 to a ect gene expression.84,85 For example, Fhl1 associates with the

RBP-J DNA binding protein to modulate gene transcription.84,85 Sdpr is a phosphatidylserine-binding protein86 that is induced during growth arrest by serum deprivation of non-transformed cells, but not transformed cells.87 Fhl1 and Sdpr expression is suppressed in some human tumors including those of the breast, kidney and prostate.32,33 Fhl1 is a functionally relevant protein that inhibits anchorage-independent growth and migration of transformed cells.33 Interestingly, in addition to blocking gap junctional communication,77,78 Src utilizes the Cas adaptor protein to suppress Fhl1 expression.33 This relationship